| The essential oil from Amomum tsaoko is a yellow oily liquid with rich fragrancy,which possesses superior antioxidant,antibacterial,antitumour,antiviral,antiasthma,and antiasthmatic efferts.Most of the Amomum tsaoko essential oils can be used as food additives with the distinct smell and good antibacterial activity.However,as a member of the plant essential oil,Amomum tsaoko essential oil is also volatile and unstable.In order to overcome the shortage of Amomum tsaoko essential oil in application,ionic cross-linking with O/W method was used to prepare the Amomum tsaoko essential oil chitosan microparticles.Optimized the preparation process,characterized the microparticles and evaluated the antibacterial activity of Amomum tsaoko essential oil chitosan microparticles.Base on the good antibacterial activity Amomum tsaoko essential oil,the scanning electron microscopy,atomic force microscopy and flow cytometry were used to investigate the changes of E.coli before and after treatment with essential oils,the antibacterial activity antibacterial mechanisms of Amomum tsaoko essential oil towards E.coli were studied.The major results were as follows:1.The ionic cross-linking with O/W method was used to prepare the Amomum tsaoko essential oil chitosan microparticles.Taken particle size as index,the preparation process of chitosan microparticles were optimized with the concentration of the chitosan,the pH in solution,the dose of the Tween-80 and the ratio of chitosan and TPP.It was showed a linear relation was between the concentration of the chitosan and the particle size,and the optimal preparation was acquired.With the optimal preparation,the encapsulation efficiency of Amomum tsaoko essential oil chitosan microparticles was decreased with the increased weight ratio of chitosan to Amomum tsaoko essential oil.At last,the size and the uniformity,the microstructure and the morphology of Amomum tsaoko essential oil chitosan microparticles was characterized.The prepared chitosan microparticles were regular and uniform size,which were demonstrated the success in the preparation of chitosan microparticles as the result.2.The cumulative release of Amomum tsaoko essential oil chitosan microparticles was studied in vitro.With the increased weight ratio of chitosan to essential oil,the cumulative release of Amomum tsaoko essential oil chitosan microparticles was decreased.Compared to the Amomum tsaoko essential oil,the Amomum tsaoko essential oil chitosan microparticles were possessed of the sustained-release activities.Take three typical pathogenic bacteria for examples,the antibacterial activities of the Amomum tsaoko essential oil chitosan microparticles were study in vitro.It was turned out that Amomum tsaoko essential oil chitosan microparticles were more sensitive to bacteria than fungi.And Amomum tsaoko essential oilchitosan microparticles were less volatile,more stable and controlled release of the antibacterial activity.The analysis of the Amomum tsaoko essential oil in chitosan microparticles was study.The compositions of the Amomum tsaoko essential oil in chitosan microparticles were same with the initial Amomum tsaoko essential oil.Therefore,the ionic cross-linking with O/W method maintained the characters and the bioactivities of the Amomum tsaoko essential oil.3.The agar diffusion method,micro-broth dilution method and time-kill curves were used to investigated the antimicrobial activities of Amomum tsaoko essential oil against three pathogenic bacterium.The results indicated that Amomum tsaoko essential oil exhibited broadspectrum antimicrobial activity.And it was more sensitivity to bacteria.The inhibition diameters of E.coli and S.aureus were larger than C.albicans’.The MIC value of Amomum tsaoko essential oil to E.coli and S.aureus were both 2.5%.And the antibacterial activity of Amomum tsaoko essential oil against E.coli was the most rapid.Scanning electron microscopy,atomic force microscopy and flow cytometry technique were used to investigate the bacterial morphology,membrane permeability and the changes of components in the cell before and after treatment with essential oils.The morphological observation showed the damage degree of bacterial body became stronger,cell wall and membrane were damaged,the bacterial body collapsed and cytoplasm overflew,the dimension of bacteria reduced with the increasing concentration.Crystal violet study showed the membrane permeability of E.coli was increased with the increasing concentration.The depolarization of membrane potential of E.coil with the increasing concentration indicated that the destruction of the cell membrane.PI uptake assay showed the increased fluorescence caused by PI–DNA interaction indicated the damage of the cell membrane the damage of the cell membrane.At last,the leakage of DNA,RNA and protein led the death of the E.coli.In conclusion,the ionic cross-linking with O/W method was used to prepare the Amomum tsaoko essential oil chitosan microparticles.It was easy operation and possessed of the sustained-release activities.Base on the good activity of the Amomum tsaoko essential oil,the antibacterial mechanism of the Amomum tsaoko essential oil was studied in vitro.The changes in permeability and integrity of the disrupted cell membranes were detected.These findings provide the theoretical data for the application of Amomum tsaoko essential oil,and Amomum tsaoko essential oil or Amomum tsaoko essential oil chitosan microparticles can be a candidate for the new food preservatives. |
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