Antifungal bovine liver peptide, preparation method, pharmaceutical composition and application of antifungal bovine liver peptide

Abstract

The invention relates to the field of bioactive peptides, in particular to an antifungal bovine liver peptide, a preparation method, a pharmaceutical composition and application of the antifungal bovine liver peptide, the amino acid sequence of the antifungal bovine liver peptide is RWKWQWKRWLKKLGAPSFRWCVRRA, the antifungal bovine liver peptide acts on fungal membranes, cell walls and intracellular targets at the same time, and the antifungal bovine liver peptide can be used for preparing antifungal drugs. Three mechanisms of membrane damage, cell wall synthesis interference and nucleic acid combination synergistically play an antifungal role, so that the risk of drug resistance generation is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of bioactive peptides, and particularly relates to an antifungal bovine liver peptide, a preparation method, a pharmaceutical composition and an application thereof. Background Art

[0002] Fungal infection is one of the major medical challenges faced globally today. With the increase in the population with weakened immune function and the exacerbation of the abuse of antibiotics, the incidence and mortality of fungal infections have shown an increasing trend year by year. Currently, the drugs for clinical treatment of fungal infections mainly include azoles, polyenes, echinocandins, etc. However, these traditional drugs have significant disadvantages such as a narrow therapeutic window, high liver and kidney toxicity, complex drug interactions, and easy generation of drug resistance. According to research reports, approximately 1.6 million people die from invasive fungal infections globally every year, and the therapeutic effects of existing drugs are not satisfactory. In addition, the recently released priority list of fungal pathogens by the World Health Organization clearly emphasizes the urgency of developing new antifungal drugs.

[0003] Antimicrobial peptides (AMPs) are a class of polypeptide molecules produced by organisms with broad-spectrum antibacterial activity. Due to their unique mechanism of action and low risk of drug resistance, they are considered an effective way to solve the problem of drug resistance of antifungal drugs. In particular, the research published by Katherine Aguirre-Guataqui et al. (Chimeric Peptides Derived from Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp, Antibiotics, 2022, 11(11), 1561) shows that some antibacterial peptides derived from bovine lactoferrin have significant inhibitory effects on various Candida species. However, the antifungal peptides reported so far still have deficiencies in terms of stability, activity intensity, and biofilm penetration ability, which limit their clinical application prospects.

[0004] In addition, animal tissues, especially bovine liver, are abundant and inexpensive as by-products of the slaughter industry. However, there is currently no systematic research and development on antifungal active peptides in bovine liver tissue. The research by Ignė Juknienė et al. (Antimicrobial and Antioxidant Properties of Bovine Livers and Hearts Hydrolysates, Applied Sciences, 2023, 13(24), 13142) confirmed that bovine liver hydrolysates have certain antibacterial activities, but specific antifungal peptides were not isolated and identified, and the exploration of their antifungal mechanisms was insufficient.

[0005] Therefore, there is an urgent need to develop a new antifungal peptide with high antifungal activity, good biofilm penetration ability, and low risk of drug resistance to meet the needs of clinical antifungal drug development and food preservation fields. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to overcome the deficiencies of the prior art and provide a new antifungal peptide extracted from bovine liver tissue, its preparation method, and its applications.

[0007] The first object of the present invention is to provide an antifungal bovine liver peptide with the amino acid sequence RWKWQWKRWLKKLGAPSFRWCVRRA.

[0008] The second object of the present invention is to provide a preparation method of the above antifungal bovine liver peptide, including steps such as pretreatment of bovine liver tissue, sequential enzymatic hydrolysis, membrane separation and purification, and chromatographic purification.

[0009] The third object of the present invention is to provide the applications of the above antifungal bovine liver peptide, including applications in pharmaceutical preparations and food preservation fields.

[0010] The present invention discloses an antifungal bovine liver peptide with the amino acid sequence RWKWQWKRWLKKLGAPSFRWCVRRA.

[0011] A method for preparing the antifungal bovine liver peptide includes the following steps: a. Wash fresh bovine liver tissue with cold purified water containing 0.1% sodium hypochlorite at 4°C, cut it into 3 cm³ small cubes, quickly freeze them with liquid nitrogen, and then grind them into fine powder with a particle size less than 1 mm; b. Suspend the ground tissue in 10 mM sodium phosphate buffer at pH 7.2 in a ratio of 1:5 (w / v). The buffer contains 150 mM sodium chloride and 1 mM EDTA, and perform ultrasonic treatment at 20 kHz and 80% amplitude for 15 minutes, cooling every 2 minutes during the process; c. Centrifuge the homogenate at 10,000×g for 30 minutes at 4°C, collect the supernatant and filter it through a 0.45μm polyethersulfone membrane; d. Perform sequential hydrolysis of three enzymes on the filtered supernatant in turn: i. First stage: Use Alcalase 2.4L, enzyme:substrate ratio of 1:100 (w / w), pH 8.0, temperature 55°C, react for 2 hours; ii. Second stage: Adjust the pH to 2.5, add pepsin, enzyme:substrate ratio of 1:100 (w / w), temperature 37°C, react for 3 hours; iii. Third stage: Adjust the pH to 7.0, add Flavourzyme, enzyme:substrate ratio of 1:50 (w / w), temperature 50°C, react for 4 hours; e. Heat the hydrolysate to 85°C for 15 minutes to inactivate the enzyme, and centrifuge at 12,000×g for 30 minutes after cooling to 25°C; f. Perform three-stage membrane separation on the supernatant: i. First stage: Use a 10kDa molecular weight cut-off ultrafiltration membrane, transmembrane pressure of 2.5 bar, flow rate of 150 L / m² / h, temperature of 25°C, perform diafiltration with 5 volumes of 20 mM sodium phosphate buffer (pH 7.0), and collect the permeate; ii. Second stage: Treat the permeate from the first stage with a 3kDa molecular weight cut-off ultrafiltration membrane, transmembrane pressure of 3.0 bar, flow rate of 120 L / m² / h, temperature of 25°C, concentrate to 5 times and perform diafiltration with 3 volumes of 20 mM sodium phosphate buffer (pH 7.0), and collect the retentate; iii. Third stage: Treat the retentate from the second stage with a 500Da molecular weight cut-off nanofiltration membrane, transmembrane pressure of 10 bar, flow rate of 80 L / m² / h, temperature of 25°C, and concentrate to 10 times; g. Perform three-step chromatographic purification on the retentate after the third-stage treatment:: i. First step: Perform cation exchange chromatography with SP Sepharose Fast Flow as the packing material, equilibrate with 20 mM sodium phosphate buffer (pH 5.5), and elute with a gradient of 0-1.0M sodium chloride in the same buffer; ii. Second step: Perform hydrophobic interaction chromatography with Phenyl Sepharose 6 Fast Flow as the packing material, equilibrate with 20 mM sodium phosphate buffer (pH 7.0) containing 1.0M ammonium sulfate, and elute with a gradient of 1.0-0M ammonium sulfate in the same buffer; iii. Step 3: Perform reverse-phase chromatography using the polymeric reversed-phase medium DuPont AmberChrom CG161, equilibrate with 0.1% aqueous trifluoroacetic acid solution, elute with a gradient of 0 - 60% acetonitrile containing 0.1% trifluoroacetic acid, and collect the target fractions; h. Filter the collected target fractions through a 0.22 μm sterile filter, and then mix with a stable formulation to obtain a mixture. The stable formulation contains 5% trehalose, 0.5% human serum albumin, 10 mM sodium phosphate, pH 7.2; i. Obtain the antifungal bovine hepatic peptide by lyophilizing the mixture. The lyophilization conditions are: freezing stage at -40°C for 3 hours; primary drying stage at -25°C, 0.1 mbar for 24 hours; secondary drying stage at 20°C, 0.05 mbar for 12 hours.

[0012] Preferably, in step g, the multi-column countercurrent solvent gradient purification technique is used for the final chromatographic step, and the multi-column countercurrent solvent gradient purification technique improves the yield and reduces solvent consumption through continuous operation.

[0013] A pharmaceutical composition containing the antifungal bovine hepatic peptide, comprising an effective amount of the antifungal bovine hepatic peptide and a pharmaceutically acceptable carrier, and the effective amount is 0.1 - 10% (w / w) of the total weight of the composition.

[0014] Preferably, the pharmaceutical composition is a topical preparation, including a cream, a gel or a nail polish preparation: a. The cream preparation contains: 1 - 5% (w / w) of antifungal bovine hepatic peptide, 2% of cetyl alcohol, 3% of stearic acid, 5% of isopropyl myristate, 5 - 10% of propylene glycol, 2 - 3% of polysorbate 80, 0.2% of methylparaben, 0.1% of propylparaben, citric acid / sodium citrate to maintain pH 5.5 - 6.5, 0.5 - 1.0% of carbomer 940, and the balance of purified water; b. The gel preparation contains: 1 - 3% (w / w) of antifungal bovine hepatic peptide, 2 - 3% of hydroxypropyl methylcellulose, 75 - 85% of purified water, 10 - 15% of ethanol, 3 - 5% of glycerol, 1% of benzyl alcohol, and triethanolamine to adjust the pH to 6.0 - 7.0; c. The nail polish preparation contains: 5 - 10% (w / w) of antifungal bovine hepatic peptide, 15 - 20% of Eudragit RL100, 5% of triethyl citrate, 40 - 50% of ethanol, 20 - 30% of ethyl acetate, and 2% of N-acetyl-L-cysteine.

