Antifungal bovine liver peptide, preparation method, pharmaceutical composition and application thereof

Abstract

The present invention relates to the field of bioactive peptides, and specifically to an antifungal bovine liver peptide, a preparation method, a pharmaceutical composition and applications thereof. The antifungal bovine liver peptide has an amino acid sequence of RWKWQWKRWLKKLGAPSFRWCVRRA, and acts simultaneously on fungal membranes, cell walls and intracellular targets, synergistically exerting antifungal effects through three mechanisms: membrane destruction, cell wall synthesis interference and nucleic acid binding, thereby greatly reducing the risk of drug resistance.

Description

Technical Field

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

[0002] Fungal infection is one of the major medical challenges facing the world today. With the increase in the number of people with weakened immune systems and the intensification of the abuse of antibiotics, the morbidity and mortality of fungal infections have shown an upward trend year by year. Currently, the drugs used in the clinical treatment of fungal infections mainly include azoles, polyenes and echinocandins, but these traditional drugs have significant disadvantages such as narrow therapeutic window, high hepatotoxicity and kidney toxicity, complex drug interactions and easy development of drug resistance. According to research reports, approximately 1.6 million people die from invasive fungal infections worldwide each year, and the treatment effects of existing drugs are unsatisfactory. In addition, the priority list of fungal pathogens recently released 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 antimicrobial activity. Due to their unique mechanism of action and low risk of drug resistance, they are considered to be an effective way to address the problem of antifungal drug resistance. In particular, a study published by Katherine Aguirre-Guataqui et al. (Chimeric Peptides Derivedfrom Bovine Lactoferricin and Buforin II: Antifungal Activity against Reference Strains and Clinical Isolates of Candida spp, Antibiotics, 2022, 11(11), 1561) showed that some antimicrobial peptides derived from bovine lactoferricin have significant inhibitory effects on a variety of Candida species. However, the antifungal peptides reported so far still have deficiencies in stability, activity intensity, and biofilm penetration ability, which limits their clinical application prospects.

[0004] In addition, animal tissues, especially beef liver, are abundant and inexpensive by-products of the slaughtering industry. However, there has been no systematic research and development of antifungal peptides from beef liver tissue. A study by Ignė Juknienė et al. (Antimicrobial and Antioxidant Properties of Bovine Livers and Hearts Hydrolysates, Applied Sciences, 2023, 13(24), 13142) confirmed that beef liver hydrolysate has certain antibacterial activity, but failed to isolate and identify specific antifungal peptides, and the exploration of their antifungal mechanism of action is insufficient.

[0005] Therefore, there is an urgent need to develop a new antifungal peptide with efficient antifungal activity, good biofilm penetration ability and low risk of drug resistance to meet the needs of clinical antifungal drug development and food preservation. 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 novel antifungal peptide extracted from bovine liver tissue, and a preparation method and application thereof.

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

[0008] The second object of the present invention is to provide a method for preparing the antifungal bovine liver peptide, which comprises the steps of 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 applications of the antifungal bovine liver peptide, including applications in the fields of pharmaceutical preparations and food preservation.

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

[0011] A method for preparing the antifungal bovine liver peptide comprises the following steps:

[0012] a. Fresh bovine liver tissue was washed with cold purified water containing 0.1% sodium hypochlorite at 4°C, cut into 3 cm³ cubes, and quick-frozen in liquid nitrogen. The resulting powder was then ground into a particle size of less than 1 mm.

[0013] b. The ground tissue was suspended in 10 mM sodium phosphate buffer at a ratio of 1:5 (w / v) at pH 7.2, containing 150 mM sodium chloride and 1 mM EDTA, and sonicated at 20 kHz and 80% amplitude for 15 minutes, cooling every 2 minutes;

[0014] c. The homogenate was centrifuged at 10,000 × g for 30 min at 4°C, and the supernatant was collected and filtered through a 0.45 μm polyethersulfone membrane;

[0015] d. The filtered supernatant was sequentially hydrolyzed with three enzymes:

[0016] i. Phase I: using Alcalase 2.4 L, enzyme: substrate ratio of 1:100 (w / w), pH 8.0, temperature 55 ° C, reaction time 2 hours;

[0017] ii. Second stage: adjust pH to 2.5, add pepsin, enzyme: substrate ratio is 1:100 (w / w), temperature 37 ° C, reaction for 3 hours;

[0018] iii. The third stage: adjust the pH to 7.0, add Flavourzyme, enzyme: substrate ratio of 1:50 (w / w), temperature 50 ° C, reaction for 4 hours;

[0019] e. The hydrolyzate was heated to 85 ° C for 15 minutes to inactivate the enzyme, cooled to 25 ° C and centrifuged at 12,000 × g for 30 minutes;

[0020] f. Perform three-stage membrane separation on the supernatant:

[0021] i. First stage: using a 10 kDa molecular weight cutoff ultrafiltration membrane, a transmembrane pressure of 2.5 bar, a flow rate of 150 L / m² / h, a temperature of 25 ° C, diafiltration with 5 volumes of 20 mM sodium phosphate buffer (pH 7.0), and collecting the permeate;

[0022] ii. Second stage: The first stage permeate was treated with a 3 kDa molecular weight cutoff ultrafiltration membrane at a transmembrane pressure of 3.0 bar, a flow rate of 120 L / m² / h, and a temperature of 25°C. The permeate was concentrated to 5 times and diafiltered with 3 volumes of 20 mM sodium phosphate buffer (pH 7.0). The retentate was collected.

[0023] iii. Third stage: The retentate from the second stage was treated using a 500 Da molecular weight cutoff nanofiltration membrane at a transmembrane pressure of 10 bar, a flow rate of 80 L / m² / h, and a temperature of 25°C, and concentrated to 10 times.

[0024] g. The retentate after the third stage treatment was subjected to three-step chromatography purification:

[0025] i. Step 1: Cation exchange chromatography was performed using SP Sepharose Fast Flow as the filler, equilibrated with 20 mM sodium phosphate buffer (pH 5.5), and eluted with a gradient of 0-1.0 M sodium chloride in the same buffer;

[0026] ii. Step 2: Hydrophobic interaction chromatography was performed using Phenyl Sepharose 6 Fast Flow as a filler, equilibrated with 20 mM sodium phosphate buffer (pH 7.0) containing 1.0 M ammonium sulfate, and eluted with a gradient of 1.0-0 M ammonium sulfate in the same buffer;

[0027] iii. Step 3: Reverse phase chromatography was performed using a polymer reverse phase medium DuPont AmberChrom CG161, equilibrated with 0.1% trifluoroacetic acid aqueous solution, and eluted with a gradient of 0-60% acetonitrile containing 0.1% trifluoroacetic acid to collect the target fractions;

[0028] h. The collected target fraction was filtered through a 0.22 μm sterilizing grade filter and then mixed with a stabilizing formulation to obtain a mixture comprising 5% trehalose, 0.5% human serum albumin, 10 mM sodium phosphate, pH 7.2;

[0029] i. The mixture was freeze-dried to obtain antifungal bovine liver peptide. The freeze-drying conditions were: freezing stage at -40 ° C for 3 hours; main drying stage at -25 ° C, 0.1 mbar for 24 hours; secondary drying stage at 20 ° C, 0.05 mbar for 12 hours.

[0030] Preferably, in step g, the final chromatography step is performed using a multi-column countercurrent solvent gradient purification technique, which improves yield and reduces solvent consumption through continuous operation.

[0031] A pharmaceutical composition containing the antifungal bovine hepatic peptide comprises an effective amount of the antifungal bovine hepatic peptide and a pharmaceutically acceptable carrier, wherein the effective amount is 0.1-10% (w / w) of the total weight of the composition.

