| Clove oil is a kind of natural, safe, nontoxic and broad spectrum antibacterial agents, but the strong smell, volatile and water insolubility limit its application in food industry. Liposome is the artificial cell membrane, and it can improve the stability and the water solubility of essential oil significantly. Meanwhile, α-hemolysin can form channel in the membrane structure of cell. Thus, the preparation of α-hemolysin responsive liposome-encapsulated clove oil can improve the antibacterial effect of essential oil in the application of food industry.In this study, the chemical composition of clove oil was analyzed by GC-MS, the MIC and MBC of clove oil against 7 kinds of bacteria were evaluated, and the antibacterial activity and mechanisms of clove against E. coli and S. aureus were determined by the spread plate method, SEM, spectrophotometry, ATP bioluminescence assay and the BCA assay. Then, using phospholipids, cholesterol, clove oil and PVP as raw materials, this study employed the thin film dispersion-ultrasonic method to prepare liposome encapsulated clove oil. PDI, particle size, Zeta potential, pH, turbidity and entrapment efficiency of liposome were studied, and the best performance was established. Meanwhile, the morphology of liposome was observed by AFM, and the stability of liposome was evaluated. Besides, selective antibacterial activity for S. aureus by utilizing α-hemolysin to trigger clove oil release from liposome was testified by the spread plate method and GC-MS, and the applications of free clove oil and clove oil encapsulated in liposome on soy products were compared by the spread plate method, sensory evaluation and instrument analysis. The main results were as follows:(1) 7 components were identified, representing 99.64% of the total amount of clove oil, and eugenol(83.73%) was the main composition. The results of MIC and MBC showed that clove oil and eugenol exhibited marked antibacterial activities against seven kinds of investigated bacteria, and the antibacterial activity of clove oil was a little stronger than that of eugenol.(2) Time-kill analysis indicated that low concentration of clove oil was effective against E. coli and S. aureus, achieving complete bacterial elimination at short incubation time. After 8 h treatment, almost 99.9999% reductions in population were achieved in E. coli and S. aureus respectively. And the mechanisms of the antibacterial action of clove oil against E. coli and S. aureus were concluded as that clove oil could disrupt the cell membrane integrity, increase the cell membrane permeability, leading to the leakage of intracellular constituents, such as the 260 nm absorbing materials, ATP, nucleic acid, DNA and protein.(3) The optimal particle size(149.2 nm), PDI(0.196), turbidity(762 NTU), pH(7.12), Zeta potential(-24.5 mV) and entrapment efficiency(20.41%) of liposome were obtained at the concentration of clove oil to 5 mg/m L. AFM image revealed that liposome was smooth vesicle-like structures and uniformly distribution, without obvious aggregation. The liposome stored 60 d had good stabilities, maintaining their original physical and chemical properties(4) Time-kill analysis showed that α-hemolysin secreted by S. aureus could trigger clove oil release from liposome, and then clove oil reacted with S. aureus, reducing the population of S. aureus. Meanwhile, GC-MS analysis also testified that α-hemolysin had the ability to release the essential oil, and the characteristic peak of eugenol was detected in the spectrogram.(5) Liposome-encapsulated clove oil had a long-term antibacterial activity. Almost 99.995% reduction in population was detected, after 5 d of liposome treatment, and liposome could maintain the quality of soy products. The results of sensory evaluation and instrument analysis showed that liposome could maintain the quality of dried tofu, and no significant changes in color, hardness, adhesiveness, resilience, cohesion, springiness, gumminess and chewiness were detected. |
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