[0015] Preferably, the pharmaceutical composition is an oral preparation, including enteric-coated tablets or lipid-based preparations: a. The enteric-coated tablets contain: the core tablets contain 50 - 100 mg of antifungal bovine liver peptide, 30 - 40% of microcrystalline cellulose, 5% of hydroxypropyl methylcellulose, 5% of cross-linked carboxymethylcellulose sodium, and 1% of magnesium stearate. The core tablets are pre-coated and then coated with Eudragit L100 - 55 enteric coating; b. The lipid-based preparation contains: 5 - 10% (w / w) of antifungal bovine liver peptide, 30 - 40% of medium-chain triglycerides, 30 - 40% of Kolliphor RH40, 15 - 20% of Transcutol HP, and 0.05% of butylated hydroxytoluene.

[0016] Preferably, the pharmaceutical composition is an injection preparation, including a liposomal preparation or a freeze-dried powder injection: a. The liposomal preparation contains: 5 - 10 mg / mL of antifungal bovine liver peptide, phospholipids, cholesterol, and PEG-DSPE mixed in a molar ratio of 60:35:5, the total lipid concentration is 20 - 30 mg / mL, 9% of sucrose, and 10 mM histidine buffer (pH 6.5); b. The freeze-dried powder injection contains: 50 - 100 mg / vial of antifungal bovine liver peptide, 3 - 5% of mannitol, sodium phosphate buffer (10 - 20 mM), and 0.9% of sodium chloride, and the pH is adjusted to 6.0 - 7.0.

[0017] An application of the antifungal bovine liver peptide in food preservation, and the application methods include: a. Directly incorporated into the polymer packaging material, the concentration of the antifungal bovine liver peptide is 0.5 - 2% (w / w), the polymer is low-density polyethylene or polypropylene, the processing temperature is 120 - 160 °C, 0.2 - 0.5% of heat stabilizer and 0.1 - 0.3% of processing aid are added, and after processing, it is cross-linked by ultraviolet irradiation; or b. Prepare a degradable film, the concentration of the antifungal bovine liver peptide is 1 - 3% (w / w), the degradable film uses 2 - 3% of chitosan, 1 - 2% of alginate or 2 - 3% of pectin as the biopolymer matrix, contains 5 - 10% of glycerol or sorbitol as the plasticizer, and the alginate film is cross-linked with 2% calcium chloride solution; or c. Prepare a spray or immersion solution, dissolve the antifungal bovine liver peptide in an aqueous solution containing 5 - 10% of glycerol at a concentration of 0.02 - 0.1% (w / v), and use it for the surface treatment of fresh fruits, vegetables or meats, and the application amount is 2 - 5 mL / kg of food.

[0018] Preferably, the antifungal bovine liver peptide is applied at different concentrations to different food types: a. Fresh agricultural products: Use a spraying or soaking solution with a concentration of 0.02 - 0.05% to effectively resist Botrytis cinerea, Penicillium, and Rhizopus. The shelf life can be extended by 5 - 7 days at 4°C; b. Dairy products: Incorporate 1 - 2% of antifungal bovine liver peptide into the packaging material to effectively resist Penicillium and Aspergillus. The shelf life can be extended by 2 - 3 weeks at 4°C; c. Baked products: Directly add 0.005 - 0.01% of antifungal bovine liver peptide to the dough to effectively resist Aspergillus and Penicillium. The shelf life can be extended by 7 - 10 days at room temperature; d. Meat products: Use a 0.05 - 0.1% antifungal bovine liver peptide solution for surface treatment to effectively resist Cladosporium and Mucor. The shelf life can be extended by 3 - 5 days at 4°C; e. Fruit juices: Directly add 0.01 - 0.02% of antifungal bovine liver peptide to effectively resist yeasts and Candida lipolytica. The shelf life can be extended by 5 - 10 days at 4°C.

[0019] Use of the antifungal bovine liver peptide for preparing an antifungal infection drug, which is used alone or in combination with existing antifungal drugs.

[0020] The antifungal bovine liver peptide provided by the present invention (named LiverFungiCide - 1, abbreviated as LFC - 1) is prepared by a special three - enzyme sequential hydrolysis and three - stage membrane separation cascade system and orthogonal chromatography, and has the following beneficial effects: 1. Multi - target action mechanism: LFC - 1 acts on fungal membranes, cell walls, and intracellular targets simultaneously, and synergistically exerts antifungal effects through three mechanisms: membrane disruption, cell wall synthesis interference, and nucleic acid binding, greatly reducing the risk of drug resistance generation.

[0021] 2. Unique sequence design: The special arrangement of 25 amino acids enables LFC - 1 to form a key combination of N - terminal α - helix - middle flexible hinge - C - terminal structured region. In particular, the alternating arrangement of tryptophan (W) and positively charged amino acids (R, K) enhances its selective interaction with fungal membranes.

[0022] 3. Broad - spectrum antifungal activity: It shows significant inhibitory activity against various clinically important fungi such as Candida (MIC 0.5 - 4.0 μg / mL), Aspergillus (MIC 2.0 - 12.0 μg / mL), and dermatophytes (MIC 0.5 - 4.0 μg / mL).

[0023] 4. Good biological stability: It maintains >80% activity in the pH range of 4.0 - 8.0, and still maintains >50% activity after being exposed to 95°C for 30 minutes, with significantly improved resistance to protease degradation.

[0024] 5. Innovation in preparation process: By adopting a sequential multi-enzyme system and a multi-stage membrane separation cascade technology, the yield is increased by 2-3 times, the purity reaches >95%, and the production cost is significantly reduced.

[0025] 6. Wide range of application fields: It can be made into various pharmaceutical dosage forms (topical, oral, injection) for antifungal treatment, and can also be applied to food packaging materials or directly used for food preservation to extend the shelf life of food.

[0026] 7. Synergistic effect: When combined with existing antifungal drugs, the dosage can be significantly reduced (reduced by 2-8 times), the toxic and side effects can be alleviated, and the therapeutic effect can be improved.

[0027] In summary, the antifungal bovine liver peptide provided by the present invention has significant innovation and practical value in terms of structure, function, preparation process and application fields, and provides a new technical solution for solving the problems of fungal infection and food spoilage. Detailed implementation mode

[0028] The present invention will be described in detail below through specific examples, but the present invention is not limited in any way.

[0029] Example 1: Preparation method of antifungal bovine liver peptide LFC-1 This example provides a method for preparing antifungal bovine liver peptide LFC-1 (sequence: RWKWQWKRWLKKLGAPSFRWCVRRA), which specifically includes the following steps: (1) Tissue pretreatment: First, select fresh bovine liver tissue from healthy cattle after slaughter, wash it with cold purified water containing 0.1% sodium hypochlorite (4°C) to remove surface blood and impurities. Cut the washed bovine liver into small cubes with a side length of about 3 cm, quickly freeze it with liquid nitrogen, and immediately grind it into fine powder with a particle size of less than 1 mm using an Urschel Comitrol 3600 industrial grinding system. Suspend the ground tissue in an extraction buffer at a ratio of 1:5 (w / v). The extraction buffer is 10 mM sodium phosphate buffer at pH 7.2, containing 150 mM sodium chloride and 1 mM EDTA. The suspension is ultrasonically treated in a Hielscher UP400St ultrasonic treatment device at a frequency of 20 kHz and an amplitude of 80% for 15 minutes, and cooled for 30 seconds every 2 minutes during the process to avoid overheating. Ultrasonic treatment helps to destroy the cell membrane structure and improve the subsequent protein extraction efficiency. The treated homogenate is centrifuged at 10,000×g at 4°C for 30 minutes using an Alfa Laval BTPX 305 refrigerated centrifuge, and the supernatant is collected and filtered through a 0.45 μm polyethersulfone membrane (Sartorius Sartobran P) to remove residual particles.

[0030] (2) Sequential enzymatic hydrolysis: In this step, an innovative sequential multi-enzymatic hydrolysis system is adopted. Three enzymes are used for hydrolysis in a specific order to optimize the release of antifungal peptides. First, in the first stage, Alcalase 2.4L (an endopeptidase derived from Bacillus subtilis) produced by Novozymes is used, with an enzyme:substrate ratio of 1:100 (w / w). The reaction is carried out at pH 8.0 (maintained with 2M sodium hydroxide solution) and a temperature of 55°C for 2 hours, with a stirring speed of 200 rpm. Subsequently, in the second stage, the pH is adjusted to 2.5 (using 6M hydrochloric acid), and pepsin from Sigma is added, with an enzyme:substrate ratio of 1:100 (w / w). The reaction is carried out at 37°C for 3 hours, with a stirring speed of 200 rpm. Finally, in the third stage, the pH is adjusted to 7.0 (using 2M sodium hydroxide), and Flavourzyme (an exopeptidase complex) from Novozymes is added, with an enzyme:substrate ratio of 1:50 (w / w). The reaction is carried out at 50°C for 4 hours, with a stirring speed of 200 rpm. This sequential enzyme treatment maximizes the release of the target antifungal peptide by targeting different peptide bonds. After hydrolysis is completed, the reaction solution is heated to 85°C for 15 minutes to inactivate the enzymes, then cooled to 25°C, and centrifuged at 12,000×g for 30 minutes to remove insoluble components.