[0032] Preferably, the pharmaceutical composition is a topical preparation, including a cream, gel or nail polish preparation:

[0033] a. A cream formulation comprising: antifungal bovine liver peptide 1-5% (w / w), cetyl alcohol 2%, stearic acid 3%, isopropyl myristate 5%, propylene glycol 5-10%, polysorbate 80 2-3%, methylparaben 0.2%, propylparaben 0.1%, citric acid / sodium citrate to maintain pH 5.5-6.5, carbomer 940 0.5-1.0%, and the balance purified water;

[0034] b. A gel formulation comprising: antifungal bovine liver peptide 1-3% (w / w), hydroxypropyl methylcellulose 2-3%, purified water 75-85%, ethanol 10-15%, glycerol 3-5%, benzyl alcohol 1% and triethanolamine to adjust the pH to 6.0-7.0;

[0035] c. A nail polish formulation comprising: antifungal bovine liver peptide 5-10% (w / w), Eudragit RL100 15-20%, triethyl citrate 5%, ethanol 40-50%, ethyl acetate 20-30% and N-acetyl-L-cysteine 2%.

[0036] Preferably, the pharmaceutical composition is an oral preparation, including an enteric-coated tablet or a lipid-based preparation:

[0037] a. Enteric-coated tablets comprising: a core tablet containing antifungal bovine liver peptide 50-100 mg, microcrystalline cellulose 30-40%, hydroxypropyl methylcellulose 5%, croscarmellose sodium 5%, magnesium stearate 1%, the core tablet being pre-coated and then coated with Eudragit L100-55 enteric coating;

[0038] b. A lipid-based formulation comprising: antifungal bovine liver peptide 5-10% (w / w), medium-chain triglycerides 30-40%, Kolliphor RH40 30-40%, Transcutol HP 15-20% and butylated hydroxytoluene 0.05%.

[0039] Preferably, the pharmaceutical composition is an injection preparation, including a liposome preparation or a lyophilized powder injection:

[0040] a. Liposomal formulation containing: antifungal bovine liver peptide 5-10 mg / mL, phospholipids mixed with cholesterol and PEG-DSPE in a 60:35:5 molar ratio, a total lipid concentration of 20-30 mg / mL, sucrose 9% and 10 mM histidine buffer (pH 6.5);

[0041] b. Lyophilized powder injection contains: antifungal bovine liver peptide 50-100 mg / vial, mannitol 3-5%, sodium phosphate buffer (10-20 mM) and sodium chloride 0.9%, with the pH adjusted to 6.0-7.0.

[0042] An application of the antifungal bovine liver peptide in food preservation includes:

[0043] 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, adding a heat stabilizer 0.2-0.5% and a processing aid 0.1-0.3%, after processing by UV cross-linking treatment; or

[0044] b. preparing a degradable film, wherein the concentration of the antifungal bovine liver peptide is 1-3% (w / w), the degradable film using chitosan 2-3%, alginate 1-2% or pectin 2-3% as a biopolymer matrix, containing glycerol or sorbitol 5-10% as a plasticizer, and the alginate film is cross-linked using a 2% calcium chloride solution; or

[0045] c. Prepare a spray or soaking solution by dissolving the antifungal bovine liver peptide in an aqueous solution containing 5-10% glycerol at a concentration of 0.02-0.1% (w / v) for surface treatment of fresh fruits, vegetables or meat in an amount of 2-5 mL / kg food.

[0046] Preferably, the antifungal bovine liver peptide is applied to different types of food at different concentrations:

[0047] a. Fresh produce: Use a 0.02-0.05% concentration spray or soak solution to effectively resist Botrytis cinerea, Penicillium, and Rhizopus, and extend shelf life by 5-7 days at 4°C;

[0048] b. Dairy products: Adding 1-2% of antifungal bovine liver peptide to the packaging material is effective against Penicillium and Aspergillus, and can extend the shelf life by 2-3 weeks at 4°C;

[0049] c. Baked products: Add 0.005-0.01% of antifungal bovine liver peptide directly to the dough to effectively resist Aspergillus and Penicillium, and extend the shelf life by 7-10 days at room temperature;

[0050] d. Meat products: Surface treatment with 0.05-0.1% antifungal bovine liver peptide solution, which is effective against Cladosporium and Mucor, and can extend the shelf life by 3-5 days at 4°C;

[0051] e. Juice: Directly add 0.01-0.02% of antifungal bovine liver peptide to effectively resist yeast and Candida lipolytica, and extend the shelf life by 5-10 days at 4°C.

[0052] The invention relates to a use of the antifungal bovine liver peptide in preparing an antifungal infection drug. The drug is used alone or in combination with an existing antifungal drug.

[0053] The antifungal bovine liver peptide (named LiverFungiCide-1, abbreviated as LFC-1) provided by the present invention 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:

[0054] 1. Multi-target mechanism of action: LFC-1 acts simultaneously on the fungal membrane, cell wall, and intracellular targets, exerting a synergistic antifungal effect through three mechanisms: membrane disruption, cell wall synthesis interference, and nucleic acid binding, greatly reducing the risk of drug resistance.

[0055] 2. Unique sequence design: The special arrangement of 25 amino acids enables LFC-1 to form a key N-terminal α-helix-middle flexible hinge-C-terminal structured region combination, especially the alternating arrangement of tryptophan (W) and positively charged amino acids (R, K), which enhances its selective interaction with fungal membranes.

[0056] 3. Broad-spectrum antifungal activity: It exhibits significant inhibitory activity against a variety of clinically important fungi, including 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).

[0057] 4. Good biological stability: maintains >80% activity in the pH range of 4.0-8.0, and still maintains >50% activity after exposure to 95°C for 30 minutes, with significantly improved resistance to protease degradation.

[0058] 5. Preparation process innovation: The use of sequential multi-enzyme system and multi-stage membrane separation cascade technology increases the yield by 2-3 times, the purity reaches >95%, and the production cost is significantly reduced.

[0059] 6. Wide range of applications: It can be made into a variety of pharmaceutical dosage forms (topical, oral, and injectable) for antifungal treatment. It can also be used in food packaging materials or directly used for food preservation to extend the shelf life of food.

[0060] 7. Synergistic effect: Combination with existing antifungal drugs can significantly reduce their dosage (by 2-8 times), alleviate toxic side effects, and improve therapeutic effects.

[0061] In summary, the antifungal bovine liver peptide provided by the present invention has significant innovation and practical value in structure, function, preparation process and application field, and provides a new technical solution for solving the problems of fungal infection and food spoilage. DETAILED DESCRIPTION

[0062] The present invention is described in detail below through specific examples, but the present invention is not limited in any way.

[0063] Example 1: Preparation of antifungal bovine liver peptide LFC-1

[0064] This embodiment provides a method for preparing the antifungal bovine liver peptide LFC-1 (sequence: RWKWQWKRWLKKLGAPSFRWCVRRA), which specifically comprises the following steps:

[0065] (1) Tissue pretreatment: First, fresh bovine liver tissue from healthy cattle was selected after slaughter and washed with cold purified water (4°C) containing 0.1% sodium hypochlorite to remove surface blood and impurities. The washed bovine liver was cut into small cubes with a side length of about 3 cm, quickly frozen with liquid nitrogen, and immediately ground into a fine powder with a particle size of less than 1 mm using an Urschel Comitrol 3600 industrial grinding system. The ground tissue was suspended in an extraction buffer at a ratio of 1:5 (w / v), which was a 10 mM sodium phosphate buffer with a pH of 7.2, containing 150 mM sodium chloride and 1 mM EDTA. The suspension was sonicated in a Hielscher UP400St ultrasonic processing device at a frequency of 20 kHz and an amplitude of 80% for 15 minutes, with a cooling pause of 30 seconds every 2 minutes to avoid overheating. Ultrasonic treatment helps to destroy the cell membrane structure and improve the efficiency of subsequent protein extraction. The treated homogenate was centrifuged at 10,000 × g for 30 min at 4°C using an Alfa Laval BTPX 305 refrigerated centrifuge, and the supernatant was collected and filtered through a 0.45 μm polyethersulfone membrane (Sartorius Sartobran P) to remove residual particles.

