| The natural preservative carvacrol has a broad-spectrum antibacterial activity,antioxidant and anti-cancer effects,but it is unstable under light and heat conditions,and direct use is easy to reduce the antibacterial effect.By combining with other antibacterial ingredients and adopting delivery vehicle embedding strategy,the above application defects of carvacrol can be eliminated to a certain extent.The combined use of carvacrol and other preservatives can reduce its concentration,while maintaining antibacterial efficiency.Delivery systems such as liposomes and cyclodextrins are non-toxic,harmless,and biocompatible.Carvacrol can be embedded in the delivery vehicle to protect the antibacterial activity of carvacrol and mask its special smell.Based on this,the synergistic antibacterial activity and mode of action of carvacrol and ?-polylysine were studied,and two differently charged ?-polylysine/S-layer protein modified carvacrol lipids were prepared characterize the two liposomes and investigate their stability,release performance,and antibacterial properties,with the aim of improving the antibacterial effect and application performance of carvacrol.The main results are as follows:The synergistic antibacterial effect and mechanism of carvacrol and ?-polylysine were studied.The minimum inhibitory concentration(MIC)of carvacrol against E.coli and S.is 320 ?g/mL,and the corresponding minimum bactericidal concentration(MBC)is 1280 ?g/mL;?-polylysine against E.coli The MICs of coli and S.aureus are 25 ?g/mL and 12.5 ?g/mL,respectively,and the corresponding MBCs are 100 ?g/mL and 50 ?g/mL,respectively.The graded bacteriostatic index(FICI)shows that carvacrol and ?-polylysine have significant synergistic bacteriostatic effects on S.aureus(FICI=0.5)and E.coli(FICI=0.375).The biofilm removal experiment confirmed that carvacrol and ?-polylysine can synergistically inhibit E.coli and S.aureus growth and biofilm formation at low concentration.Through UV-Vis,FCM,SEM and other means to reveal the antibacterial mechanism of carvacrol and ?-poly lysine are:destruction of bacterial cell membranes,inhibition of bacterial respiratory activity,and damage to the permeability of cell membranes leading to the outflow of intracellular substances,causing bacteria death.?-polylysine-coated?-CD-carvacrol liposomes(?-pL-?-CD-Car-LP)were prepared and their antibacterial properties were studied.The suitable preparation conditions of?-cyclodextrin-carvacrol(?-CD-Car)inclusion compound are:the mass ratio of ?-CD-Car is 1:9,the inclusion temperature is 55?,and the magnetic stirring time is 2 h The average inclusion rate of?-CD-Car prepared under this condition was 65.45 ±2.51%,and the embedding rate was 82.33±1.69%.?-CD-Car is 10 mg/mL,the particle size of ?-pL-?-CD-Car-LP is moderately 377.2± 1.96 nm,the minimum PDI is 0.223 ± 0.019,the maximum encapsulation rate of essential oil is 74.28 ±2.17%,The ?-pL adsorption rate is 79.59±1.23%.The liposome has the best storage stability at 4?,the smallest change is within the acceptable range.The release rate in vitro of?-pL-?-CD-Car-LP was 8%in the first 6 hours,and it had a good sustained-release function.The experimental results showed that the MICs of carvacrol and ?-polylysine in liposomes were 20 ?g/mL and 3.125 ?g/mL,respectively,which were higher than those of carvacrol and ?-polylysine when used in combination.4 times and 2 times,can significantly reduce the use of carvacrol and achieve antibacterial effect.It shows that ?-pL-?-CD-Car-LP has a long-acting release antibacterial effect.The ?-CD-Car-LP coated with S-layer protein was prepared and its antibacterial properties were studied.Application method was used to extract L.buchneri 20023 surface protein,SDS-PAGE was used to identify the band size between 45?66.7 kDa,and the two bands a and b were tapped and identified as S-layer protein by MALDI-TOF-MS.The molecular weights are 55 kDa and 64 kDa respectively;the suitable preparation conditions of protein-modified liposomes are:when the concentration of S-layer protein is 30 ?g/mL,the liposomes obtain the best average particle size of 229.1 ±6.81 nm,PDI The value was 0.139 and the Zeta potential was 27.9 mV;it was confirmed by testing the release rate of carvacrol in liposomes before and after Triton X100 treatment.S-layer protein coating significantly enhanced the stability of liposome membrane,S-layer protein coating The in vitro release rate of coated ?-CD-Car-LP was 4.1%lower than that of?-cyclodextrin-carvacrol,indicating that S-layer protein coating reduced the burst effect of liposomes.The minimum inhibitory concentration experiment further confirmed that the double-layer embedding of ?-cyclodextrin/liposome system can improve the utilization rate of carvacrol,and the MIC value of carvacrol was reduced from 320 ?g/mL to 40 ?g/mL.The above research shows that ?-CD-Car-LP coated with S-layer protein has a long-acting antibacterial effect. |
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