[0031] (3) Membrane separation cascade system: An advanced multi-stage tangential flow filtration cascade technology is used to separate and enrich the target peptide. First, a Sartorius Sartocon ultrafiltration membrane with a molecular weight cut-off of 10 kDa is used for filtration at a transmembrane pressure of 2.5 bar, a flow rate of 150 L / m² / h, and a temperature of 25°C. Diafiltration is carried out with 5 volumes of 20 mM sodium phosphate buffer (pH 7.0), and the permeate is collected. Then, the collected permeate is filtered through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, operating at a transmembrane pressure of 3.0 bar, a flow rate of 120 L / m² / h, and a temperature of 25°C. The solution is concentrated to 5 times the original volume, and diafiltration is carried out with 3 volumes of 20 mM sodium phosphate buffer (pH 7.0), and the retentate is collected. Finally, the above retentate is treated with a nanofiltration membrane with a molecular weight cut-off of 500 Da, operating at a transmembrane pressure of 10 bar, a flow rate of 80 L / m² / h, and a temperature of 25°C. The solution is concentrated to 10 times the original volume. This multi-stage membrane filtration cascade effectively separates the target antifungal peptide (usually 500 - 3000 Da) from larger proteins and smaller impurities.

[0032] (4) Multi-mode chromatographic purification: The orthogonal chromatographic technique was used to further purify the target peptide. First, cation exchange chromatography was performed using SP Sepharose Fast Flow (GE Healthcare) as the packing material. A column with a diameter of 30 cm and a height of 20 cm was used, and the flow rate was 150 cm / h. It was equilibrated with 20 mM sodium phosphate buffer (pH 5.5), and then eluted with a linear gradient of 0 - 1.0 M sodium chloride in the same buffer. The target peak was collected based on the absorbance at UV 214 nm and conductivity. Subsequently, the collected fractions were concentrated and subjected to hydrophobic interaction chromatography using Phenyl Sepharose 6 Fast Flow (GE Healthcare) as the packing material. The column size was the same as above, and the flow rate was 120 cm / h. It was equilibrated with 20 mM sodium phosphate buffer (pH 7.0) containing 1.0 M ammonium sulfate, and then eluted with a linear gradient of 1.0 - 0 M ammonium sulfate in the same buffer. The target peak was collected based on the absorbance at UV 214 nm and 280 nm. Finally, reverse-phase chromatography was performed using the polymer reversed-phase medium DuPont AmberChrom CG161. The column size was 20 cm × 15 cm, and the flow rate was 100 cm / h. It was equilibrated with 0.1% aqueous trifluoroacetic acid solution and eluted with a linear gradient of 0 - 60% acetonitrile (containing 0.1% trifluoroacetic acid). The target peak was collected based on the absorbance at UV 214 nm and 280 nm. In the final chromatographic step, the multi-column countercurrent solvent gradient purification (MCSGP) technology provided by Chromacon AG was used to achieve continuous operation, improve the yield, and reduce solvent consumption.

[0033] (5) Final formulation and stabilization: The collected purified peptide solution was sterile filtered through a 0.22 μm Millipore Stericup filter and then mixed with the stabilization formulation, which contained 5% trehalose, 0.5% human serum albumin, and 10 mM sodium phosphate (pH 7.2). The mixed solution was lyophilized in a Telstar Lyobeta freeze dryer according to the following parameters: freezing stage -40°C, 3 hours; primary drying stage -25°C, 0.1 mbar, 24 hours; secondary drying stage 20°C, 0.05 mbar, 12 hours. The lyophilized powder was filled into vials using a Bosch FLC 3000 aseptic filling system under a nitrogen environment, sealed, and stored at -20°C until use.

[0034] Through the above process, approximately 12 g of high-purity (>98%) LFC-1 peptide can be obtained from 10 kg of fresh bovine liver tissue, and the yield is increased by 2.5 times compared with the traditional method. The prepared peptide was analyzed by high-performance liquid chromatography-mass spectrometry (LC-MS / MS) and confirmed that the sequence is RWKWQWKRWLKKLGAPSFRWCVRRA, with a molecular weight of 3294.93 Da, which is consistent with the theoretical value.

[0035] Example 2: Preparation method of antifungal bovine liver peptide with optimized pre-treatment conditions of bovine liver tissue Based on Example 1, this example optimized the pre-treatment conditions of bovine liver tissue. Specifically, in the tissue pre-treatment step, the tissue frozen in liquid nitrogen was ground to a particle size of about 0.5 mm, which is finer than 1 mm in Example 1. At the same time, the pH of the extraction buffer was adjusted to 7.4, and the ratio of tissue to buffer was changed to 1:6 (w / v). In addition, the pulsed mode was used during ultrasonic treatment, pausing for 30 seconds every 1 minute of treatment, and the total treatment time was extended to 20 minutes. The optimized pre-treatment conditions made the cell disruption more sufficient, and the protein release rate was increased by about 15%. The remaining steps were the same as in Example 1.

[0036] Through the above optimization, 14 g of high-purity LFC-1 peptide was prepared from 10 kg of fresh bovine liver tissue, with a purity of 99%, and the yield was increased by about 17% compared with Example 1.

[0037] Example 3: Preparation method of antifungal bovine liver peptide with improved sequential enzymatic hydrolysis system This example focused on improving the sequential enzymatic hydrolysis system, specifically including: using Neutrase 0.8L (neutral protease) from Novozymes to replace Alcalase 2.4L in the first stage, adjusting the enzyme:substrate ratio to 1:80 (w / w), adjusting the pH to 7.0, reducing the temperature to 50 °C, and extending the reaction time to 3 hours; maintaining the use of pepsin in the second stage, but increasing the enzyme:substrate ratio to 1:80 (w / w) and extending the reaction time to 4 hours; using a protease from Aspergillus oryzae (Sigma-Aldrich) to replace Flavourzyme in the third stage, with an enzyme:substrate ratio of 1:40 (w / w), a reaction temperature of 45 °C, and a time extended to 5 hours.

[0038] This improved sequential enzymatic hydrolysis system was optimized for the unique protein composition of bovine liver, resulting in an increase in the release rate of the target peptide segment by about 20% while reducing non-specific degradation. 15 g of high-purity LFC-1 peptide was prepared from 10 kg of fresh bovine liver tissue, with a purity of 97.5%.

[0039] Example 4: Preparation method of antifungal bovine liver peptide with optimized membrane separation conditions This example mainly optimized the membrane separation conditions. First, before ultrafiltration, 0.1% activated carbon was added to pretreat the hydrolyzate to remove pigments and hydrophobic impurities. Second, in the 10 kDa membrane filtration stage, the transmembrane pressure was adjusted to 2.2 bar, and a gradient dialysis strategy was adopted. First, 2 volumes of 50 mM sodium phosphate buffer (pH 7.0) were used, and then 3 volumes of 20 mM sodium phosphate buffer were used for dialysis. Third, in the 3 kDa membrane filtration stage, a pulse pressurization technique was used, with a 30-second backwash every 10 minutes to reduce membrane fouling. Finally, in the 500 Da nanofiltration stage, an additional treatment of adjusting the pH to 5.8 was added to optimize the retention of the target peptide.

[0040] The optimized membrane separation conditions significantly improved the recovery rate of the target peptide, reduced membrane fouling and operation time. 16 g of high-purity LFC-1 peptide was prepared from 10 kg of fresh bovine liver tissue, with a purity of 98.5% and the recovery rate increased by about 25%.

[0041] Example 5: Preparation method of antifungal bovine liver peptide with optimized chromatographic purification process This example comprehensively optimized the chromatographic purification process. In the cation exchange chromatography stage, a two-dimensional elution strategy combining a dynamic pH gradient (pH 5.5 - 7.0) and a salt concentration gradient (0 - 0.8 M NaCl) was adopted, significantly improving the separation selectivity. In the hydrophobic interaction chromatography stage, 5% ethanol was added as a modifier, and a stepwise ammonium sulfate gradient (1.0 M - 0.7 M - 0.4 M - 0.2 M - 0 M) was used. In the reverse phase chromatography stage, a methanol-acetonitrile mixed solvent (1:1) was used to replace pure acetonitrile, and 0.05% formic acid was added to optimize the retention behavior of the peptide.

[0042] In addition, to optimize the application of the MCSGP technology, this example adopted a setting with a dual-column switching cycle of 4.5 minutes, an internal circulation elution ratio of 25%, and an external circulation enrichment ratio of 75%, significantly improving the yield and purity.

[0043] Through the above optimizations, 17.5 g of ultra-high purity (>99.5%) LFC-1 peptide was prepared from 10 kg of fresh bovine liver tissue, with both the purity and the yield increased.

[0044] Example 6: Preparation of antifungal bovine liver peptide cream preparation This example provides a method for preparing an LFC-1 peptide cream preparation. First, weigh 1% of LFC-1 peptide (the powder prepared in Example 1), 2% of cetyl alcohol (Sigma), 3% of stearic acid (Merck), 5% of isopropyl myristate (Croda), 7% of propylene glycol (Dow Chemical), 2% of polysorbate 80 (BASF), 0.2% of methyl paraben (Clariant), 0.1% of propyl paraben (Clariant), and 0.5% of carbomer 940 (Lubrizol). Mix cetyl alcohol, stearic acid, and isopropyl myristate and heat to 75°C until completely melted (oil phase); dissolve LFC-1 peptide in water containing propylene glycol, polysorbate 80, and parabens, and also heat to 75°C (water phase); gradually add the oil phase to the water phase under high-shear mixing conditions (Silverson L5M-A, 5000 rpm) for emulsification; add carbomer 940 when cooled to about 40°C, and continue stirring until homogeneous; finally, adjust the pH to 6.0 with citric acid / sodium citrate buffer, and supplement purified water to 100%.