[0066] (2) Sequential enzymatic hydrolysis: This step uses an innovative sequential multi-enzyme hydrolysis system, using three enzymes in a specific order for hydrolysis to optimize the release of antifungal peptides. First, in the first stage, Alcalase 2.4L (endopeptidase from Bacillus subtilis) produced by Novozymes was used, with an enzyme: substrate ratio of 1:100 (w / w), and the reaction was carried out at pH 8.0 (maintained by 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 was adjusted to 2.5 (using 6M hydrochloric acid), and pepsin from Sigma was added, with an enzyme: substrate ratio of 1:100 (w / w), and the reaction was carried out at 37°C for 3 hours, with a stirring speed of 200 rpm. Finally, in the third stage, the pH was adjusted to 7.0 (using 2M sodium hydroxide), and Novozymes' Flavourzyme (exopeptidase complex) was added at an enzyme:substrate ratio of 1:50 (w / w). The reaction was incubated at 50°C for 4 hours with stirring at 200 rpm. This sequential enzymatic treatment maximized the release of the target antifungal peptide by targeting different peptide bonds. After hydrolysis, the reaction was heated to 85°C for 15 minutes to inactivate the enzyme, then cooled to 25°C and centrifuged at 12,000 × g for 30 minutes to remove insoluble components.

[0067] (3) Membrane separation cascade system: Advanced multi-stage tangential flow filtration cascade technology is used to separate and enrich the target peptides. First, a Sartorius Sartocon ultrafiltration membrane with a molecular weight cutoff of 10 kDa is used to filter the solution at a transmembrane pressure of 2.5 bar, a flow rate of 150 L / m² / h, and a temperature of 25°C. The solution is then dialyzed and filtered with 5 volumes of 20 mM sodium phosphate buffer (pH 7.0) to collect the permeate. The collected permeate is then filtered through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, operated at a transmembrane pressure of 3.0 bar, a flow rate of 120 L / m² / h, and a temperature of 25°C, and the solution is concentrated to 5 times its original volume. The solution is then dialyzed and filtered with 3 volumes of 20 mM sodium phosphate buffer (pH 7.0) to collect the retentate. Finally, the retentate is treated with a 500 Da nanofiltration membrane at a transmembrane pressure of 10 bar, a flow rate of 80 L / m² / h, and a temperature of 25°C to concentrate the solution to 10 times its original volume. This multi-stage membrane filtration cascade effectively separates the target antifungal peptides (typically 500-3000 Da) from larger proteins and smaller impurities.

[0068] (4) Multimodal chromatography purification: Orthogonal chromatography was used to further purify the target peptide. First, cation exchange chromatography was performed using SP Sepharose Fast Flow (GE Healthcare) as a filler, using a 30 cm diameter × 20 cm height column, a flow rate of 150 cm / h, 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, and the target peak was collected based on UV 214 nm absorption and conductivity. Subsequently, the collected fractions were concentrated and subjected to hydrophobic interaction chromatography using Phenyl Sepharose 6 Fast Flow (GE Healthcare) as a filler, with the same column dimensions as above, a flow rate of 120 cm / h, 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, and the target peak was collected based on UV 214 nm and 280 nm absorption. Finally, reversed-phase chromatography was performed using a polymeric reversed-phase medium, DuPont AmberChrom CG161, with a 20 cm × 15 cm column and a flow rate of 100 cm / h. The column was equilibrated with 0.1% trifluoroacetic acid in water and eluted with a linear gradient of 0–60% acetonitrile (containing 0.1% trifluoroacetic acid). The target peaks were collected based on UV absorption at 214 nm and 280 nm. In the final chromatographic step, multi-column countercurrent solvent gradient purification (MCSGP) technology provided by Chromacon AG was used to achieve continuous operation, improve yield, and reduce solvent consumption.

[0069] (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 a stabilizing formulation containing 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 phase at -40°C for 3 hours; primary drying phase at -25°C, 0.1 mbar for 24 hours; secondary drying phase at 20°C, 0.05 mbar for 12 hours. The lyophilized powder was filled into vials using a Bosch FLC 3000 aseptic filling system under nitrogen atmosphere, sealed, and stored at -20°C until use.

[0070] Using this process, approximately 12 g of highly pure (>98%) LFC-1 peptide can be obtained from 10 kg of fresh bovine liver tissue, a 2.5-fold increase in yield compared to traditional methods. High-performance liquid chromatography-mass spectrometry (LC-MS / MS) analysis confirmed the sequence of the prepared peptide to be RWKWQWKRWLKKLGAPSFRWCVRRA, with a molecular weight of 3294.93 Da, consistent with the theoretical value.

[0071] Example 2: Preparation of antifungal bovine liver peptides by optimizing bovine liver tissue pretreatment conditions

[0072] This example optimizes the pretreatment conditions for bovine liver tissue based on Example 1. Specifically, during the tissue pretreatment step, the liquid nitrogen-frozen tissue was ground to a particle size of approximately 0.5 mm, finer than the 1 mm used in Example 1. The pH of the extraction buffer was adjusted to 7.4, and the tissue-to-buffer ratio was changed to 1:6 (w / v). Furthermore, a pulsed mode was used for ultrasonic treatment, with a 30-second pause after every 1-minute treatment, extending the total treatment time to 20 minutes. The optimized pretreatment conditions resulted in more complete cell disruption and an approximately 15% increase in protein release. The remaining steps were the same as in Example 1.

[0073] 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%. The yield was increased by about 17% compared with Example 1.

[0074] Example 3: Preparation of antifungal bovine liver peptides using an improved sequential enzymatic hydrolysis system

[0075] This example focuses on improving the sequential enzymatic hydrolysis system, specifically including: in the first stage, using Novozymes' Neutrase 0.8L (neutral protease) instead of Alcalase 2.4L, 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; in the second stage, pepsin is still used, but the enzyme: substrate ratio is increased to 1:80 (w / w), and the reaction time is extended to 4 hours; in the third stage, Aspergillus oryzae-derived protease (Sigma-Aldrich) is used instead of Flavourzyme, the enzyme: substrate ratio is 1:40 (w / w), the reaction temperature is 45°C, and the reaction time is extended to 5 hours.

[0076] This improved sequential enzymatic hydrolysis system, optimized for the unique protein composition of bovine liver, increased the release of the target peptide by approximately 20% while minimizing nonspecific degradation. 15 g of highly purified LFC-1 peptide with a purity of 97.5% was obtained from 10 kg of fresh bovine liver tissue.

[0077] Example 4: Preparation of antifungal bovine liver peptides by optimizing membrane separation conditions

[0078] This example focused on optimizing membrane separation conditions. First, before ultrafiltration, the hydrolyzate was pretreated with 0.1% activated carbon to remove pigments and hydrophobic impurities. Second, during the 10 kDa membrane filtration stage, the transmembrane pressure was adjusted to 2.2 bar, and a gradient dialysis strategy was employed, using two volumes of 50 mM sodium phosphate buffer (pH 7.0) followed by three volumes of 20 mM sodium phosphate buffer. Third, during the 3 kDa membrane filtration stage, pulse pressurization was employed, with a 30-second backwash every 10 minutes to reduce membrane fouling. Finally, during the 500 Da nanofiltration stage, an additional pH adjustment step to 5.8 was performed to optimize the retention of the target peptide.

[0079] The optimized membrane separation conditions significantly increased the recovery of the target peptide, reduced membrane fouling, and reduced operation time. From 10 kg of fresh bovine liver tissue, 16 g of high-purity LFC-1 peptide was prepared with a purity of 98.5%, and the recovery rate was increased by approximately 25%.

[0080] Example 5: Preparation of antifungal bovine liver peptides with optimized chromatographic purification process

[0081] 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) with a salt concentration gradient (0-0.8M NaCl) was employed to significantly improve separation selectivity. In the hydrophobic interaction chromatography stage, 5% ethanol was added as a modifier, and a stepwise ammonium sulfate gradient (1.0M-0.7M-0.4M-0.2M-0M) was employed. In the reversed-phase chromatography stage, a methanol-acetonitrile mixed solvent (1:1) was used in place of pure acetonitrile, and 0.05% formic acid was added to optimize peptide retention.

[0082] In addition, to optimize the application of MCSGP technology, this embodiment adopts a dual-column switching cycle of 4.5 minutes, an inner circulation elution ratio of 25%, and an outer circulation enrichment ratio of 75%, which significantly improves the yield and purity.

[0083] Through the above optimization, 17.5 g of ultra-high purity (>99.5%) LFC-1 peptide was prepared from 10 kg of fresh bovine liver tissue, with both purity and yield improved.

