Studies On Antifungal Activity, Mechanism Against Food Spoilage Fungi And Development Of A Natural Preservative Of Essential Oil From Dill (Anethum Graveolens L.)

Fungal contamination has caused serious damage to many aspects of human’s life. Mold species, especially the Aspergillus flavus, can not only cause direct infections and systematic diseases in humans, but also infect food, plant and herb resulting in the change of nutritional and medicinal ingredient and the lost of economic value due to deterioration, which leads to serious economic losses. With the development of science and technology and the improvement of people’s living standard, more and more attention of consumers has been paid on the concerns of health and food safety. The application of synthetic preservatives has led to a number of health problems because they are themselves carcinogenic, teratogenic, and highly toxic, the public demands more acceptable natural food preservatives that are safe, effective and economical. Essential oil is one of secondary metabolites in plants. It is reported that essential oil has antibacterial insecticide, anti-inflammatory, antiviral, antioxidant, antitumor, antipyretic and analgesic, expectorant cough and other biological activity. Moreover, the majority of the essential oils are classified as Generally Recognized As Safe (GRAS). Thus, essential oil is a potential source of natural food preservative. A. graveolens (dill), one of species of Umbelliferae, is a traditional Chinese herb. Dill seed essential oil have antioxidant, antimicrobial and other biological activity.In this thesis, we describes the studies on antifungal activity, mechanism against food spoilage fungi and development of a natural preservative of essential oil from dill. The main results are as following:1. The essential oil from the seed of dill was extracted by hydrodistillation and collected by Clevenger-type apparatus, with the average extraction rate of3.5%. GC-MS analyses of the dill oil led to the identification of23different components, representing99.4%of the total oil. The major components detected in oil were carvone (41.5%), limonene (32.6%), and apiol (16.8%). After oral administration of different dose of dill oil, the LD50is3861.5mg/kg with the95%confidence intervals of3555.1～4194.2mg/kg, counted by the method of Bliss.2. We studied in vitro and in vivo activity of essential oil from dill against food spoilage fungi. Dill oil showed pronounced antifungal efficacy against common pathogens in food contamination, including in Aspergillus flavus, Aspergillus oryzae, Aspergillus niger and Alternaria alternata. We evaluated its effects on mycelial growth and the biomass of mycelium, and minimum inhibitory concentration against all the tested fungi was2.0μL/mL. We also assessed its effects on spore germination and growth kinetics. A. niger using light and scanning electron microscopes revealed degenerative alterations in morphology after oil treatment, including distorted conidial heads, decreased hyphal diameters, shriveled hyphal aggregates, and swelling of the hyphal wall. Furthermore, the oil exhibited a noticeable inhibition on the synthesis of aflatoxin B1(AFB1) by A. flavus, completely restraining AFB1production at2.0μL/mL. The effect of dill oil on inhibition of decay development in artificially inoculated and naturally infected cherry tomatoes was tested in vivo, which showed dill oil can control food spoilage at120μL/mL and100μL/mL concentrations, respectively.3. The technology for preparation of solid sublimation agent was studied. The optimal conditions for preparation of solid sublimation agent were obtained as follows: the optimum HLB was ascertained to be8.2, adding15-20%the essential oil, adding emulsifier (mixture of Span80and Tween80at the ratio of16:9), phase inversion emulsification under60℃, then homogenizing with1%agar solution. According to classical constant temperature text, with a method of headspace gas chromatography, the decomposition rate constant of the essential oil of solid sublimation agent is K25=0.00044662, half-life of the product is1551.7h (about65days) and it can be storaged for236.0h (about10days) in normal temperature.4. The plasma membrane and mitochondria in A. flavus were taken as potential targets for antifungal mechanism of action. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The damage of plasma membrane led to the loss of cytoplasmic contents. We assayed the mitochondrial membrane potential (MMP) using rhodamine123, determined the pH value by acidification of external medium and detected the antivities of mitochondrial ATPase and dehydrogenases by spectrophotometry. Besides, the mitochondrial dysfunction-induced reactive oxygen species (ROS) production was determined by a fluorometric assay. The results showed that exposure to dill oil resulted in an elevation of MMP and inhibition of the mitochondrial dehydrogenases. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine (Cys) resulting in the inhibition of antifungal activity, which reveals that ROS is a key mediator of the antifungal action of dill oil.5. The ROS is an important regulator to A. flavus apoptosis. Thus, the further study was conducted to study whether dill oil induces apoptosis in A. flavus. The ultrastructure was observed by transmission electron microscopy, the nuclear fragmentation was investigated by DAPI staining, and the expore of phosphatidylserine at the outer layer of the plasma membrane was examined by the FITC-Annexin V staining. In addition, the activity of metacaspase was assayed by FITC-VAD-FMK staining, and the release of cytochrome C (Cyt C) from mitochondria was also measured by spectrophotometric method. Moreover, the efficacy of antioxidant Cys on dill oil-induced apoptosis in A. flavus was also studied. The results indicated that cells treated with dill oil exhibited typical markers of apoptosis, such as phosphatidylserine exposure, nuclear fragmentation, chromatin condensation. Additionly, dill oil induced the release of Cyt C and activated the metacaspase, which led to A. flavus apoptosis. The addition of Cys prevented dill oil-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, revealing the ROS was a key mediator of dill oil-induced apoptosis. To sum up, dill oil induced apoptosis in A. flavus through a metacaspase-dependent apoptotic pathway.