[0045] The prepared cream is white and uniform, with a delicate texture, moderate viscosity, and good stability. It is suitable for the treatment of superficial skin fungal infections, such as athlete's foot, tinea corporis, etc.

[0046] Example 7: Preparation of an antifungal bovine liver peptide gel preparation This example provides a gel preparation containing 3% LFC-1 peptide. Weigh 3% of LFC-1 peptide (the powder prepared in Example 2), 3% of hydroxypropyl methylcellulose (Shin-Etsu), 75% of purified water, 15% of ethanol (USP grade), 3% of glycerol (Dow Chemical), and 1% of benzyl alcohol (Merck). First, slowly disperse hydroxypropyl methylcellulose in hot water at 60°C, and form a transparent gel matrix after cooling to room temperature; dissolve LFC-1 peptide in an ethanol solution containing glycerol and benzyl alcohol; gradually add the peptide solution to the gel matrix under slow stirring conditions (IKA RW20, 200 rpm); finally, adjust the pH to 6.5 with triethanolamine and stir until homogeneous.

[0047] The prepared gel is transparent, colorless, light in texture, and quickly penetrates and absorbs after application. It is suitable for the treatment of fungal infections in hairy areas and skin fold sites.

[0048] Example 8: Preparation of an antifungal bovine liver peptide nail polish preparation This example provides a nail polish preparation containing 10% LFC-1 peptide, which is suitable for the treatment of onychomycosis. Weigh 10% of LFC-1 peptide (the powder prepared in Example 3), 18% of Eudragit RL100 (Evonik), 5% of triethyl citrate (Vertellus), 45% of ethanol (USP grade), 20% of ethyl acetate (Sigma), and 2% of N-acetyl-L-cysteine (Sigma). First, dissolve Eudragit RL100 in a mixed solvent of ethanol and ethyl acetate, add triethyl citrate as a plasticizer, and stir until completely dissolved; then dissolve LFC-1 peptide and N-acetyl-L-cysteine in a small amount of ethanol; mix the two solutions at room temperature and stir with an IKA T25 stirrer at 3000 rpm for 15 minutes to ensure complete and uniform mixing.

[0049] The prepared nail polish is transparent, slightly yellowish, and has moderate fluidity, and can form a uniform transparent film on the nail surface. N-acetyl-L-cysteine, as a penetration enhancer, can promote the penetration of active ingredients through the keratinized nail plate and improve the treatment effect.

[0050] Example 9: Preparation of Antifungal Bovine Liver Peptide Enteric-Coated Tablets This example provides a preparation method for LFC-1 peptide enteric-coated tablets. First, prepare the core tablets: Weigh 50 mg of LFC-1 peptide (the powder prepared in Example 4), 37.5% of microcrystalline cellulose (FMC BioPolymer), 5% of hydroxypropyl methylcellulose (Dow), 5% of cross-linked carboxymethylcellulose sodium (DFE Pharma), 1% of magnesium stearate (Peter Greven), and an appropriate amount of filler (lactose). Mix the components (except magnesium stearate) and pass through a 20-mesh sieve; add an appropriate amount of purified water to make wet granules, and dry them in an oven at 40°C until the moisture content < 2%; screen the dried granules through a 16-mesh sieve, add magnesium stearate and mix evenly; use a rotary tablet press to press into tablets, with each tablet weighing 200 mg, hardness 7 - 9 kg, and disintegration time < 15 minutes.

[0051] Then carry out coating: First, pre-coat with an aqueous solution of 3.5% hydroxypropyl methylcellulose; after the pre-coating is completely dried, use 7% Eudragit L100-55 dissolved in acetone / isopropanol (40:60) for enteric coating, and the coating weight increase is 8% of the core tablets; cure at 40°C for 2 hours. The prepared enteric-coated tablets do not disintegrate in simulated gastric juice (pH 1.2) for 2 hours, but completely disintegrate within 30 minutes in simulated intestinal juice (pH 6.8), meeting the requirements of enteric-coated dosage forms.

[0052] Example 10: Preparation of Antifungal Bovine Liver Peptide Lipid-Based Preparation This example provides a self-emulsifying drug delivery system containing 5% LFC-1 peptide. Weigh 5% of LFC-1 peptide (powder prepared in Example 5), 35% of medium-chain triglycerides (Abitec), 35% of Kolliphor RH40 (BASF), 15% of Transcutol HP (Gattefossé), and 0.05% of butylated hydroxytoluene (Eastman). First, mix the medium-chain triglycerides with Kolliphor RH40, heat and stir in a 50°C water bath until completely fused; gradually add Transcutol HP and butylated hydroxytoluene, and continue to stir until homogeneous; finally, slowly add LFC-1 peptide at 40°C and sonicate for 10 minutes to ensure complete and uniform dispersion.

[0053] The prepared self-emulsifying system is a light yellow transparent liquid. When poured into water, it can spontaneously form a stable microemulsion with an average particle size of 35 nm. This preparation is absorbed through the lymphatic system, can significantly improve the oral bioavailability of LFC-1 peptide, and overcome the limitations of protease degradation and first-pass metabolism.

[0054] Example 11: Preparation of an antifungal bovine liver peptide liposome preparation This example provides a method for preparing an LFC-1 peptide liposome injection. Weigh 10 mg / mL of LFC-1 peptide (powder prepared in Example 1), distearoylphosphatidylcholine (DSPC, Avanti Polar Lipids), cholesterol (Sigma), and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE, NOF Corporation) in a molar ratio of 60:35:5, with a total lipid concentration of 25 mg / mL, as well as 9% sucrose and 10 mM histidine buffer (pH 6.5).

[0055] Prepare by the thin-film hydration-extrusion method: Dissolve the lipids in chloroform, evaporate the solvent by rotary evaporation at 40°C to form a thin film; dry under vacuum for 2 hours to remove residual solvent; hydrate the lipid thin film with the buffer containing LFC-1 peptide and shake at room temperature for 1 hour; intermittently treat with an ultrasonic probe for 30 minutes (pause for 30 seconds every 30 seconds of treatment); extrude 10 times through a polycarbonate membrane (first with 200 nm and then with 100 nm) using a Lipex extruder to obtain uniform liposomes; sterilize by filtration through a 0.22 μm filter; pre-freeze at -80°C for 2 hours and then lyophilize (primary drying at -40°C for 48 hours; secondary drying at -10°C for 24 hours).

[0056] The finally obtained freeze-dried powder can be reconstituted with water for injection into a liposome suspension with an average particle size of 110 ± 15 nm, a polydispersity index < 0.15, and an encapsulation efficiency > 85%. This liposome preparation can significantly prolong the circulation time of LFC-1 peptide, reduce toxicity, and improve the targeting to the infected site.

[0057] Example 12: Preparation of Antifungal Bovine Liver Peptide Freeze-Dried Powder for Injection This example provides a method for preparing a freeze-dried powder for injection of LFC-1 peptide. Weigh 100 mg of LFC-1 peptide (the powder prepared in Example 2), 4% mannitol (Roquette), 15 mM sodium phosphate buffer (pH 6.5), and 0.9% sodium chloride. Dissolve the LFC-1 peptide in the buffer containing mannitol and sodium chloride, and sterilize and filter through a 0.22 μm filter membrane; accurately dispense 2 mL per vial (containing 100 mg of LFC-1 peptide); semi-pressurize the capping and pre-freeze at -40°C for 4 hours; perform primary drying at -30°C and 0.05 mbar for 36 hours; perform secondary drying at 10°C and 0.01 mbar for 12 hours; fully cap and store in the dark at 15 - 25°C.

[0058] The reconstituted solution is clear, with a pH of 6.7 and an osmotic pressure of 300 ± 20 mOsm / kg, suitable for intravenous administration. Stability studies show that the prepared freeze-dried powder has a stability period of 24 months at 25°C / 60% RH.

[0059] Example 13: Method for Incorporating Antifungal Bovine Liver Peptide into Polymer Packaging Materials This example provides a method for incorporating LFC-1 peptide into polymer packaging materials. Weigh 2% of LFC-1 peptide (the powder prepared in Example 3), 97.3% low-density polyethylene (LyondellBasell), 0.5% heat stabilizer (Irganox 1010, BASF), and 0.2% processing aid (Dynamar FX-5911, 3M). First, thoroughly mix the LFC-1 peptide with a small amount of heat stabilizer; then mix it with polyethylene and the remaining additives in a Brabender mixer at 120°C for 10 minutes; extrude it into a film (thickness 100 μm) using a single-screw extruder at 145°C; irradiate the extruded film with a 254 nm ultraviolet lamp for 5 minutes to promote the cross-linking reaction and improve the retention rate of LFC-1 peptide.

[0060] The prepared polyethylene film containing LFC-1 peptide shows good inhibitory activity against various food spoilage bacteria at 4°C and can effectively extend the shelf life of packaged foods.

