A Study On The Fractionation And Identification,structure-function Relationships And The Pilot-scale Preparations Of Cottonseed Protein Antibacterial Peptides

Cotton is recognized as the most important economic and oil crops in China,which can produce more than three million tons of defatted cotton seed protein powder.It is a very important natural plant protein,which contains more than 50%of the protein.In recent years,under the influence of"anti-antibiotic"movement all over the world,China has also issued relevant policies to reduce and ban antibiotics in feed.Therefore,to explore and develop a safe and efficient substitute for antibiotics-antimicrobial peptides has become the focus of people's research.Moreover,it is urgent to realize the industrial production of antimicrobial peptides.This paper explored the use of defatted cottonseed protein powder as raw material.The antimicrobial peptides were separated and identified quickly and accurately using ion exchange chromatography,reverse high performance liquid chromatography and mass spectrometry.Moreover,the antibacterial mechanism was studied.On this basis,a pilot-scal production line of antimicrobial peptides was designed and established.The main conclusions are as follows:First of all,the enzymatic hydrolysis of defatted cottonseed protein powder was investigated by using three kinds of proteases.It was proved that the enzymatic hydrolysis effect of alkaline protease was better than that of other enzyme.The fraction F₂ isolated using sephadex C-25 showed antibacterial activity against Staphylococcus Aureus,Escherichia coli and Streptococcus.Comparing with the pre-separation component F₀,the basic amino acid content of fraction F₂ was increased by 18.3%,and the arginine content increased by 11.7%.Sensitivity analysis of environmental conditions revealed antibacterial peptides were not influenced by temperature and p H,but sensitive to Ca²⁺.When the concentration of Ca²⁺increased from 0.05%to 5%,its antibacterial activity decreased by 40%.There was no hemolysis when the concentration of fraction F₂ increased from 2.0 mg/mL to 64.0 mg/mL.Secondly,the components F3,F3-3 and F3-3-C were obtained by step-by-step separation and purification,and the most active component F3-3-C was analyzed by LC-MS/MS.Three peptides were identified,among which the peptide His-His-Arg-Arg-Phe-Ser-Leu-Tyr?HHRRFSLY?had the highest IC₅₀ value of 0.26 mg/mL.In contrast,four fractions were separated and purified using the characteristic reaction of arginine and benzaldehyde,of which the highest inhibition rate was 62.3%.Seven peptides were identified from fraction P5-3 by using MALDI-TOF-MS/MS,of which Lys-Asp-Phe-Gly-Arg-Arg?KDFGRR?has the highest antibacterial activity with IC₅₀ value of 0.62 mg/m L.The interaction mechanism between antibacterial peptides and Escherichia coli cell membrane was studied.Flow cytometry analysis showed that 20.6%and 82.5%cells of Escherichia coli cells were infected after 20 min and 120 min,respectively.While only 5.8%of cells in the control group were infected.Fluorescent double labeling technique demonstrated the binding site was the cell membrane.Moreover,the microscopic morphology technique demonstrated that the peptides destroyed the permeability of cell membrane and caused cytoplasmic fluid leakage.The molecular docking technique was used to verify that HHRRLFSY was connected to the Asp107 residue of OmpF through the hydrogen bond of 2.88?.In addition,the gel block experiment proved that HHRRLFSY could combine with DNA and delay the migration of DNA.The relationship between the antibacterial activity and the characteristic parameters of antimicrobial peptides was analyzed,considering the structural characteristics of antimicrobial peptides.The IC₅₀ value of antimicrobial peptides decreased with the increase of theoretical isoelectric point and net positive charge.When the theoretical isoelectric point increased from pH 9.88 to pH 12.88,the IC₅₀ value decreased from 2.3 mg/mL to 0.5 mg/mL.when the number of net positive charge increased from 2 to 5,its antibacterial activity increased by 4.8 times.The position of basic amino acid residues in the peptide also had a certain effect on the antibacterial activity.The results of path analysis showed that the fitted linear regression equation is y=0.892-0.106x₁-0.799x₂-0.019x₃.The results of significance test showed that there was a significant difference between independent variables and dependent variables.Finally,the pilot production line of antimicrobial peptides was established,including enzymatic hydrolysis,separation,evaporation,and spray drying system.The pilot-scale products were successfully used in feed instead of antibiotics.The product parameters included acid-soluble protein??90.0%?,IC₅₀ value??3.0 mg/mL?,lead??5.0 mg/kg?,arsenic??2.0mg/kg?,and colonial morphology??1000 CFU/g?.