| Citrus essential oil is a kind of secondary metabolite extracted from citrus peel,which has a broad spectrum of antibacterial effect.However,the nature of citrus essential oil is unstable,easy to volatilize,easy to oxidize,and insoluble in water,so it is not easy to be used in actual production and life.Preparation of citrus essential oil into nanoemulsions can not only overcome the shortcomings of essential oil,but also improve the antibacterial activity of essential oil,which has a certain practical significance.Staphylococcus aureus is a common food-borne bacteria,which can cause food spoilage and food poisoning,the preparation of citrus essential oil nanoemulsions can not only promote the rational utilization of citrus peel,but also inhibit Staphylococcus aureus in a stable and green way,which is expected to replace the traditional bacteriostatic agents,and has a broad application prospect.In this study,essential oil was extracted from 10 kinds of common citrus,and citrus essential oil with good antibacterial effect against Staphylococcus aureus was selected to prepare nanoemulsion.The stability and antibacterial activity of nanoemulsion were comprehensively evaluated.The main results were as follows.1.The the inhibition zone test showed that 10 kinds of citrus essential oils had different levels of bacteriostatic effect on Staphylococcus aureus.The lemon(15.80±0.30 mm),pomelo(14.66±0.89 mm)and sweet orange(14.65±0.70 mm)with better bacteriostatic effect were selected for subsequent experiments.Gas chromatography-mass spectrometry(GC-MS)showed that the main components of the three citrus essential oils were D-limonene,?-terpinene,?-myrcene and?-pinene,which generally had good antibacterial effects.The nanoemulsions preparation test showsed that when the surfactant-to-oil rate(SOR)was 9:1,8:2,6:4,5:5,the three citrus essential oils all had phase transition points,which could form a nanoemulsions system,according to the Tindall effect,the particle size distribution of the emulsions formed were between 1-100 nm,the Sudan III dyeing test could determine that the emulsions were the type of oil-in-water.2.The average particle size and polydispersity index(PDI)test results of nanoemulsion showed that When the SOR value was 8:2,the three citrus essential oil nanoemulsions had the largest oil carrying capacity,and the average particle size was 14.93±0.53 nm for lemon,15.29±0.57 nm for sweet orange,16.39±0.48 nm for honey pomelo,the PDI value was0.244±0.031 for lemon,0.314±0.028 for sweet orange,0.353±0.024 for honey pomelo,the particle size distribution was uniform and the stability was high of the nanoemulsions.Therefore,the formulations of the three nanoemulsions were uniformly set as essential oil6%,Tween 80 24%,and water 70%.The sustained release test of the nanoemulsions showed that the volatilization rate of the three citrus essential oil nanoemulsions was significantly lower than the other two control groups under a continuous high temperature(100?)environment,it showed that the nanoemulsions had a sustained-release effect.The nanoemulsions storage test showed that the three nanoemulsions kept the system stable during storage at various temperatures(4?,25?,40?)for 14 days,and the reduction of sample retention rate was lower than that of the control group under the same treatment,indicating that the prepared nanoemulsions had good thermal stability;there was no significant difference in the sample retention rate of the three nanoemulsions under light and dark conditions after continuous storage for 14 d,indicating that the prepared nanoemulsions had good light stability.4.The antibacterial activity test of the nanoemulsions showed that compared with pure essential oils,the inhibition zone diameter of the three citrus essential oil nanoemulsions was significantly increased,and the minimal inhibit concentration(MIC)and minimum bactericidal concentration(MBC)were all reduced,indicating that the preparation of nanoemulsions improved the antibacterial properties of the essential oils,among them,the antibacterial effect of the lemon essential oil nanoemulsions was the most obvious,the MIC and MBC values were 1.875 mg/m L,which were about 4 times that of pure essential oil.Scanning electron microscopy experiments observed that the Staphylococcus aureus treated with lemon essential oil nanoemulsions became distorted,deformed,and adhered,the surface of the bacteria became extremely rough,the cell membrane was severely damaged,which showed that the nanoemulsions had a greater degree of damage to the morphology and a stronger damaging effect on the cell membrane of Staphylococcus aureus than pure essential oils.Transmission electron microscopy experiments showed that the cells in the nanoemulsions treatment group had hollows and holes,the cell walls were segmented or completely disappeared,a large amount of cell fragments were produced,and the cytoplasm became uneven,indicating that the nanoemulsions might destroy the structure of the cell wall of Staphylococcus aureus,caused the dissolution and loss of the cell wall,thereby affecting the permeability of the cell membrane and causing the leakage of intracellular material.Cell membrane permeability test and intracellular substance release test showed that nanoemulsions might be closely combined with cell membrane,changing the cell membrane permeability,destroying the homeostasis of the cell environment,and causing a large amount of leakage of intracellular substances such as nucleic acid,protein and K~+,which made the bacteria die. |
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