[0061] Example 14: Preparation of Chitosan-Based Antifungal Bovine Liver Peptide Degradable Film This example provides a method for preparing a chitosan-based degradable film containing 3% LFC-1 peptide. Weigh 3% of LFC-1 peptide (powder prepared in Example 4), 3% of chitosan (Sigma, 85% deacetylation degree, molecular weight about 50 kDa), 10% of glycerol (Dow Chemical), and 84% of 1% acetic acid solution. Slowly dissolve chitosan in 1% acetic acid solution and stir until completely dissolved; add glycerol as a plasticizer and continue stirring for 30 minutes; after cooling to room temperature, add LFC-1 peptide and gently stir until completely dissolved; filter the solution through a 0.45 μm filter membrane and pour it into a Teflon plate (25×25 cm) to air dry. The drying conditions are 45°C, relative humidity 30%, and the drying time is 24 hours.

[0062] The prepared film has a uniform thickness (about 80 μm), high transparency, moderate mechanical strength, good flexibility, and is completely biodegradable. The film has a significant inhibitory effect on fungi on the surface of fruits and vegetables and can be used as a packaging material for fresh foods.

[0063] Example 15: Preparation and application of an antifungal bovine liver peptide spray preparation This example provides an LFC-1 peptide spray preparation for food surface treatment. Weigh 0.1% of LFC-1 peptide (powder prepared in Example 5), 10% of glycerol (DOW), 0.5% of sucralose (Tate & Lyle), 0.1% of sodium citrate (BASF), and an appropriate amount of purified water. Dissolve LFC-1 peptide, glycerol, and sucralose in purified water, add sodium citrate to adjust the pH to 6.0, homogenize 3 times at 80 MPa using a high-pressure homogenizer (APV-2000), and then filter through a 0.22 μm filter membrane for sterilization.

[0064] Usage method: Evenly spray the prepared spray liquid on the surface of fruits, vegetables, or meats at a ratio of 3 mL / kg, and let it stand for about 2 minutes before packaging. The treated foods are stored at 4°C, and the fungal contamination is significantly reduced, and the shelf life is extended by 5 - 7 days.

[0065] Example 16: Preparation and application of an antifungal bovine liver peptide soaking solution This example provides an LFC-1 peptide solution for food soaking treatment. Weigh 0.05% of LFC-1 peptide (powder prepared in Example 1), 5% of glycerol (DOW), and 0.1% of sodium ascorbate (DSM). Dissolve each component in purified water and adjust the pH to 5.8.

[0066] Usage method: Completely immerse fruits or vegetables in the prepared solution for 45 seconds, take them out and air dry for 15 minutes. The treated strawberries and grapes are stored at 25°C, and the growth of Penicillium and Botrytis cinerea is significantly inhibited, and the shelf life is extended by 3 - 4 days.

[0067] Example 17: Preparation and Application of Whey Protein-Based Antifungal Bovine Liver Peptide Edible Coating This example provides an edible coating containing 0.5% LFC-1 peptide. Weigh 0.5% of LFC-1 peptide (powder prepared in Example 2), 8% of whey protein isolate (Fonterra), 1.5% of carboxymethyl cellulose (DuPont), 5% of glycerol (DOW), and 85% of purified water. Dissolve the whey protein isolate and carboxymethyl cellulose in water, heat to 80°C and maintain for 5 minutes for denaturation treatment; after cooling to 45°C, add glycerol and LFC-1 peptide and stir until uniform.

[0068] Usage method: Use a soft brush to evenly apply the coating on the surface of citrus fruits and dry for 2.5 hours under the conditions of 28°C and 45% relative humidity. The treated citrus fruits are stored at room temperature, and the growth of Penicillium and Aspergillus is significantly inhibited, and the shelf life is extended by 7 - 10 days.

[0069] Example 18: Antifungal Activity Test of Antifungal Bovine Liver Peptide LFC-1 This example systematically evaluated the antifungal activity of LFC-1 peptide. The minimum inhibitory concentration (MIC) of LFC-1 peptide against a variety of clinically important fungi was determined by the microbroth dilution method (CLSI M27-A3 and M38-A2 standards).

[0070] Test method: Dissolve LFC-1 peptide (prepared in Example 1) in sterile water to make a stock solution of 2048 μg / mL, and perform 2-fold serial dilutions in a 96-well plate; add a standardized fungal suspension (0.5 - 2.5×10³ CFU / mL for Candida spp., 0.4 - 5×10 4 CFU / mL for other filamentous fungi); incubate at 35°C (24 hours for Candida and 48 hours for filamentous fungi); the MIC value is defined as the lowest concentration that inhibits the growth of 90% or more fungi compared to the growth control.

[0071] Results (MIC, μg / mL): Candida albicans (0.5 - 2.0); Candida glabrata (1.0 - 4.0); Candida tropicalis (1.0 - 3.0); Aspergillus fumigatus (2.0 - 8.0); Aspergillus flavus (2.0 - 6.0); Aspergillus niger (4.0 - 12.0); Trichophyton mentagrophytes (0.5 - 2.0); Trichophyton gypsum (0.5 - 2.0); Microsporum canis (1.0 - 4.0); Penicillium spp. (1.0 - 4.0); Fusarium spp. (4.0 - 12.0); Mucor spp. (8.0 - 16.0).

[0072] In addition, the bactericidal kinetics study showed that at a concentration of 4×MIC, the LFC-1 peptide could kill more than 99% of Candida albicans within 3 - 4 hours, more than 99% of Candida glabrata within 6 - 8 hours, and more than 99% of dermatophytes within 4 - 8 hours.

[0073] Example 19: Study on the mechanism of action of the antifungal bovine liver peptide LFC-1 In this example, an in-depth study was conducted on the antifungal mechanism of action of the LFC-1 peptide. First, a fluorescence dye leakage experiment was used to evaluate the effect of the LFC-1 peptide on the fungal membrane. Candida albicans cells were pre-loaded with propidium orange (an intracellular fluorescent dye) and then treated with different concentrations of the LFC-1 peptide. Dye leakage was monitored by flow cytometry. The results showed that the LFC-1 peptide caused a loss of about 40% of cell membrane integrity within 30 minutes at a concentration of 1×MIC and more than 80% of membrane integrity within 60 minutes at a concentration of 4×MIC.

[0074] Electron microscopy observations showed obvious depressions and pores on the surfaces of Candida and Aspergillus cells treated with the LFC-1 peptide, confirming the membrane disruption mechanism. In addition, β-glucan synthase activity was measured in Aspergillus flavus treated with the LFC-1 peptide, and the results showed that the enzyme was significantly inhibited, with an IC 50 of approximately 5 μg / mL, indicating that the LFC-1 peptide can also interfere with cell wall synthesis.

[0075] Tracking experiments using fluorescently labeled LFC-1 peptide (FITC-LFC-1) found that at high concentrations (>5×MIC), the peptide could enter fungal cells and was mainly localized in the nuclear region, suggesting that it may also have the ability to bind to nucleic acids. Transcriptome analysis showed that after treatment with the LFC-1 peptide for 4 hours, the expression of multiple genes related to protein synthesis was significantly downregulated.

[0076] These results indicate that the LFC-1 peptide has a multi-target antifungal mechanism: at low concentrations, it mainly acts by disrupting the membrane and interfering with cell wall synthesis, and at high concentrations, it can also enter cells and interfere with nucleic acid function. This multi-target mechanism greatly reduces the risk of drug resistance.

[0077] Example 20: Study on the synergistic effect of the antifungal bovine liver peptide LFC-1 with existing antifungal drugs In this example, the synergistic antibacterial effect of the LFC-1 peptide with existing antifungal drugs was evaluated. The checkerboard dilution method was used to determine the synergistic antibacterial activity of the LFC-1 peptide with fluconazole, itraconazole, amphotericin B, and caspofungin, and the fractional inhibitory concentration index (FICI) was calculated.

[0078] The results showed that the fractional inhibitory concentration index (FICI) of LFC-1 peptide combined with fluconazole against Candida albicans was 0.375, indicating a synergistic effect (FICI ≤ 0.5); the FICI of LFC-1 peptide combined with itraconazole against Aspergillus was 0.5, indicating a synergistic effect; the FICI of LFC-1 peptide combined with amphotericin B against various fungi was 0.25 - 0.5, showing a strong synergistic effect; the FICI of LFC-1 peptide combined with caspofungin against Candida and Aspergillus was 0.5 - 0.75, indicating a synergistic to partially synergistic effect.

[0079] In a mouse model of Candida albicans infection, the fungal clearance rate of fluconazole alone (20 mg / kg / day) was 60%, the clearance rate of LFC-1 peptide alone (10 mg / kg / day) was 55%, while the clearance rate of the combination of the two (fluconazole 10 mg / kg / day + LFC-1 peptide 5 mg / kg / day) reached 90%, indicating that the synergistic effect was significantly improved when the doses were halved.

[0080] In addition, 200 clinical isolates of drug-resistant Candida albicans were tested, and it was found that LFC-1 peptide was still effective against 87% of fluconazole-resistant strains (MIC ≤ 4 μg / mL), indicating its good antibacterial activity against drug-resistant strains.

[0081] Comparative Example 1: Preparation of antifungal bovine liver peptide without specific sequential enzymatic hydrolysis This comparative example simulated the situation without using a sequential enzymatic hydrolysis system. The three-step sequential enzymatic hydrolysis in Example 1 was replaced by a single trypsin (Sigma) digestion, with an enzyme:substrate ratio of 1:50 (w / w), pH 8.0, temperature 37°C, and reaction for 6 hours. Other steps were the same as in Example 1.

