Study On Antibacterial And Synergistic Effect Of Lysozyme-eugenol-casein Nanoparticles

Eugenol,as a natural phytochemical essential oil,has been found effective antibacterial,antiinflammatory,antiviral and other biological activities,and is widely used as a natural preservative in the preservative food and medicine industry.However,the antibacterial efficiency of eugenol is substantially limited for its high volatility,and low water solubility.In recent years,the application of nano-encapsulation technology has significantly improved its physicochemical stability,achieved controllable release,and significantly enhanced its antibacterial properties.In particular,the cell wall polysaccharide of pathogenic gram-positive bacteria significantly hampered the permeability of eugenol,resulting in a significant decrease of the antibacterial efficiency.In this study,a lysozyme-eugenol-casein nanoparticle(Ly-EL-CS-NPs)was constructed by self-assembly of eugenol-casein and lysozyme modification.The effects of process conditions and formulation on the particle size,polydispersity indexes(PDI),zeta potential,entrapment efficiency,eugenol loading and antibacterial activity were investigated.The sustained-release and stability were compared between the lysozyme-eugenol-casein nanoparticle(Ly-EL-CS-NPs)and eugenol-casein nanoparticle(EL-CS-NPs)groups.Furthermore,the enhancement of eugenol antibacterial activities against gram-positive bacteria by a lysozymeeugenol-casein nanosystem was further explored.With the average particle size,zeta potential,entrapment efficiency(EE)and drug loading rate as the indices,the best preparation process conditions of EL-CS-NPs and Ly-EL-CS-NPs were thoroughly investigated by means of dynamic light dynamic(DLS)and high-performance liquid chromatography(HPLC).Results showed that when casein concentration was 20 mg/m L and eugenol concentration was up to 5 mg/m L,the EE of eugenol in EL-CS-NPs was up to 89.67%with the average particle size of 133.7 nm.when casein concentration was 20 mg/m L and eugenol concentration was up to 5 mg/m L,as well as the content of lysozyme was 0.05 mg/m L,the EE of eugenol in Ly-EL-CS-NPs was up to 92.20% with the average particle size of 151.90 nm.ELCS-NPs and Ly-EL-CS-NPs nanocomposites had excellent storage stability and freeze-drying stability.Compared with EL-CS-NPs,Ly-EL-CS-NPs have better storage stability: the shelf life of EL-CS-NPs nanocomposite was 14 days and the shelf life of Ly-EL-CS-NPs nanocomposite was 180 days.The results of slow-release kinetics analysis showed that both EL-CS-NPs and LyEL-CS-NPs nanocomposites had a good slow-release trend.The lower the temperature,the slower the release rate of eugenol,and at the same temperature,the slower-release rate was: LyEL-CS-NPs>EL-CS-NPs>EL(free).Under the conditions of 20? and 37?,the free eugenol is almost completely released within 48 h,and the eugenol in the EL-CS-NPs and Ly-EL-CS-NPs system still remained above 70%.Further measurement of the particle size change within 48 h showed that,The Ly-EL-CS-NPs system could be realized the slow and sustained release of eugenol.At the same time,the volatilization of eugenol was effectively inhibited by Ly-EL-CSNPs system,confirming a better protection of eugenol.In order to study the antibacterial activity of eugenol on gram-positive bacteria,the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of eugenol against gram-positive bacteria,were determined by liquid dilution method and agar diffusion method.The results showed that the MIC and MBC of EL-CS-NPs for Bacillus subtilis were 0.24mg/m L and 0.48 mg/m L.The MIC and MBC of Ly-EL-CS-NPs for Bacillus subtilis were 0.12mg/m L and 0.40 mg/m L.The MIC and MBC of EL-CS-NPs for Staphylococcus aureus were0.24 mg/m L and 0.28 mg/m L.The MIC and MBC of Ly-EL-CS-NPs for Staphylococcus aureus were 0.16 mg/m L and 0.24 mg/m L.Compared with EL-CS-NPs,Ly-EL-CS-NPs have lower MIC and MBC,and show higher antibacterial activity against gram-positive bacteria.According to cell morphology analysis,Ly-EL-CS-NPs nanocomposite promoted the penetration of eugenol into the cell membrane structure and it achieved the purpose of effective sterilization by jointly destroying the cell wall,changing the permeability of the cell membrane,overflowing the contents and apoptosis.