Perilla protein polypeptide and its application in treating poultry infectious diseases

Abstract

This application provides a perilla protein polypeptide obtained by enzymatic hydrolysis of perilla protein using ficin. Prepared from the safe food ingredient perilla, the perilla protein polypeptide exhibits significant antibacterial effects against a variety of Gram-negative and Gram-positive avian pathogens, including Enterococcus gallinarum, Staphylococcus aureus, and Salmonella gallinarum, providing a new treatment option for infectious diseases in poultry.

Description

Field of the invention:

[0001] The present application belongs to the field of proteins and anti-infective drugs. Specifically, the present application provides perilla protein polypeptides and their application in treating poultry infectious diseases. Background technology:

[0002] Perilla is a herbaceous plant of the genus Perilla in the Lamiaceae family, primarily found in East and South Asia. It has a long history of medicinal and edible use, including in the preparation of oils and spices. In Traditional Chinese Medicine, it is believed to have anti-cold properties, promote qi circulation, alleviate fullness, and stabilize pregnancy. Modern medical research has revealed that perilla and many of its components possess antibacterial, anti-inflammatory, anti-tumor, and analgesic properties.

[0003] The most common use of perilla is oil extraction. The remaining perilla meal, which contains a large amount of perilla protein, is currently used primarily as feed and fertilizer, sometimes even discarded. Given perilla's known medicinal uses, further processing and functional exploration of perilla protein is a viable approach to further developing this plant resource. Summary of the invention:

[0004] In view of the above situation, in order to further develop the application of perilla protein in the treatment of poultry diseases, on the one hand, the present application provides a perilla protein polypeptide, which is obtained by enzymatic hydrolysis of perilla protein with ficin.

[0005] Furthermore, the preparation method of perilla protein is as follows: crushing defatted perilla meal and adding it into water with a mass 20 times that of the defatted perilla meal, mixing; adjusting the pH to 10; treating in a 60-70°C water bath for 1 hour; centrifuging to obtain the supernatant; adjusting the pH of the supernatant to 4.5; centrifuging to obtain a precipitate; washing the precipitate with water and freeze-drying it to obtain perilla protein.

[0006] Furthermore, the enzymatic hydrolysis method comprises: using water as a solvent to prepare an enzymatic hydrolysis composition comprising 100 mg / mL perilla protein, 500 U / mL ficin, 5 mg / mL Tween 20 or Span 20, and pH 5.5-6.5; and enzymatically hydrolyzing the enzymatic hydrolysis composition at 40-50° C. for 4-5 hours.

[0007] Furthermore, the perilla protein polypeptide is a polypeptide having a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] On the other hand, the present application provides the use of the above-mentioned perilla protein polypeptide in the preparation of a drug for treating poultry infectious diseases.

[0009] Furthermore, the poultry infectious disease is caused by Staphylococcus aureus infection.

[0010] Furthermore, the poultry infectious disease is caused by Enterococcus gallinarum infection.

[0011] Furthermore, the poultry infectious disease is caused by infection with Salmonella Gallinarum.

[0012] Preferably, the poultry infectious disease is a chicken infectious disease. The chicken may be a broiler or a laying hen.

[0013] On the other hand, the present application provides a drug for treating poultry infectious diseases, wherein the drug comprises the above-mentioned perilla protein polypeptide.

[0014] Furthermore, the drug is an oral drug.

[0015] Furthermore, the medicine also includes excipients acceptable in veterinary medicine.

[0016] Those skilled in the art can determine the specific types of acceptable excipients in the veterinary drugs based on veterinary medicine books and relevant regulatory provisions for veterinary drugs and poultry products, including but not limited to fillers, dispersants, adhesives, lubricants, flavorings, coating agents, solvents, cosolvents, pH regulators, osmotic pressure regulators, etc.

[0017] The perilla protein peptide used in this application is derived from the food ingredient perilla, which is safe and has potential medicinal properties in Traditional Chinese Medicine, such as dispelling cold and promoting qi circulation and soothing the stomach. The perilla protein peptide exhibits significant antibacterial effects against a variety of Gram-negative and Gram-positive avian pathogens, including Enterococcus gallinarum, Staphylococcus aureus, and Salmonella gallinarum, providing a new drug option for the treatment of various poultry infectious diseases. Description of the drawings:

[0018] Figure 1 This is the mass spectrum of the synthesized ZS-1.

[0019] Figure 2 This is the mass spectrum of the synthesized ZS-2. Specific implementation method:

[0020] How to consider antibacterial performance:

[0021] The strains used included Enterococcus gallinarum (CGMCC 1.9125, G+), Staphylococcus aureus (CGMCC 1.2386, G+), and Salmonella gallinarum (ATCC 13036, G-).

[0022] The inhibition zone method was used for the preliminary screening: wells were punched on the LB plates prepared with each strain, 50 μL of a 20 mg / mL protein solution was added to each well, and the plates were cultured at 37°C for 24 hours to observe the presence and size of the inhibition zone.

[0023] Minimum inhibitory concentration test: The bacterial suspension of each strain in the logarithmic growth phase was adjusted to 1×10 6CFU / mL, 100 μL of bacterial solution, as well as 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, and 2048 μg / mL of antimicrobial peptide / protein solutions and antibiotic positive and negative controls were added to each well, and OD600 was measured after incubation at 37°C for 12 hours.