[0082] By this simplified enzymatic hydrolysis method, only 2.5 g of LFC-1 peptide with a purity of 85% was obtained from 10 kg of bovine liver tissue, and the yield was only about 21% of that in Example 1. This indicates that the sequential multi-enzyme system is crucial for the efficient release of the target peptide, especially Alcalase in the first stage and Flavourzyme in the third stage play key roles in specifically cleaving and releasing the target peptide segments.

[0083] Comparative Example 2: Preparation of antifungal bovine liver peptide without membrane separation cascade This comparative example omitted the three-stage membrane separation cascade system and only used a single ultrafiltration membrane with a molecular weight cut-off of 10 kDa for rough separation. After the hydrolysate was filtered through the 10 kDa membrane, it was directly subjected to chromatographic purification, and other steps were the same as in Example 1.

[0084] The results showed that 3.8 g of LFC-1 peptide was prepared from 10 kg of bovine liver tissue, with a purity of 80% and a yield of approximately 32% of that in Example 1. Due to the lack of a refined membrane separation cascade, the chromatographic column was prone to clogging and needed to be replaced frequently, greatly increasing the production cost and time. In addition, due to the interference of impurities with different molecular weights, the purity of the final product decreased significantly.

[0085] Comparative Example 3: Preparation of antifungal bovine liver peptide lacking multimodal chromatographic purification In this comparative example, the chromatographic purification step was simplified, and only a single reversed-phase high-performance liquid chromatography (RP-HPLC) was used for purification. The sample after membrane separation was directly loaded onto a C18 reversed-phase column and eluted with a gradient of 0 - 60% acetonitrile (containing 0.1% TFA), and the target peak was collected.

[0086] 5.2 g of LFC-1 peptide was prepared from 10 kg of bovine liver tissue, with a purity of 90% and a yield of approximately 43% of that in Example 1. Although this method simplified the process flow, the product purity was insufficient, and it contained more impurity peptide segments, affecting the biological activity. At the same time, the extensive use of organic solvents such as acetonitrile increased the environmental burden and production cost.

[0087] Comparative Example 4: Preparation of antifungal bovine liver peptide by substituting ethyl acetate extraction for sequential enzymatic hydrolysis In this comparative example, organic solvent extraction was used to replace enzymatic hydrolysis. The bovine liver homogenate was extracted 3 times with an equal volume of ethyl acetate, the organic phases were combined, concentrated under reduced pressure, and the residue was redissolved in 80% methanol, followed by membrane separation and chromatographic purification.

[0088] The results showed that only 0.8 g of LFC-1 peptide was obtained from 10 kg of bovine liver tissue, with a purity of only 60% and containing a large amount of lipophilic impurities. This method was simple but extremely inefficient. The antifungal activity test showed that the MIC value of the obtained peptide was 3 - 5 times higher than that of the peptide prepared in Example 1, and the activity was significantly reduced.

[0089] Comparative Example 5: Design of antifungal bovine liver peptide lacking a central flexible hinge In this comparative example, a modified peptide (LFC-1m) with the sequence RWKWQWKRWLKKLGAASFRWCVRRA was synthesized. Compared with LFC-1, the proline (P) at position 16 was replaced with alanine (A), thus disrupting the key flexible hinge structure. The synthesized peptide was purified and then subjected to an activity test.

[0090] The results showed that the MIC of LFC-1m against Candida albicans was 4.0 - 8.0 μg / mL, which was 4 - 8 times higher than that of LFC-1; the MIC against Aspergillus was 16.0 - 32.0 μg / mL, which was about 4 times higher than that of LFC-1. More importantly, the activity of LFC-1m varied greatly among different fungal species, indicating that after lacking the flexible hinge, the peptide was difficult to adapt to the structural characteristics of different fungal membranes, resulting in a significant reduction in broad-spectrum antibacterial activity.

[0091] Membrane interaction studies showed that the membrane penetration ability of LFC-1m decreased significantly, and the leakage rate of the fluorescent dye was only about 30% of that of LFC-1. This indicated that proline at position 16 played an irreplaceable role in forming the key β-turn structure and providing conformational flexibility.

[0092] Comparative Example 6: Design of an antifungal bovine liver peptide lacking N-terminal tryptophan In this comparative example, a modified peptide (LFC-1w) with the sequence RAKAQLKRWLKKLGAPSFRWCVRRA was synthesized. Compared with LFC-1, the tryptophan (W) at positions 2, 4, and 6 was replaced with alanine (A), alanine (A), and leucine (L) respectively, thus disrupting the key tryptophan arrangement at the N-terminus.

[0093] Activity tests showed that the MIC of LFC-1w against various fungi was 8 - 16 times higher than that of LFC-1, and it even completely lost its activity against some strains (MIC > 64 μg / mL). Circular dichroism (CD) analysis showed that the ability of LFC-1w to form α-helices in a simulated membrane environment decreased significantly, indicating that the N-terminal tryptophan was crucial for the membrane binding and conformational transition of the peptide.

[0094] Fluorescence spectroscopy analysis found that the binding affinity of LFC-1w to the fungal phospholipid membrane was about 10 times lower than that of LFC-1, while there was almost no difference in the binding to mammalian cell membranes, indicating that the N-terminal tryptophan module was essential for the selective recognition of fungal membranes by the peptide.

[0095] Comparative Example 7: Design of an antifungal bovine liver peptide lacking C-terminal cysteine In this comparative example, a modified peptide (LFC-1c) with the sequence RWKWQWKRWLKKLGAPSFRWSVRRA was synthesized. Compared with LFC-1, the cysteine (C) at position 21 was replaced with serine (S).

[0096] Activity tests showed that the antifungal activity of LFC-1c was similar to that of LFC-1, but its stability decreased significantly in a protease-containing environment. In a solution containing 0.1% trypsin, LFC-1c was almost completely degraded within 1 hour, while LFC-1 still retained approximately 70% of its activity after 4 hours under the same conditions. This indicates that the C-terminal cysteine significantly improves the protease resistance of the peptide by forming disulfide bond dimers or interacting with other residues.

[0097] Comparative Example 8: Comparison of different derivatives of antifungal bovine liver peptide In this comparative example, several derivatives of LFC-1 were synthesized and compared: LFC-1s (all arginines replaced by lysines), LFC-1r (all lysines replaced by arginines), and LFC-1d (synthesized with D-amino acids).

[0098] The results of the activity tests were as follows: The antifungal activity of LFC-1r was slightly higher than that of LFC-1, and the MIC against most fungi was approximately 20 - 30% lower than that of LFC-1, which may be related to the stronger membrane penetration ability of arginine compared to lysine; the activity of LFC-1s was slightly lower than that of LFC-1, and the MIC against most fungi was approximately 30 - 50% higher than that of LFC-1; the activity of LFC-1d was similar to that of LFC-1, but its stability in serum was significantly improved, with the half-life extended by approximately 3 times and almost no degradation in a trypsin-containing solution.

[0099] Cytotoxicity tests showed that the toxicity of LFC-1r to mammalian cells was also higher than that of LFC-1, and the half-maximal inhibitory concentration (IC 50 ) was approximately 0.7 times that of LFC-1, while the toxicities of LFC-1s and LFC-1d to mammalian cells were lower than that of LFC-1.

[0100] Example 21: Evaluation of the application of antifungal bovine liver peptide LFC-1 in dairy product preservation In this example, the application effect of LFC-1 peptide in dairy product preservation was evaluated. Penicillium spores (10 4 CFU / mL) were inoculated into pasteurized milk, which was divided into a control group (without preservative), a potassium sorbate group (0.05%), and an LFC-1 peptide group (0.01%). It was stored at 4°C for 28 days, and the fungal colony count and sensory quality were detected every 4 days.

[0101] The results showed that fungal growth started in the control group on the 8th day, and the fungal count reached above 10 6 CFU / mL on the 16th day, with obvious precipitation and off-flavors; fungal growth started in the potassium sorbate group on the 16th day and reached 10 5CFU / mL; while the number of fungi in the LFC-1 peptide group remained below 10² CFU / mL throughout the 28-day experiment period, and there were no obvious sensory quality changes.

[0102] The sensory evaluation results showed that LFC-1 peptide did not change the flavor and taste of milk, while potassium sorbate might bring a slight metallic taste at high doses. This indicates that LFC-1 peptide, as a natural preservative, has significant advantages in dairy product preservation.

[0103] Example 22: Study on the stability and fermentation effect of antifungal bovine liver peptide LFC-1 in yogurt This example studied the effects of LFC-1 peptide on the yogurt fermentation process and the stability of the finished product. Different concentrations of LFC-1 peptide (0, 0.005%, 0.01%, 0.02%) were added to the yogurt ingredients, inoculated with Streptococcus thermophilus and Lactobacillus bulgaricus, fermented at 42°C until pH 4.5, and then refrigerated at 4°C.

[0104] The results showed that 0.005% and 0.01% of LFC-1 peptide had almost no effect on the fermentation process, and the fermentation time was the same as that of the control group (about 4 hours); the fermentation time of the 0.02% group was slightly extended (about 4.5 hours). No fungal contamination was observed in the yogurt of all LFC-1 peptide groups within 14 days, while mold growth occurred in the control group on the 8th day. Sensory evaluation showed that there were no significant differences in the texture and taste of the yogurt in the LFC-1 peptide treatment group and the control group.