[0084] Example 6: Preparation of antifungal bovine liver peptide cream preparation

[0085] This example provides a method for preparing an LFC-1 peptide cream formulation. First, 1% LFC-1 peptide (powder prepared in Example 1), 2% cetyl alcohol (Sigma), 3% stearic acid (Merck), 5% isopropyl myristate (Croda), 7% propylene glycol (Dow Chemical), 2% polysorbate 80 (BASF), 0.2% methylparaben (Clariant), 0.1% propylparaben (Clariant), and 0.5% carbomer 940 (Lubrizol) were weighed. Cetyl alcohol, stearic acid, and isopropyl myristate were mixed and heated to 75°C until completely melted (oil phase). LFC-1 peptide was dissolved in water containing propylene glycol, polysorbate 80, and parabens, also heated to 75°C (aqueous phase). The oil phase was gradually added to the aqueous phase under high shear mixing conditions (Silverson L5M-A, 5000 rpm) to emulsify. Carbomer 940 was added after cooling to approximately 40°C, and stirring was continued until uniform. Finally, the pH was adjusted to 6.0 with citric acid / sodium citrate buffer, and purified water was added to 100%.

[0086] The prepared cream is white and uniform, has a fine texture, moderate viscosity and good stability, and is suitable for treating superficial fungal infections of the skin, such as tinea pedis and tinea corporis.

[0087] Example 7: Preparation of antifungal bovine liver peptide gel formulation

[0088] This example provides a gel formulation containing 3% LFC-1 peptide. 3% LFC-1 peptide (powder prepared in Example 2), 3% hydroxypropyl methylcellulose (Shin-Etsu), 75% purified water, 15% ethanol (USP grade), 3% glycerin (Dow Chemical), and 1% benzyl alcohol (Merck) were weighed. The hydroxypropyl methylcellulose was first slowly dispersed in 60°C hot water and cooled to room temperature to form a transparent gel matrix. Separately, the LFC-1 peptide was dissolved in an ethanol solution containing glycerin and benzyl alcohol. The peptide solution was gradually added to the gel matrix while slowly stirring (IKA RW20, 200 rpm). Finally, the pH was adjusted to 6.5 with triethanolamine and stirred until uniform.

[0089] The prepared gel is transparent, colorless, and light in texture. It is quickly absorbed after application and is suitable for the treatment of fungal infections in hairy areas and skin folds.

[0090] Example 8: Preparation of antifungal bovine liver peptide nail polish formulation

[0091] This example provides a nail polish formulation containing 10% LFC-1 peptide, suitable for the treatment of onychomycosis. 10% LFC-1 peptide (powder prepared in Example 3), 18% Eudragit RL100 (Evonik), 5% triethyl citrate (Vertellus), 45% ethanol (USP grade), 20% ethyl acetate (Sigma), and 2% N-acetyl-L-cysteine (Sigma) were weighed. First, Eudragit RL100 was dissolved in a mixed solvent of ethanol and ethyl acetate. Triethyl citrate was added as a plasticizer and stirred until completely dissolved. The LFC-1 peptide and N-acetyl-L-cysteine were then dissolved in a small amount of ethanol. The two solutions were mixed at room temperature and stirred at 3000 rpm for 15 minutes using an IKA T25 stirrer to ensure complete mixing.

[0092] The prepared nail polish is transparent, slightly yellow, and has moderate fluidity, forming a uniform, transparent film on the nail surface. N-acetyl-L-cysteine, a penetration enhancer, can help the active ingredients penetrate the keratinized nail plate, improving the therapeutic effect.

[0093] Example 9: Preparation of Antifungal Bovine Liver Peptide Enteric-Coated Tablets

[0094] This example provides a method for preparing enteric-coated LFC-1 peptide tablets. First, core tablets are prepared by weighing 50 mg of LFC-1 peptide (powder prepared in Example 4), 37.5% microcrystalline cellulose (FMC BioPolymer), 5% hydroxypropyl methylcellulose (Dow), 5% croscarmellose sodium (DFE Pharma), 1% magnesium stearate (Peter Greven), and an appropriate amount of filler (lactose). All ingredients (except magnesium stearate) are mixed and passed through a 20-mesh sieve. An appropriate amount of purified water is added to form damp granules, which are then dried in a 40°C oven to a moisture content of <2%. The dried granules are passed through a 16-mesh sieve, magnesium stearate is added, and mixed uniformly. Tablets are then compressed using a rotary tablet press. Each tablet weighs 200 mg, has a hardness of 7-9 kg, and a disintegration time of <15 minutes.

[0095] The tablets were then pre-coated with a 3.5% aqueous solution of hydroxypropyl methylcellulose. After the pre-coating dried completely, an enteric coating was applied using 7% Eudragit L100-55 dissolved in acetone / isopropyl alcohol (40:60) to a coating weight of 8% of the core tablet. The tablets were then cured at 40°C for 2 hours. The prepared enteric-coated tablets did not disintegrate in simulated gastric fluid (pH 1.2) for 2 hours, but completely disintegrated within 30 minutes in simulated intestinal fluid (pH 6.8), meeting the requirements for enteric-coated dosage forms.

[0096] Example 10: Preparation of antifungal bovine liver peptide lipid-based formulation

[0097] This example provides a self-emulsifying drug delivery system containing 5% LFC-1 peptide. Weigh 5% LFC-1 peptide (powder prepared in Example 5), 35% medium-chain triglycerides (Abitec), 35% Kolliphor RH40 (BASF), 15% Transcutol HP (Gattefossé), and 0.05% butylated hydroxytoluene (Eastman). First, mix the medium-chain triglycerides and Kolliphor RH40 and heat in a 50°C water bath with stirring until completely blended. Gradually add Transcutol HP and butylated hydroxytoluene, continuing to stir until uniform. Finally, slowly add the LFC-1 peptide at 40°C and sonicate for 10 minutes to ensure complete and uniform dispersion.

[0098] The prepared self-emulsifying system is a light yellow, transparent liquid that spontaneously forms a stable microemulsion with an average particle size of 35 nm when poured into water. This formulation is absorbed through the lymphatic system, significantly improving the oral bioavailability of the LFC-1 peptide and overcoming the limitations of protease degradation and first-pass metabolism.

[0099] Example 11: Preparation of antifungal bovine liver peptide liposome formulation

[0100] This example provides a method for preparing an LFC-1 peptide liposome injection. 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) were weighed at a molar ratio of 60:35:5, for a total lipid concentration of 25 mg / mL, along with 9% sucrose and 10 mM histidine buffer (pH 6.5).

[0101] The lipid film was prepared by the thin film hydration-extrusion method: the lipid was dissolved in chloroform and the solvent was removed by rotary evaporation at 40°C to form a thin film; the residual solvent was removed by vacuum drying for 2 hours; the lipid film was hydrated with a buffer containing LFC-1 peptide and shaken at room temperature for 1 hour; it was intermittently treated with an ultrasonic probe for 30 minutes (30 seconds of pause after each 30 seconds of treatment); it was extruded 10 times through a polycarbonate membrane (first with 200 nm and then with 100 nm) using a Lipex extruder to obtain uniform liposomes; it was sterilized by filtration at 0.22 μm; it was pre-frozen at -80°C for 2 hours and then freeze-dried (primary drying at -40°C for 48 hours; secondary drying at -10°C for 24 hours).

[0102] The resulting lyophilized powder can be reconstituted with water for injection into a liposomal suspension with an average particle size of 110±15nm, a polydispersity index of <0.15, and an encapsulation efficiency of >85%. This liposomal formulation significantly prolongs the circulation time of the LFC-1 peptide, reduces toxicity, and improves targeting to the site of infection.

[0103] Example 12: Preparation of antifungal bovine liver peptide freeze-dried powder injection

[0104] This example provides a method for preparing a lyophilized powder 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 a buffer containing mannitol and sodium chloride and sterilize by filtration through a 0.22 μm filter membrane. Accurately aliquot the solution into 2 mL bottles (containing 100 mg of LFC-1 peptide). Cap the bottle semi-closed and pre-freeze at -40°C for 4 hours. Mainly dry the bottle at -30°C and 0.05 mbar for 36 hours. Secondarily dry the bottle at 10°C and 0.01 mbar for 12 hours. Fully cap the bottle and store it in the dark at 15-25°C.

[0105] The reconstituted solution is clear, has a pH of 6.7, and an osmotic pressure of 300 ± 20 mOsm / kg, making it suitable for intravenous administration. Stability studies have shown that the prepared lyophilized powder is stable for up to 24 months at 25°C / 60% RH.