[0024] Example 1 Preparation of Perilla Protein

[0025] Perilla protein was produced using defatted perilla meal from an oil production company as raw material. The defatted perilla meal was crushed and added to water (20 times the mass of the defatted perilla meal), mixed, and the pH was adjusted to 10. The mixture was placed in a 60-70°C water bath for 1 hour, centrifuged, and the supernatant was collected. The pH of the supernatant was adjusted to 4.5, and the precipitate was collected by centrifugation. The precipitate was washed with water and freeze-dried to obtain perilla protein.

[0026] Example 2 Enzymatic Hydrolysis of Perilla Protein

[0027] The perilla protein obtained in Example 1 was enzymatically hydrolyzed using papain, bromelain, ficin, and trypsin (Xiasheng, food grade), and the specific conditions were as follows:

[0028] Table 1 Enzymatic hydrolysis conditions

[0029]

[0030] The results showed that under the experimental conditions, undigested perilla protein exhibited only a small inhibition zone against Staphylococcus aureus. Among the enzymatically digested perilla proteins, trypsin produced similar results to undigested proteins, while bromelain hydrolyzates exhibited increased resistance to Enterococcus gallinarum. Only the ficin hydrolyzate exhibited a significant antibacterial zone against all three bacteria. Therefore, the ficin hydrolyzate was selected for further investigation of enzymatic conditions.

[0031] Considering that the raw material perilla is rich in oil, the aggregation mode of protein in aqueous solution may vary greatly. Therefore, the applicant added a surfactant (domestic, food grade) to the enzymatic hydrolysis system:

[0032] Table 2 Improved enzymatic hydrolysis conditions

[0033]

[0034] The MIC results of the enzymatic hydrolysates are shown in Table 3

[0035] Table 3 MIC test results of enzymatic hydrolysates

[0036]

[0037] The results showed that the addition of Tween or Span indeed promoted the enzymatic hydrolysis process and improved the antibacterial properties of the enzymatic hydrolysates.

[0038] Example 3 Identification, synthesis and antimicrobial effect verification of antimicrobial peptides

[0039] For the sake of stability, resistance to digestion and ease of preparation, the target product is a short peptide with a smaller molecular weight. Therefore, the enzymatic hydrolysate is centrifuged using a 3kDa ultrafiltration tube to obtain a perilla protein polypeptide less than 3kDa.

[0040] After desalting, the above-mentioned small molecule perilla protein peptide was separated and identified by LC-MS / MS method: the liquid phase was Shimadzu LC-20AD (self-assembled C18 column 75μm×25cm; mobile phase 1: 2% ACN, 0.2% formic acid, mobile phase 2: 98% ACN, 0.2% formic acid; column flow rate 300nL / min, column temperature 40℃), directly connected to the mass spectrometer (Thermo Fisher MALDI-TOP, primary scan range: 200-1800m / z, AGC target: 3e 6 , resolution: 70000, maximum injection time: 50ms):

[0041] Mass spectrometry results were analyzed using MaxQuant 1.5.3.30 and identified using Mascot 2.3.02. R3 The researchers used the random forest algorithm and the antiBP2 database (support vector machine algorithm) to predict antimicrobial peptides, resulting in three perilla protein peptides predicted to be antimicrobial peptides. ToxinPrep (support vector machine algorithm) was used to predict toxicity and synthesis difficulty, eliminating one peptide. The remaining two peptides, ZS-1 (AIRKNLL, SEQ ID NO. 1, molecular weight 827.02) and ZS-2 (ELGLSN, SEQ ID NO. 2, molecular weight 631.67), both showed promising potential toxicity and digestibility, with acceptable solubility.

[0042] NovoPro solid phase resin method (Fmoc) was commissioned to synthesize ZS-1 and ZS-2 (mass spectrum as shown in Figure 1 and Figure 2 The MICs of the two antimicrobial peptides were further tested, and the results are shown in Table 4:

[0043] Table 4 MIC test results of synthetic antimicrobial peptides

[0044] antimicrobial peptides Enterococcus gallinarum Staphylococcus aureus Salmonella Gallinarum ZS-1:AIRKNLL 32 16 16 ZS-2:ELGLSN 32 8 16

[0045] The results showed that the two peptides achieved significant antibacterial activity at significantly higher concentrations than their enzymatic hydrolysates, demonstrating efficacy against three typical poultry Gram-positive and Gram-negative bacteria. Given the relatively low synthesis cost of the two peptides (approximately 500 yuan per 50mg during the experimental phase), they hold promise for use as standalone antibacterial agents in poultry farming.

Claims

1. Use of a perilla protein polypeptide in preparing a medicament for treating poultry infectious diseases; the amino acid sequence of the perilla protein polypeptide is SEQ ID NO.1 or SEQ ID NO.2; the poultry infectious disease is caused by infection with Staphylococcus aureus, Enterococcus gallinarum or Salmonella gallinarum.

2. A drug for treating poultry infectious diseases, characterized in that: The medicine contains a perilla protein polypeptide, and the amino acid sequence of the perilla protein polypeptide is SEQ ID NO.1 or SEQ ID NO.2; the poultry infectious disease is caused by infection with Staphylococcus aureus, Enterococcus gallinarum or Salmonella gallinarum. The drug according to claim 2 , which is an oral drug.

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