Synergistic Antibacterial Mechanism Of Ultrasonication With Thyme Essential Oil Nanoemulsion And Their Application

Plant-derived essential oil is a new natural and safe food bactericide.However,due to their high hydrophobicity,poor stability,pungent odor and other shortcomings,the application in food is limited.In order to improve the bactericidal performance of essential oils and reduce their impact on the sensory quality of foods,most of the current researches use nanotechnology to encapsulated plant essential oils in a nano-delivery system.To the best our knowledge,current published research mainly focuses on emulsion properties and bactericidal effects,and related research on bactericidal mechanism,while few reports has paid attention on their sterilization mechanism.In this regard,this thesis used thyme oil as the material,and firstly used high-pressure homogenization(HPH)and ultrasonic(US)technology to prepare thyme essential oil nanoemulsion(TEON),and mainly compared the effects of different preparation methods on the physical properties of the emulsion,as well as the effects on Escherichia coli O157:H7(E.coli O157:H7)and Staphylococcus aureus(S.aureus)for their bactericidal effects and mechanism of action.In order to further improve the bactericidal effect of TEON and reduce its dosage and the impact on food sensory quality.Ultrasound and TEON were combined to explore the bactericidal effect on E.coli O157:H7 and S.aureus,and to explore ultrasound synergistic sterilization mechanism of field and TEON.In addition,the two are combined to clean the surface of purple cabbage to make a preliminary study on the antibacterial effect and the effect on the quality of purple cabbage.The main findings are as follows.(1)Preparation of TEON and research its bactericidal performanceAnd the physical and chemical properties of TEONs were analyzed,such as particle size,Zeta-potential,and storage stability.In addition,the antibacterial effect and mechanism of TEON against E.coli O157:H7 and S.aureus were analyzed.The results showed that TEON prepared by ultrasound had the best physical and chemical properties,with the smallest particle size of 8.96 nm and the largest Zeta-potential value of 60.38 m V.After 15 days of storage at4°C,the particle size of the emulsion was less than 32 nm.The study of bactericidal kinetics found that E.coli O157:H7 and S.aureus decreased by 0.48 and 3.43 log CFU/m L after treated by TEON-US for 20 min,indicated that TEON-US had stronger bactericidal effect against both strains than TEON-HPH.Observed by electron microscope,compared with the crude emulsion treatment group,after TEON treatment,E.coli O157:H7 cells were damaged and pores increased.At the same time,through fluorescence measurement,that TEON reduced the fluidity of the cell membrane and depolarized the cell membrane,resulting in damage to the cell membrane function.In addition,gas chromatography-mass spectrometry and fourier infrared spectroscopy analysis found that the degree of changes in cell membrane fatty acid and other components and structure increased after TEON treatment.(2)Synergistic bactericidal effect and mechanism of ultrasound and TEONThe bactericidal effect and mechanism of TEON on E.coli O157:H7 and S.aureus under ultrasonic field were analyzed.TEON(0.375 mg/m L)and US(255 W/cm~2)reduced E.coli O157:H7 bacteria by 4.13 and 0.69 log CFU/m L after treated with 9 min,respectively.While TEON+US reduced E.coli O157:H7 7.42 log CFU/m L at the same treat conditions.When the power density was 191 W/cm~2 and the treatment time was 3 min,the combined treatment increased the lethality of S.aureus(an increase of 1.33 log CFU/m L)compared with the sum of the individual treatments,indicating that US and TEON had a synergistic effect in the process of killing bacteria.Observation by electron microscope showed that after TEON+US treatment,cell damage was more serious,some cells were lysed,and the leakage of intracellular macromolecular substances increased significantly.After TEON+US treatment,E.coli O157:H7 and S.aureus shrunk,some cells were lysed,and the leakage of intracellular macromolecular substances increased significantly.Analysis the fluidity,potential of cell membrane,and membrane components of E.coli O157:H7 and S.aureus,found that cell membrane fluidity was significantly reduced after TEON+US,the degree of membrane potential depolarization,and cell membrane components(proteins,phospholipids,etc.).The degree of change was significantly higher than that of the single treatment group.It was speculated that ultrasonic treatment may increase the permeability of cell membranes,promote the thyme oil nanoemulsion to enter bacterial cells,and act on intracellular macromolecules,ultimately killing somatic cells.(3)Study on the application of ultrasound combined with TEON in the cleaning of purple cabbageThe bactericidal effect of the combined treatment of ultrasound and TEON on E.coli O157:H7 inoculated on the surface of purple cabbage,and its inhibitory effect on the mold and yeast in purple cabbage under the storage conditions of 4?and 25?,as well as its effect on anthocyanin content,color and weight,etc.The results showed that the TEON concentration was 0.03125 mg/m L,the ultrasonic power density was 63.75 W/cm~2,and the combination of the two could reduce E.coli O157:H7 bacteria by 3.85 log CFU/m L when the purple cabbage was treated for 6 min.Under this treatment condition,we found that the amount of mold and yeast in purple cabbage were significantly reduced during storage after treating by TEON+US.Compared with the control group,TEON+US treatment had no significant effect on the anthocyanin content,hardness,and antioxidant properties of purple cabbage(p>0.05),but could significantly reduce the weight loss of purple cabbage(storage condition 4?)(p<0.05).It can be concluded that the TEON+US treatment can not only inhibit the growth of mold and yeast during the storage period,but also had no negative impact on the sensory quality of purple cabbage.