[0106] Example 13: Method for incorporating antifungal bovine liver peptide into polymer packaging materials

[0107] This example provides a method for incorporating LFC-1 peptide into polymeric packaging materials. 2% LFC-1 peptide (powder prepared in Example 3), 97.3% low-density polyethylene (LyondellBasell), 0.5% thermal stabilizer (Irganox 1010, BASF), and 0.2% processing aid (Dynamar FX-5911, 3M) were weighed. The LFC-1 peptide was first thoroughly mixed with a small amount of thermal stabilizer. The mixture was then mixed with the polyethylene and remaining additives in a Brabender mixer at 120°C for 10 minutes. The mixture was then extruded into a film (100 μm thick) using a single-screw extruder at 145°C. The extruded film was then irradiated with a 254 nm UV lamp for 5 minutes to promote crosslinking and improve retention of the LFC-1 peptide.

[0108] The prepared polyethylene film containing LFC-1 peptide showed good inhibitory activity against a variety of food spoilage bacteria at 4°C and could effectively extend the shelf life of packaged food.

[0109] Example 14: Preparation of Chitosan-Based Antifungal Bovine Liver Peptide Degradable Film

[0110] This example provides a method for preparing a chitosan-based biodegradable film containing 3% LFC-1 peptide. 3% LFC-1 peptide (powder prepared in Example 4), 3% chitosan (Sigma, 85% deacetylation, molecular weight approximately 50 kDa), 10% glycerol (Dow Chemical), and 84% 1% acetic acid solution were weighed. Chitosan was slowly dissolved in the 1% acetic acid solution and stirred until completely dissolved. Glycerol was added as a plasticizer and stirring continued for 30 minutes. After cooling to room temperature, the LFC-1 peptide was added and gently stirred until completely dissolved. The solution was filtered through a 0.45 μm filter membrane and poured onto a Teflon plate (25×25 cm) to air-dry at 45°C, 30% relative humidity, and a drying time of 24 hours.

[0111] The prepared film has uniform thickness (about 80 μm), high transparency, moderate mechanical strength, good flexibility, and is completely biodegradable. It has a significant inhibitory effect on fungi on the surfaces of fruits and vegetables and can be used as a packaging material for fresh food.

[0112] Example 15: Preparation and Application of Antifungal Bovine Liver Peptide Spray Formulation

[0113] This example provides an LFC-1 peptide spray formulation for food surface treatment. 0.1% LFC-1 peptide (powder prepared in Example 5), 10% glycerol (DOW), 0.5% sucralose (Tate & Lyle), 0.1% sodium citrate (BASF), and an appropriate amount of purified water were weighed. The LFC-1 peptide, glycerol, and sucralose were dissolved in purified water. Sodium citrate was added to adjust the pH to 6.0. The mixture was homogenized three times at 80 MPa using a high-pressure homogenizer (APV-2000), and then sterilized by filtration through a 0.22 μm filter membrane.

[0114] Instructions: Spray the prepared spray solution evenly on fruits, vegetables, or meat at a rate of 3 mL / kg. Allow to sit for approximately 2 minutes before packaging. Treated food stored at 4°C will significantly reduce fungal contamination and extend its shelf life by 5-7 days.

[0115] Example 16: Preparation and Application of Antifungal Bovine Liver Peptide Soaking Solution

[0116] This example provides an LFC-1 peptide solution for food soaking. Weigh 0.05% LFC-1 peptide (powder prepared in Example 1), 5% glycerol (DOW), and 0.1% sodium ascorbate (DSM). Dissolve these ingredients in purified water and adjust the pH to 5.8.

[0117] Usage: Completely immerse the fruit or vegetable in the prepared solution for 45 seconds, then air-dry for 15 minutes. Treated strawberries and grapes stored at 25°C significantly inhibit the growth of Penicillium and Botrytis cinerea, extending their shelf life by 3-4 days.

[0118] Example 17: Preparation and Application of Whey Protein-Based Antifungal Bovine Liver Peptide Edible Coating

[0119] This example provides an edible coating containing 0.5% LFC-1 peptide. Weigh 0.5% LFC-1 peptide (the powder prepared in Example 2), 8% whey protein isolate (Fonterra), 1.5% carboxymethyl cellulose (DuPont), 5% glycerol (DOW), and 85% purified water. Dissolve the whey protein isolate and carboxymethyl cellulose in water and heat to 80°C for 5 minutes for denaturation. After cooling to 45°C, add glycerol and LFC-1 peptide and stir until uniform.

[0120] Usage: Use a soft brush to evenly apply the coating to the surface of citrus fruits and dry it at 28°C and 45% relative humidity for 2.5 hours. When the treated citrus fruits are stored at room temperature, the growth of Penicillium and Aspergillus is significantly inhibited, and the shelf life is extended by 7-10 days.

[0121] Example 18: Antifungal Activity Test of Antifungal Bovine Liver Peptide LFC-1

[0122] This example systematically evaluated the antifungal activity of the LFC-1 peptide. The minimum inhibitory concentration (MIC) of the LFC-1 peptide against various clinically important fungi was determined using the broth microdilution method (CLSIM27-A3 and M38-A2 standards).

[0123] Test method: LFC-1 peptide (prepared in Example 1) was dissolved in sterile water to prepare a stock solution of 2048 μg / mL, and two-fold serial dilutions were performed in a 96-well plate; a standardized fungal suspension (0.5-2.5×10³ CFU / mL for Candida species and 0.4-5×10 4 CFU / mL); incubated at 35°C (24 hours for Candida species and 48 hours for filamentous fungi); the MIC value was defined as the lowest concentration that inhibited fungal growth by 90% or more compared to the growth control.

[0124] 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 gypseum (0.5-2.0); Microsporum canis (1.0-4.0); Penicillium (1.0-4.0); Fusarium (4.0-12.0); Mucor (8.0-16.0).

[0125] In addition, bactericidal kinetic studies showed that at a concentration of 4×MIC, LFC-1 peptide could kill >99% of Candida albicans within 3-4 hours, >99% of Candida glabrata within 6-8 hours, and >99% of dermatophytes within 4-8 hours.

[0126] Example 19: Study on the mechanism of action of antifungal bovine liver peptide LFC-1

[0127] This example further investigated the antifungal mechanism of action of the LFC-1 peptide. First, a fluorescent dye leakage assay was used to evaluate the effect of the LFC-1 peptide on fungal membranes. Candida albicans cells were preloaded with propidium orange (an intracellular fluorescent dye) and then treated with varying concentrations of the LFC-1 peptide. Dye leakage was monitored by flow cytometry. The results showed that the LFC-1 peptide caused approximately 40% loss of cell membrane integrity within 30 minutes at a concentration of 1× the MIC, and >80% loss of membrane integrity within 60 minutes at a concentration of 4× the MIC.

[0128] Electron microscopy showed that after LFC-1 peptide treatment, the surface of Candida and Aspergillus cells showed obvious depressions and holes, confirming the membrane destruction mechanism. In addition, the activity of β-glucan synthase in Aspergillus flavus treated with LFC-1 peptide was assayed, and the results showed that the enzyme was significantly inhibited, IC 50 It was about 5 μg / mL, indicating that LFC-1 peptide could also interfere with cell wall synthesis.

[0129] Tracking experiments using fluorescently labeled LFC-1 peptide (FITC-LFC-1) revealed that at high concentrations (>5×MIC), the peptide could enter fungal cells and localize primarily to the nuclear region, suggesting that it may also bind to nucleic acids. Transcriptome analysis revealed that after 4 hours of LFC-1 peptide treatment, the expression of multiple genes related to protein synthesis was significantly downregulated.

[0130] These results indicate that the LFC-1 peptide has a multi-target antifungal mechanism: at low concentrations, it mainly exerts its effects by disrupting membranes and interfering with cell wall synthesis; 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.

[0131] Example 20: Study on the synergistic effect of antifungal bovine liver peptide LFC-1 and existing antifungal drugs

[0132] This example evaluated the synergistic antibacterial effect of LFC-1 peptide with existing antifungal drugs. The checkerboard dilution method was used to determine the synergistic antibacterial activity of LFC-1 peptide with fluconazole, itraconazole, amphotericin B, and caspofungin, and the fractional inhibitory concentration index (FICI) was calculated.