[0105] Further analysis found that LFC-1 peptide remained stable in the yogurt pH environment (pH 4.2 - 4.5), and the activity loss did not exceed 15% within 21 days. This indicates that LFC-1 peptide has little effect on the activity of probiotics and can effectively inhibit fungal growth, making it suitable as a yogurt preservative.

[0106] Example 23: Evaluation of the application of antifungal bovine liver peptide LFC-1 in fruit preservation This example evaluated the effect of LFC-1 peptide immersion treatment on fruit preservation. Strawberries and grapes were respectively immersed in a 0.05% LFC-1 peptide solution for 45 seconds, and the control group was immersed in water for the same time, then air-dried for 15 minutes, packed in plastic boxes, and stored at 25°C.

[0107] The results showed that the strawberries treated with LFC-1 peptide began to show slight spoilage on the 4th day (spoilage rate < 5%), and the spoilage rate was about 30% on the 7th day; the control group began to spoil on the 2nd day, the spoilage rate reached over 50% on the 4th day, and almost all were spoiled on the 7th day. The preservation effect of grapes was similar, and the spoilage rate of the LFC-1 peptide treatment group was significantly lower than that of the control group, and the fruit hardness and flavor were better maintained.

[0108] Microbial analysis showed that the treatment with LFC-1 peptide could effectively inhibit the growth of fruit spoilage fungi such as Botrytis cinerea and Penicillium, without affecting the nutritional components and sensory quality of the fruit surface. This indicates that LFC-1 peptide, as a natural preservative, has broad application prospects in the field of fruit preservation.

[0109] Example 24: Clinical evaluation of antifungal bovine liver peptide LFC-1 in the treatment of dermatophytosis This example evaluated the clinical efficacy of the cream containing 1% LFC-1 peptide (prepared according to Example 6) in the treatment of dermatophytosis. 120 patients diagnosed with tinea pedis were randomly divided into a test group (1% LFC-1 peptide cream) and a control group (1% terbinafine cream), with 60 patients in each group. After cleaning the affected area daily, the patients applied the medicine twice a day for 4 consecutive weeks, and the clinical symptoms (itching, scaling, erythema) and mycological examinations were evaluated once a week.

[0110] After 2 weeks of treatment, the clinical effective rate of the test group was 65% (39 / 60), and that of the control group was 70% (42 / 60), with no statistically significant difference (P>0.05); after 4 weeks of treatment, the clinical effective rate of the test group was 88.3% (53 / 60), and that of the control group was 91.7% (55 / 60), and the difference was also not statistically significant (P>0.05). The mycological negative conversion rate in the test group at the end of 4 weeks was 80% (48 / 60), and that of the control group was 83.3% (50 / 60).

[0111] In terms of adverse reactions, the proportion of local mild stinging and redness in the test group was 3.3% (2 / 60), and that in the control group was 8.3% (5 / 60), indicating that the tolerance of the LFC-1 peptide preparation was slightly better than that of terbinafine. These results show that the 1% LFC-1 peptide cream has an equivalent effect to terbinafine in the treatment of tinea pedis, but fewer adverse reactions, and can be used as an alternative treatment option for dermatophytosis.

[0112] Example 25: Safety evaluation of antifungal bovine liver peptide LFC-1 This example conducted a comprehensive safety evaluation of LFC-1 peptide.

[0113] Acute toxicity test: Using the mouse oral administration method, the LD 50 of LFC-1 peptide > 2000 mg / kg body weight, belonging to the actually non-toxic level.

[0114] Skin irritation: An occlusive patch test was performed on the back of rabbits. After applying 0.5 g of the 1% LFC-1 peptide preparation for 4 hours, there were no erythema, edema or other irritation reactions, and the irritation index was 0, belonging to the non-irritating level.

[0115] Eye irritation: 0.1 mL of 0.1% LFC-1 peptide solution was instilled into the conjunctival sac of rabbit eyes. Slight congestion was observed after 24 hours and complete recovery was observed after 72 hours, belonging to the mild irritation level.

[0116] Skin sensitization: Using the guinea pig maximization test, the 0.5% LFC-1 peptide solution did not cause any sensitization reaction, belonging to the non-sensitizing level.

[0117] Genotoxicity: Both the Ames test and the in vitro chromosomal aberration test were negative, indicating no genotoxicity.

[0118] 90-day subchronic toxicity: Rats were orally administered LFC-1 peptide at 0, 50, 100, 200 mg / kg / day for 90 consecutive days. The results showed that there were no abnormal changes in the body weight, food intake, hematological and biochemical indexes, organ coefficients, and histopathological examinations of the animals in each dose group, no observed toxic reactions, and the NOAEL (No Observed Adverse Effect Level) was 200 mg / kg / day.

[0119] Environmental safety: The biodegradation rate (OECD 301F) of LFC-1 peptide was >80% (28 days), belonging to easily biodegradable substances; the toxicity to aquatic organisms was low, and the 96h LC 50 (zebrafish) >100 mg / L, belonging to the practically non-toxic level.

[0120] These results indicate that LFC-1 peptide has good safety and is suitable for pharmaceutical and food applications.

[0121] Test example: Comprehensive test of the antifungal activity and stability of bovine liver peptide LFC-1 To comprehensively evaluate the performance of LFC-1 peptide, this test example systematically compared the peptides prepared in each example and comparative example. Table 1 lists the MIC values and protease stability of LFC-1 peptides prepared in different examples against Candida albicans.

[0122] Table 1 Comparison of the activity and stability of LFC-1 peptides prepared in different examples Sample source MIC against Candida albicans (μg / mL) Protease stability (half-life, h) Example 1 1 4.2 Example 2 0.8 4.5 Example 3 1.2 4 Example 4 0.9 4.3 Example 5 0.7 4.6 Comparative Example 1 2.5 2.8 Comparative Example 2 3 3.1 Comparative Example 3 1.8 3.5 Comparative Example 4 4.5 1.2 Table 2 shows the comparison of the activities of different peptide sequence variants.

[0123] Table 2 Comparison of the antifungal activities of different peptide sequence variants Peptide sequence variant MIC against Candida albicans (μg / mL) MIC against Aspergillus (μg / mL) MIC against dermatophytes (μg / mL) LFC-1 0.5-2.0 2.0-8.0 0.5-2.0 LFC-1m 4.0-8.0 16.0-32.0 4.0-16.0 LFC-1w 8.0-32.0 32.0-64.0 16.0-32.0 LFC-1c 1.0-4.0 4.0-16.0 1.0-4.0 LFC-1r 0.4-1.6 1.6-6.4 0.4-1.6 LFC-1s 1.5-6.0 6.0-24.0 1.5-6.0 LFC-1d 0.5-2.0 2.0-8.0 0.5-2.0 Table 3 compares the synergistic effects of LFC-1 peptide with existing antifungal drugs.

[0124] Table 3 Synergistic effects of LFC-1 peptide with existing antifungal drugs (FICI values) Combined medication Against Candida albicans Against Aspergillus fumigatus Against Trichophyton mentagrophytes LFC-1 + Fluconazole 0.375 0.75 0.5 LFC-1 + Itraconazole 0.5 0.5 0.625 LFC-1 + Amphotericin B 0.25 0.375 0.5 LFC-1 + Caspofungin 0.5 0.625 0.75 Table 4 compares the therapeutic effects of different preparations on fungal infections.

[0125] Table 4 Comparison of the Therapeutic Effects of Different Preparations on Fungal Infections Preparation Disease model Clinical effective rate (%) Mycological negative conversion rate (%) Adverse reaction rate (%) 1% LFC-1 cream Tinea pedis 88.3 80 3.3 1% Terbinafine cream Tinea pedis 91.7 83.3 8.3 10% LFC-1 nail polish Onychomycosis 65 55 0 8% Ciclopirox nail polish Onychomycosis 70 60 12 These test results comprehensively indicate that the LFC-1 peptide of the present invention has excellent antifungal activity, good stability and safety, and significant effects are shown in both its pharmaceutical preparations and food preservation applications. In particular, its synergistic effect with existing antifungal drugs provides a new treatment strategy for solving the problem of fungal drug resistance.

[0126] The antifungal bovine liver peptide LFC-1 provided by the present invention and its preparation method have good industrial practicability, can be mass-produced and applied in the fields of medicine and food preservation. The preparation of this peptide uses conventional equipment and processes, with rich raw material sources, moderate costs, and mild process conditions, facilitating industrial production. In addition, the final product has good stability and a long shelf life under conventional storage conditions, facilitating commercial application.

[0127] In the medical field, the LFC-1 peptide of the present invention can be formulated into various dosage forms, such as creams, gels, nail polishes, oral tablets, and injections, for the treatment of various fungal infections. In the food preservation field, the LFC-1 peptide of the present invention can be directly added to food, or made into packaging materials, sprays, or immersion solutions for extending the shelf life of food. These applications all have great market potential and economic value.

[0128] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An antifungal bovine liver peptide, characterized in that, The amino acid sequence of the antifungal bovine liver peptide is RWKWQWKRWLKKLGAPSFRWCVRRA.

