Preparation Of Epsilon-poly-lysine Nanoliposomes And Its Antibacterial Mechanism Against Alicyclobacillus Spp.

As a natural cationic antibacterial peptide,ε-Polylysine(EPL)is produced by Streptomyces spp.through aerobic fermentation.Due to the excellent safety and antibacterial properties,Chinese government has approved EPL to be included in the list of food additives.According to the Chinese national standard(GB 2760-2014),EPL could be applied to baking foods,cooked meat,fruit and vegetable juice.However,some studies found that EPL will cause turbidity and precipitation when applying to fruit and vegetable juice.Meanwhile,the antibacterial activity will decline.This problem restricts EPL’s application in the juice industry.Therefore,this study aims to prepare a liposome delivery system loaded with EPL,which can be used in fruit juice and maintain stable and efficient antibacterial activity.Especially,the liposome won’t cause adverse effects or safety problems on the food products.The main research contents and results are as follows:(1)The preparation parameters of EPL nanoliposomes were optimized by single factor rotation method and the nanoliposomes were modified with chitosan.The morphology and structure of the nanoliposomes were characterized by Atomic Force Microscope(AFM),Transmission Electron Microscope(TEM),Fourier Transform Infrared Spectrometer(FTIR),and X-ray Diffractometer(XRD).Finally,the stability and sustained release properties of the nanoliposomes were evaluated through storage and release experiments.The optimization results showed that when the concentrations of EPL was 0.4 g/L,the addition of cholesterol was 5%,the ultrasound power was 340 W,the ultrasound time was 5 min,and the p H value was 6.5,the encapsulation efficiency,particle size,polydispersity index(PDI)and zeta potential of the primary EPL nanoliposomes was 55.41±3.37%,71.96±0.42 nm,0.20±0.01,and-7.55±0.43 m V,respectively.The modification results showed that the encapsulation efficiency of chitosan coated EPL nanoliposomes(CS-EPLLP)was 83.01±4.00%,significantly higher than that of EPL nanoliposomes without chitosan(EPLLP).The characterization results showed that the micro morphology of nanoliposomes was spherical vesicular structure,and there was an obvious coating on the surface of CS-EPLLP.EPL could be encapsulated into nanoliposomes,while chitosan was attached to the surface of nanoliposomes through hydrogen bonding.The storage and release results showed that there was no significant change in the particle size and PDI value of the nanoliposomes within 15 days storage.Compared to free EPL,EPL encapsulated with nanoliposomes had a slow-release characteristic,and chitosan coating could effectively avoid the "sudden release" phenomenon at the initial release stage.In summary,this study successfully prepared EPL nanoliposomes modified with chitosan,revealing its construction mechanism,and confirming that chitosan modification can effectively maintain the complete structure of EPL nanoliposomes and improve encapsulation efficiency.(2)The minimal inhibitory concentration(MIC)values of chitosan,EPL,EPLLP and CS-EPLLP against three strains of Alicyclobacillus spp.(A.acidoterrestris DSM 3923,A.acidocaldarius SD-4,A.fructus DSM 112018)were determined by two-fold dilution method.The growth inhibition of EPL,EPLLP and CS-EPLLP at different concentrations against three strains of Alicyclobacillus spp.was studied in 5 days.In the culture medium and apple juice system,the minimum bactericidal concentration(MBC)values of EPL,EPLLP and CS-EPLLP against Alicyclobacillus spp.were determined by two-fold dilution method.Finally,the inhibition mechanisms of EPL,EPLLP and CS-EPLLP against three strains of Alicyclobacillus spp.were evaluated,with the methods of measuring reactive oxygen species(ROS)release,protein leakage,ATP production and observation of cell morphology changes.The MIC determination and growth inhibition results showed that the MIC values of chitosan,EPL,EPLLP,CS-EPLLP were 5 mg/L,2.5 mg/L,1.25～2.5 mg/L,and 0.625～1.25mg/L,respectively.At the same dosage,EPL nanoliposomes showed better slow-release inhibition and killing effects than free EPL.The MBC determination results showed that that when the addition of EPLLP and CS-EPLLP was 5 mg/L and 2.5 mg/L,respectively,A.acidocaldarius SD-4 and A.fructus DSM 112018 could be killed completely.The inhibition mechanisms results showed that EPL nanoliposomes could increase cell membrane permeability,cause the surface damage and severe oxidative stress,result in cell death eventually.In summary,the EPL nanoliposomes prepared in this study have strong antibacterial activity and are less affected by the juice environment.Especially,those can be used for the control of Alicyclobacillus spp.in fruit juice.(3)The storage and processing stability of EPL nanoliposomes in a simulated juice system was evaluated,and the effects of different amounts of nanoliposomes on the quality of apple juice were measured.Finally,the biological safety of nanoliposomes was verified through cytotoxicity experiments.The results of storage and processing stability showed that low temperature and chitosan modification were more conducive to the stability of EPL nanoliposomes.High-temperature short-time thermal sterilization and pulsed light sterilization didn’t have a significant impact on nanoliposomes.However,compared to the untreated group,after 90 minutes of UV sterilization,the particle sizes of EPLLP and CS-EPLLP increased by 35.46% and 57.09%,respectively,and the structure of nanoliposomes was destroyed.When the homogenization pressure exceeded 30 MPa,the PDI value significantly increased to above 0.35,which was due to the accelerated aggregation of vesicular fragments caused by system temperature rise,resulting in a decrease in stability.The evaluation results of fruit juice showed that the addition of nanoliposomes had almost no impact on the main aromatic components and centrifugal precipitation rate,but had a small impact on the transmittance,and had a significant impact on the color of fruit juice.With the changes of addition of nanoliposomes,ΔE value was between 1.00 and 3.25.The results of cytotoxicity experiments showed that when the addition of EPLLP and CS-EPLLP were 5 mg/L and 20 mg/L,respectively,there was no significant difference between the treatment group and the control group.In summary,UV sterilization and high-pressure homogenization aren’t recommended for use in the processing of fruit juices containing EPL nanoliposomes.Adding an appropriate amount of EPL nanoliposomes to fruit juices won’t have a negative impact on the food quality and safety.This study mainly prepared a novel type of biomaterial,chitosan-coated EPL nanoliposomes.Its basic physicochemical properties and structural characteristics were evaluated.Some special properties,such as stability,slow-release,bacteriostasis and safety,were systematically analyzed.As an effective EPL delivery system,EPL nanoliposomes were potential to solve the problem of precipitation and bacteriostasis failure in fruit and vegetable juice.