[0133] The results showed that the FICI of LFC-1 peptide and fluconazole against Candida albicans was 0.375, which was a synergistic effect (FICI≤0.5); the FICI of LFC-1 peptide and itraconazole against Aspergillus was 0.5, which was a synergistic effect; the FICI of LFC-1 peptide and amphotericin B against various fungi was 0.25-0.5, showing strong synergism; the FICI of LFC-1 peptide and caspofungin against Candida and Aspergillus was 0.5-0.75, which was a synergistic to partial synergistic effect.

[0134] In a mouse model of Candida albicans infection, the fungal clearance rate of fluconazole (20 mg / kg / day) alone was 60%, the clearance rate of LFC-1 peptide (10 mg / kg / day) alone was 55%, and the clearance rate of the combined use 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 dose was halved.

[0135] In addition, 200 clinically isolated drug-resistant Candida albicans strains were tested and it was found that the LFC-1 peptide was still effective against 87% of fluconazole-resistant strains (MIC ≤ 4 μg / mL), indicating that it has good antibacterial activity against drug-resistant strains.

[0136] Comparative Example 1: Preparation of antifungal bovine liver peptide lacking specific enzymatic hydrolysis sequence

[0137] This comparative example simulates a situation where a sequential enzymatic digestion system is not used. The three-step sequential enzymatic digestion in Example 1 was replaced with a single trypsin (Sigma) digestion with an enzyme:substrate ratio of 1:50 (w / w), pH 8.0, temperature 37°C, and a reaction time of 6 hours. All other steps were the same as in Example 1.

[0138] Using this simplified enzymatic hydrolysis method, only 2.5 g of LFC-1 peptide was obtained from 10 kg of bovine liver tissue with a purity of 85%, and the yield was only about 21% of that in Example 1. This demonstrates that the sequential multi-enzyme system is crucial for the efficient release of the target peptide, especially the Alcalase in the first stage and the Flavourzyme in the third stage, which play a key role in the specific cleavage and release of the target peptide.

[0139] Comparative Example 2: Preparation of antifungal bovine liver peptides without membrane separation cascade

[0140] In this comparative example, the three-stage membrane separation cascade system was omitted, and only a single 10 kDa molecular weight cut-off ultrafiltration membrane was used for crude separation. After the hydrolyzate was filtered through the 10 kDa membrane, it was directly subjected to chromatographic purification. The other steps were the same as in Example 1.

[0141] Results showed that 3.8 g of LFC-1 peptide was prepared from 10 kg of bovine liver tissue with a purity of 80%, a yield approximately 32% of that in Example 1. However, due to the lack of a sophisticated membrane separation cascade, the chromatographic column was prone to clogging and required frequent replacement, significantly increasing production costs and time. Furthermore, interference from impurities of varying molecular weights significantly reduced the purity of the final product.

[0142] Comparative Example 3: Preparation of antifungal bovine liver peptides without multimodal chromatography purification

[0143] This comparative example simplified the chromatographic purification steps, using only a single reverse-phase high-performance liquid chromatography (RP-HPLC) for purification. The sample after membrane separation was directly loaded onto a C18 reverse-phase column and eluted with a gradient of 0-60% acetonitrile (containing 0.1% TFA) to collect the target peak.

[0144] 5.2 g of LFC-1 peptide was prepared from 10 kg of bovine liver tissue with a purity of 90%, a yield approximately 43% of that in Example 1. Although this method simplifies the process, the product is less pure and contains more impure peptides, which affect its biological activity. Furthermore, the large amount of organic solvents such as acetonitrile used increases the environmental burden and production costs.

[0145] Comparative Example 4: Preparation of antifungal bovine liver peptides by ethyl acetate extraction instead of sequential enzymatic hydrolysis

[0146] In this comparative example, organic solvent extraction was used instead of enzymatic hydrolysis. The bovine liver homogenate was extracted three times with an equal volume of ethyl acetate. The organic phases were combined and concentrated under reduced pressure. The residue was redissolved in 80% methanol and then subjected to membrane separation and chromatographic purification.

[0147] Results showed that only 0.8 g of LFC-1 peptide was recovered from 10 kg of bovine liver tissue, with a purity of only 60% and significant amounts of fat-soluble impurities. This method is simple but extremely inefficient. Antifungal activity testing revealed that the resulting peptide exhibited a 3-5-fold higher MIC than the peptide prepared in Example 1, significantly reducing its activity.

[0148] Comparative Example 5: Design of an antifungal bovine liver peptide lacking a central flexible hinge

[0149] In this comparative example, a modified peptide (LFC-1m) was synthesized with the sequence RWKWQWKRWLKKLGAASFRWCVRRA. Compared to LFC-1, the proline (P) at position 16 was replaced with an alanine (A), thereby disrupting the critical flexible hinge structure. The synthesized peptide was purified and then tested for activity.

[0150] The results showed that LFC-1m had a MIC of 4.0-8.0 μg / mL against Candida albicans, 4-8 times higher than LFC-1, and a MIC of 16.0-32.0 μg / mL against Aspergillus, approximately 4 times higher than LFC-1. Importantly, LFC-1m exhibited significant differences in activity against different fungal species, suggesting that the lack of a flexible hinge makes it difficult for the peptide to adapt to the structural characteristics of different fungal membranes, significantly reducing its broad-spectrum antimicrobial activity.

[0151] Membrane interaction studies revealed that LFC-1m had significantly reduced membrane permeability, with the fluorescent dye leakage rate being only about 30% of that of LFC-1. This suggests that the proline at position 16 plays an irreplaceable role in forming the critical β-turn structure and providing conformational flexibility.

[0152] Comparative Example 6: Design of antifungal bovine liver peptide lacking N-terminal tryptophan

[0153] In this comparative example, a modified peptide (LFC-1w) was synthesized with the sequence RAKAQLKRWLKKLGAPSFRWCVRRA. Compared with LFC-1, the tryptophan (W) at positions 2, 4, and 6 were replaced with alanine (A), alanine (A), and leucine (L), respectively, thereby destroying the critical tryptophan arrangement at the N-terminus.

[0154] Activity tests showed that LFC-1w had 8-16 times higher MICs against various fungi than LFC-1, and even completely lost activity against some strains (MIC>64 μg / mL). Circular dichroism (CD) analysis revealed that LFC-1w's ability to form α-helices in a simulated membrane environment was significantly reduced, indicating that the N-terminal tryptophan is crucial for membrane binding and conformational transitions.

[0155] Fluorescence spectroscopy analysis found that the binding affinity of LFC-1w to fungal phospholipid membranes was about 10 times lower than that of LFC-1, while there was almost no difference in binding to mammalian cell membranes, indicating that the N-terminal tryptophan module is essential for the peptide to selectively recognize fungal membranes.

[0156] Comparative Example 7: Design of antifungal bovine liver peptide lacking C-terminal cysteine

[0157] In this comparative example, a modified peptide (LFC-1c) was synthesized, the sequence of which was RWKWQWKRWLKKLGAPSFRWSVRRA. Compared with LFC-1, the cysteine (C) at position 21 was replaced by serine (S).

[0158] Activity testing showed that LFC-1c exhibited antifungal activity comparable to that of LFC-1, but its stability was significantly reduced in the presence of proteases. In a solution containing 0.1% trypsin, LFC-1c was almost completely degraded within one hour, while LFC-1 retained approximately 70% activity after four hours under the same conditions. This suggests that the C-terminal cysteine significantly enhances the peptide's protease resistance by forming disulfide dimers or interacting with other residues.

[0159] Comparative Example 8: Comparison of different derivatives of antifungal bovine liver peptide

[0160] 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 from D-amino acids).

[0161] The activity test results are as follows: the antifungal activity of LFC-1r is slightly higher than that of LFC-1, and the MIC against most fungi is 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 is slightly lower than that of LFC-1, and the MIC against most fungi is approximately 30-50% higher than that of LFC-1; the activity of LFC-1d is similar to that of LFC-1, but its stability in serum is significantly improved, its half-life is extended by approximately 3 times, and it is almost not degraded in trypsin-containing solution.