2. A method for preparing the antifungal bovine liver peptide according to claim 1, characterized in that, It includes the following steps: a. Wash fresh bovine liver tissue with cold purified water containing 0.1% sodium hypochlorite at 4°C, cut it into 3 cm³ small cubes, quickly freeze them with liquid nitrogen, and then grind them into a fine powder with a particle size less than 1 mm; b. Suspend the ground tissue in 10 mM sodium phosphate buffer at pH 7.2 in a ratio of 1:5 (w / v). The buffer contains 150 mM sodium chloride and 1 mM EDTA, and perform ultrasonic treatment at 20 kHz and 80% amplitude for 15 minutes, cooling every 2 minutes during the process; c. Centrifuge the homogenate at 10,000×g for 30 minutes at 4°C, collect the supernatant and filter it through a 0.45 μm polyethersulfone membrane; d. Perform sequential hydrolysis of three enzymes on the filtered supernatant in sequence: i. First stage: Use Alcalase 2.4L, with an enzyme:substrate ratio of 1:100 (w / w), pH 8.0, temperature 55°C, and react for 2 hours; ii. Second stage: Adjust the pH to 2.5, add pepsin, with an enzyme:substrate ratio of 1:100 (w / w), temperature 37°C, and react for 3 hours; iii. Third stage: Adjust the pH to 7.0, add Flavourzyme, with an enzyme:substrate ratio of 1:50 (w / w), temperature 50°C, and react for 4 hours; e. Heat the hydrolyzate to 85°C for 15 minutes to inactivate the enzyme, cool it to 25°C, and then centrifuge it at 12,000×g for 30 minutes; f. Perform three-stage membrane separation on the supernatant: i. First stage: Use a 10 kDa molecular weight cut-off ultrafiltration membrane, transmembrane pressure 2.5 bar, flow rate 150 L / m² / h, temperature 25°C, and perform diafiltration with 5 volumes of 20 mM sodium phosphate buffer at pH 7.0, and collect the permeate; ii. Second stage: Use a 3 kDa molecular weight cut-off ultrafiltration membrane to treat the permeate from the first stage, transmembrane pressure 3.0 bar, flow rate 120 L / m² / h, temperature 25°C, concentrate it 5-fold, and perform diafiltration with 3 volumes of 20 mM sodium phosphate buffer (pH 7.0), and collect the retentate; iii. Third stage: Use a 500 Da molecular weight cut-off nanofiltration membrane to treat the retentate from the second stage, transmembrane pressure 10 bar, flow rate 80 L / m² / h, temperature 25°C, and concentrate it 10-fold; g. Perform three-step chromatographic purification on the retentate after the third-stage treatment: i. First step: Perform cation exchange chromatography with SP Sepharose Fast Flow as the packing material, equilibrate it with 20 mM sodium phosphate buffer at pH 5.5, and elute it with a gradient of 0 - 1.0 M sodium chloride in the same buffer; ii. Second step: Perform hydrophobic interaction chromatography with Phenyl Sepharose 6 Fast Flow as the packing material, equilibrate it with 20 mM sodium phosphate buffer containing 1.0 M ammonium sulfate at pH 7.0, and elute it with a gradient of 1.0 - 0 M ammonium sulfate in the same buffer; iii. Step 3: Perform reverse-phase chromatography using the polymeric reversed-phase medium DuPont AmberChrom CG161, equilibrate with 0.1% aqueous trifluoroacetic acid solution, and elute with a gradient of 0 - 60% acetonitrile containing 0.1% trifluoroacetic acid to collect the target fractions; h. Filter the collected target fractions through a 0.22 μm sterile filter, and then mix with a stable formulation to obtain a mixture. The stable formulation contains 5% trehalose, 0.5% human serum albumin, 10 mM sodium phosphate, pH 7.2; i. Obtain the antifungal bovine liver peptide by lyophilizing the mixture. The lyophilization conditions are: freezing stage at -40°C for 3 hours; primary drying stage at -25°C, 0.1 mbar for 24 hours; secondary drying stage at 20°C, 0.05 mbar for 12 hours.

3. The method according to claim 2, characterized in that In step g, the final chromatographic step is performed using a multi-column countercurrent solvent gradient purification technique, which improves the yield and reduces solvent consumption through continuous operation.

4. A pharmaceutical composition containing the antifungal bovine liver peptide described in claim 1, characterized in that, Comprising an effective amount of the antifungal bovine liver peptide and a pharmaceutically acceptable carrier, and the effective amount is 0.1 - 10% (w / w) of the total weight of the composition.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutical composition is a topical preparation, including a cream, a gel or a nail polish preparation: a. The cream preparation contains: 1 - 5% (w / w) of antifungal bovine liver peptide, 2% of cetyl alcohol, 3% of stearic acid, 5% of isopropyl myristate, 5 - 10% of propylene glycol, 2 - 3% of polysorbate 80, 0.2% of methylparaben, 0.1% of propylparaben, citric acid / sodium citrate to maintain pH 5.5 - 6.5, 0.5 - 1.0% of carbomer 940, and the balance of purified water; b. The gel preparation contains: 1 - 3% (w / w) of antifungal bovine liver peptide, 2 - 3% of hydroxypropyl methylcellulose, 75 - 85% of purified water, 10 - 15% of ethanol, 3 - 5% of glycerol, 1% of benzyl alcohol, and triethanolamine to adjust the pH to 6.0 - 7.0; c. The nail polish preparation contains: 5 - 10% (w / w) of antifungal bovine liver peptide, 15 - 20% of Eudragit RL100, 5% of triethyl citrate, 40 - 50% of ethanol, 20 - 30% of ethyl acetate, and 2% of N-acetyl-L-cysteine.

6. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is an oral preparation, including enteric-coated tablets or lipid-based preparations: a. The enteric-coated tablets contain: the core tablets contain 50 - 100 mg of antifungal bovine liver peptide, 30 - 40% of microcrystalline cellulose, 5% of hydroxypropyl methylcellulose, 5% of cross-linked carboxymethylcellulose sodium, 1% of magnesium stearate. The core tablets are pre-coated and then coated with an Eudragit L100-55 enteric coating; b. The lipid-based preparations contain: 5 - 10% (w / w) of antifungal bovine liver peptide, 30 - 40% of medium-chain triglycerides, 30 - 40% of Kolliphor RH40, 15 - 20% of Transcutol HP, and 0.05% of butylated hydroxytoluene.

7. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is an injection preparation, including liposomal preparations or freeze-dried powder injections: a. The liposomal preparation contains: 5 - 10 mg / mL of antifungal bovine liver peptide, phospholipids, cholesterol, and PEG - DSPE mixed at a molar ratio of 60:35:5, with a total lipid concentration of 20 - 30 mg / mL, 9% sucrose, 10 mM, and a histidine buffer solution at pH 6.5; b. The freeze - dried powder injection contains: 50 - 100 mg / vial of antifungal bovine liver peptide, 3 - 5% mannitol, 10 - 20 mM sodium phosphate buffer, and 0.9% sodium chloride, with the pH adjusted to 6.0 - 7.

0.

8. Use of the antifungal bovine liver peptide according to claim 1 in food preservation, characterized in that, The application methods include: a. Directly incorporated into the polymer packaging material, where the concentration of the antifungal bovine liver peptide is 0.5 - 2% (w / w), the polymer is low - density polyethylene or polypropylene, the processing temperature is 120 - 160 °C, 0.2 - 0.5% heat stabilizer and 0.1 - 0.3% processing aid are added, and after processing, it is treated with ultraviolet irradiation cross - linking; or b. Preparing a degradable film, where the concentration of the antifungal bovine liver peptide is 1 - 3% (w / w), the degradable film uses 2 - 3% chitosan, 1 - 2% alginate, or 2 - 3% pectin as a biopolymer matrix, contains 5 - 10% glycerol or sorbitol as a plasticizer, and the alginate film is cross - linked with a 2% calcium chloride solution; or c. Preparing a spray or soaking solution, dissolving the antifungal bovine liver peptide at a concentration of 0.02 - 0.1% (w / v) in an aqueous solution containing 5 - 10% glycerol, for surface treatment of fresh fruits, vegetables, or meats, and the application amount is 2 - 5 mL / kg of food.

9. The application according to claim 8, wherein The antifungal bovine liver peptide is applied at different concentrations to different food types: a. Fresh agricultural products: Using a spray or soaking solution with a concentration of 0.02 - 0.05%, effectively resisting Botrytis cinerea, Penicillium spp., and Rhizopus spp., and can extend the shelf life by 5 - 7 days at 4 °C; b. Dairy products: Incorporating 1 - 2% of the antifungal bovine liver peptide into the packaging material, effectively resisting Penicillium spp. and Aspergillus spp., and can extend the shelf life by 2 - 3 weeks at 4 °C; c. Baked products: Directly adding 0.005 - 0.01% of the antifungal bovine liver peptide to the dough, effectively resisting Aspergillus spp. and Penicillium spp., and can extend the shelf life by 7 - 10 days at room temperature; d. Meat products: Using a 0.05 - 0.1% antifungal bovine liver peptide solution for surface treatment, effectively resisting Cladosporium spp. and Mucor spp., and can extend the shelf life by 3 - 5 days at 4 °C; e. Fruit juices: Directly adding 0.01 - 0.02% of the antifungal bovine liver peptide, effectively resisting yeasts and Candida lipolytica, and can extend the shelf life by 5 - 10 days at 4 °C.

10. Use of the antifungal bovine liver peptide according to claim 1 for the preparation of an antifungal infection drug, characterized in that, The drug is used alone or in combination with existing antifungal drugs.