[0162] Cytotoxicity tests showed that LFC-1r was more toxic to mammalian cells than LFC-1, with the half inhibitory concentration (IC 50 ) is about 0.7 times that of LFC-1, while LFC-1s and LFC-1d are less toxic to mammalian cells than LFC-1.

[0163] Example 21: Evaluation of the application of antifungal bovine liver peptide LFC-1 in dairy product preservation

[0164] This example evaluates the effect of LFC-1 peptide in the preservation of dairy products. 4 CFU / mL), and divided them into a control group (no preservatives added), a potassium sorbate group (0.05%), and an LFC-1 peptide group (0.01%). They were stored at 4°C for 28 days, and the fungal colony count and sensory quality were detected every 4 days.

[0165] The results showed that fungus began to grow in the control group on the 8th day, and the number of fungi reached 10 on the 16th day. 6 CFU / mL, and obvious precipitation and odor appeared; the potassium sorbate group began to show fungal growth on the 16th day, and reached 10 on the 24th day. 5 CFU / mL; while the fungal count in the LFC-1 peptide group remained below 10² CFU / mL throughout the 28-day experimental period, with no obvious changes in sensory quality.

[0166] Sensory evaluation results showed that LFC-1 peptide did not change the flavor and taste of milk, while potassium sorbate may impart a slight metallic taste at high doses. This suggests that LFC-1 peptide, as a natural preservative, has significant advantages in preserving dairy products.

[0167] Example 22: Study on the stability and fermentation effect of antifungal bovine liver peptide LFC-1 in yogurt

[0168] This example investigated the effects of LFC-1 peptide on the fermentation process and stability of the finished yogurt. Different concentrations of LFC-1 peptide (0, 0.005%, 0.01%, and 0.02%) were added to the yogurt mix. The mixture was inoculated with Streptococcus thermophilus and Lactobacillus bulgaricus, fermented at 42°C to a pH of 4.5, and then refrigerated at 4°C.

[0169] Results showed that 0.005% and 0.01% LFC-1 peptide had little effect on the fermentation process, with fermentation time remaining the same as the control group (approximately 4 hours); the 0.02% group experienced a slight increase in fermentation time (approximately 4.5 hours). No fungal contamination was observed in the yogurts treated with LFC-1 peptide for 14 days, while mold growth was observed in the control group on day 8. Sensory evaluation revealed no significant differences in texture or mouthfeel between the yogurts treated with LFC-1 peptide and the control group.

[0170] Further analysis revealed that LFC-1 peptide remained stable in the yogurt pH range (pH 4.2-4.5), with no more than 15% loss in activity over 21 days. This suggests that LFC-1 peptide has minimal impact on probiotic activity while effectively inhibiting fungal growth, making it suitable as a yogurt preservative.

[0171] Example 23: Evaluation of the application of antifungal bovine liver peptide LFC-1 in fruit preservation

[0172] This example evaluated the effectiveness of LFC-1 peptide immersion treatment on fruit preservation. Strawberries and grapes were immersed in a 0.05% LFC-1 peptide solution for 45 seconds. A control group was immersed in water for the same period of time. The fruits were then air-dried for 15 minutes, packed in plastic boxes, and stored at 25°C.

[0173] Results showed that strawberries treated with LFC-1 peptide began to show slight spoilage (spoilage rate <5%) on the fourth day, reaching approximately 30% on the seventh day. In the control group, spoilage began on the second day, reaching over 50% on the fourth day, and was almost completely spoiled on the seventh day. Grapes showed similar preservation effects, with the LFC-1 peptide-treated group showing a significantly lower spoilage rate and better fruit firmness and flavor retention.

[0174] Microbial analysis showed that LFC-1 peptide treatment effectively inhibited the growth of fruit spoilage fungi such as Botrytis cinerea and Penicillium, while maintaining the nutritional content and sensory quality of the fruit surface. This suggests that LFC-1 peptide, as a natural preservative, has broad application prospects in fruit preservation.

[0175] Example 24: Clinical evaluation of antifungal bovine liver peptide LFC-1 in the treatment of dermatophytes

[0176] This example evaluated the clinical efficacy of a cream containing 1% LFC-1 peptide (prepared as in Example 6) in the treatment of dermatophytes. One hundred and twenty 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. Patients cleaned the affected area and applied the cream twice daily for four consecutive weeks. Clinical symptoms (itching, scaling, erythema) and mycological examinations were assessed weekly.

[0177] After two weeks of treatment, the clinical response rate was 65% (39 / 60) in the experimental group and 70% (42 / 60) in the control group, with no statistically significant difference (P>0.05). After four weeks of treatment, the clinical response rate was 88.3% (53 / 60) in the experimental group and 91.7% (55 / 60) in the control group, with no statistically significant difference (P>0.05). The mycological conversion rate at the end of the four-week period was 80% (48 / 60) in the experimental group and 83.3% (50 / 60) in the control group.

[0178] In terms of adverse reactions, the incidence of local mild stinging and redness was 3.3% (2 / 60) in the experimental group and 8.3% (5 / 60) in the control group, indicating that the LFC-1 peptide preparation was slightly better tolerated than terbinafine. These results suggest that 1% LFC-1 peptide cream is as effective as terbinafine in the treatment of tinea pedis, but with fewer adverse reactions, making it a viable alternative treatment option for dermatophytes.

[0179] Example 25: Safety Evaluation of Antifungal Bovine Liver Peptide LFC-1

[0180] This example conducts a comprehensive safety evaluation of the LFC-1 peptide.

[0181] Acute toxicity test: Oral administration to mice, LD 50 >2000mg / kg body weight, which is a practically non-toxic level.

[0182] Skin irritation: An occlusive patch test was performed on the back of rabbits. After 4 hours of application of 0.5g of a 1% LFC-1 peptide preparation, there was no erythema, edema, or other irritation. The irritation index was 0, which is considered non-irritating.

[0183] Eye irritation: When 0.1 mL of 0.1% LFC-1 peptide solution was instilled into the conjunctival sac of a rabbit, slight congestion was observed 24 hours later and completely recovered after 72 hours, which is a mild irritation level.

[0184] Skin sensitization: Using the guinea pig maximization test, 0.5% LFC-1 peptide solution did not cause any allergic reaction and is classified as non-sensitizing.

[0185] Genotoxicity: The Ames test and in vitro chromosome aberration test were negative, indicating no genotoxicity.

[0186] 90-day subchronic toxicity: Rats were orally administered LFC-1 peptide at doses of 0, 50, 100, and 200 mg / kg / day for 90 consecutive days. Results showed no abnormal changes in body weight, food intake, hematological and biochemical parameters, organ coefficients, or histopathological examinations across all dose groups. No toxic reactions were observed, and the NOAEL (no observed adverse effect level) was 200 mg / kg / day.

[0187] Environmental safety: The biodegradation rate of LFC-1 peptide (OECD 301F) is >80% (28 days), which is a readily biodegradable substance; it has low toxicity to aquatic organisms, with a 96h LC 50 (Zebrafish)>100mg / L, which is a practically non-toxic level.

[0188] These results indicate that LFC-1 peptide has good safety and is suitable for pharmaceutical and food applications.

[0189] Test example: Comprehensive test of activity and stability of antifungal bovine liver peptide LFC-1

[0190] To comprehensively evaluate the performance of the LFC-1 peptide, this test example systematically compared the peptides prepared in various examples and comparative examples. Table 1 lists the MIC values and protease stability of the LFC-1 peptides prepared in different examples against Candida albicans.

[0191] Table 1 Comparison of activity and stability of LFC-1 peptides prepared in different examples

[0192] 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

[0193] Table 2 shows the activity comparison of different peptide sequence variants.

[0194] Table 2 Comparison of antifungal activity of different peptide sequence variants

[0195] Peptide sequence variants 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

[0196] Table 3 compares the synergistic effects of LFC-1 peptide with existing antifungal drugs.

[0197] Table 3 Synergistic effect of LFC-1 peptide and existing antifungal drugs (FICI value)

[0198] Combination therapy Candida albicans Aspergillus fumigatus 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

[0199] Table 4 compares the therapeutic effects of different preparations on fungal infections.

[0200] Table 4 Comparison of therapeutic effects of different preparations on fungal infections

[0201] preparation Disease models Clinical efficacy (%) 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 Olamine Nail Polish Onychomycosis 70 60 12

[0202] These test results demonstrate that the LFC-1 peptide of the present invention possesses excellent antifungal activity, good stability, and safety, and demonstrates significant efficacy in pharmaceutical formulations and food preservation applications. In particular, its synergistic effect with existing antifungal drugs provides a new therapeutic strategy for addressing fungal resistance.

[0203] The antifungal bovine liver peptide LFC-1 and its preparation method provided by the present invention have excellent industrial applicability and can be produced on a large scale for applications in medicine and food preservation. The peptide is prepared using conventional equipment and processes, with abundant raw material sources, moderate costs, and mild process conditions, making it suitable for industrial production. Furthermore, the final product exhibits excellent stability and a long shelf life under standard storage conditions, facilitating commercial applications.

[0204] In the pharmaceutical field, the LFC-1 peptide of the present invention can be formulated into a variety of dosage forms, such as creams, gels, nail polishes, oral tablets, and injections, for treating various fungal infections. In the field of food preservation, the LFC-1 peptide of the present invention can be added directly to food or formulated into packaging materials, sprays, or soaking solutions to extend the shelf life of food. These applications have enormous market potential and economic value.

[0205] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection 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: The following steps are involved: a. Fresh bovine liver tissue was washed with cold purified water containing 0.1% sodium hypochlorite at 4°C, cut into 3 cm³ cubes, and quick-frozen in liquid nitrogen. The resulting powder was then ground into a particle size of less than 1 mm. b. The ground tissue was suspended in 10 mM sodium phosphate buffer at a ratio of 1:5 (w / v) at pH 7.2, containing 150 mM sodium chloride and 1 mM EDTA, and sonicated at 20 kHz and 80% amplitude for 15 minutes, cooling every 2 minutes; c. The homogenate was centrifuged at 10,000 × g for 30 min at 4°C, and the supernatant was collected and filtered through a 0.45 μm polyethersulfone membrane; d. The filtered supernatant was sequentially hydrolyzed with three enzymes: i. Phase I: using Alcalase 2.4 L, enzyme: substrate ratio of 1:100 (w / w), pH 8.0, temperature 55 ° C, reaction time 2 hours; ii. Second stage: adjust pH to 2.5, add pepsin, enzyme: substrate ratio is 1:100 (w / w), temperature 37 ° C, reaction for 3 hours; iii. The third stage: adjust the pH to 7.0, add Flavourzyme, enzyme: substrate ratio of 1:50 (w / w), temperature 50 ° C, reaction for 4 hours; e. The hydrolyzate was heated to 85 ° C for 15 minutes to inactivate the enzyme, cooled to 25 ° C and centrifuged at 12,000 × g for 30 minutes; f. Perform three-stage membrane separation on the supernatant: i. First stage: using a 10 kDa molecular weight cut-off ultrafiltration membrane, a transmembrane pressure of 2.5 bar, a flow rate of 150 L / m² / h, a temperature of 25°C, diafiltration with 5 volumes of 20 mM, pH 7.0 sodium phosphate buffer, and collecting the permeate; ii. Second stage: The first stage permeate was treated with a 3 kDa molecular weight cutoff ultrafiltration membrane at a transmembrane pressure of 3.0 bar, a flow rate of 120 L / m² / h, and a temperature of 25°C. The permeate was concentrated to 5 times and diafiltered with 3 volumes of 20 mM sodium phosphate buffer (pH 7.0). The retentate was collected. iii. Third stage: The retentate from the second stage was treated using a 500 Da molecular weight cutoff nanofiltration membrane at a transmembrane pressure of 10 bar, a flow rate of 80 L / m² / h, and a temperature of 25°C, and concentrated to 10 times. g. The retentate after the third stage treatment is subjected to three-step chromatography purification: i. Step 1: Cation exchange chromatography was performed using SP Sepharose Fast Flow as the filler, equilibrated with 20 mM sodium phosphate buffer, pH 5.5, and eluted with a gradient of 0-1.0 M sodium chloride in the same buffer; ii. Step 2: Hydrophobic interaction chromatography was performed using Phenyl Sepharose 6 Fast Flow as the filler, equilibrated with 20 mM sodium phosphate buffer containing 1.0 M ammonium sulfate, pH 7.0, and eluted with a gradient of 1.0-0 M ammonium sulfate in the same buffer; iii. Step 3: Reverse phase chromatography was performed using a polymer reverse phase medium DuPont AmberChrom CG161, equilibrated with 0.1% trifluoroacetic acid aqueous solution, and eluted with a gradient of 0-60% acetonitrile containing 0.1% trifluoroacetic acid to collect the target fractions; h. The collected target fraction was filtered through a 0.22 μm sterilizing grade filter and then mixed with a stabilizing formulation to obtain a mixture comprising 5% trehalose, 0.5% human serum albumin, 10 mM sodium phosphate, pH 7.2; i. The mixture was freeze-dried to obtain antifungal bovine liver peptide. The freeze-drying conditions were: freezing stage at -40 ° C for 3 hours; main 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 chromatography step is performed using a multi-column countercurrent solvent gradient purification technique, which improves yield and reduces solvent consumption through continuous operation.

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

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

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. Enteric-coated tablets comprising: a core tablet containing 50-100 mg of antifungal bovine liver peptide, 30%-40% microcrystalline cellulose, 5% hydroxypropyl methylcellulose, 5% cross-linked sodium carboxymethyl cellulose, and 1% magnesium stearate, the core tablet being pre-coated and then coated with Eudragit L100-55 enteric coating; b. Lipid-based formulation containing: antifungal bovine liver peptide 5%-10% (w / w), medium-chain triglycerides 30%-40%, Kolliphor RH40 30%-40%, Transcutol HP 15%-20% and butylated hydroxytoluene 0.05%.

7. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is an injection preparation, including a liposome preparation or a lyophilized powder injection: a. Liposomal formulation containing: antifungal bovine liver peptide 5-10 mg / mL, phospholipids mixed with cholesterol and PEG-DSPE in a 60:35:5 molar ratio, a total lipid concentration of 20-30 mg / mL, sucrose 9% and 10 mM, histidine buffer at pH 6.5; b. Lyophilized powder injection contains: antifungal bovine hepatic peptide 50-100 mg / vial, mannitol 3%-5%, sodium phosphate buffer 10-20 mM, and sodium chloride 0.9%, 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: 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, adding a heat stabilizer 0.2%-0.5% and a processing aid 0.1%-0.3%, after processing by UV radiation cross-linking treatment; or b. preparing a degradable film, wherein 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 using a 2% calcium chloride solution; or c. Prepare a spray or soaking solution by dissolving the antifungal bovine liver peptide in an aqueous solution containing 5%-10% glycerol at a concentration of 0.02%-0.1% (w / v) for surface treatment of fresh fruits, vegetables or meat in an application amount of 2-5 mL / kg food.

9. The use according to claim 8, characterized in that The antifungal bovine liver peptide is applied to different food types at different concentrations: a. Fresh produce: Use a 0.02%-0.05% concentration spray or soak solution to effectively resist Botrytis cinerea, Penicillium, and Rhizopus, and extend the shelf life by 5-7 days at 4°C; b. Dairy products: Adding 1%-2% of antifungal bovine liver peptide to the packaging material is effective against Penicillium and Aspergillus, and can extend the shelf life by 2-3 weeks at 4°C; c. Baked products: Add 0.005%-0.01% of antifungal bovine liver peptide directly to the dough to effectively resist Aspergillus and Penicillium, and extend the shelf life by 7-10 days at room temperature; d. Meat products: Surface treatment with 0.05%-0.1% antifungal bovine liver peptide solution, which is effective against Cladosporium and Mucor, and can extend the shelf life by 3-5 days at 4°C; e. Juice: Directly add 0.01%-0.02% antifungal bovine liver peptide to effectively resist yeast and Candida lipolytica, and extend the shelf life by 5-10 days at 4°C.

10. Use of the antifungal bovine liver peptide according to claim 1 for preparing an antifungal infection drug, characterized in that: The drug is used alone or in combination with other antifungal drugs, and the antifungal type of the drug is any one of Candida albicans, Aspergillus and Trichophyton mentagrophytes.