Study On The Antimicrobial Components, Mechanism Of Impatiens Balsamina L. Stems And Effects On Citrus Fruit Preservation

Citrus industry is the largest fruit industry in the world due to the cultivation area and production, and China is the major player in citrus market. Thereby the development of citrus production plays an important role in economy and social development in China. In recent years, extensive research has investigated the potential application of safe and eco-friendly technology in citrus preservation. But only a little achievements were gained for a series of problems on methods and ways. The overall objective of this study were to prepare botanical citrus preservatives with natural derived antimicrobial agents from plant origin, which may be a viable alternative to chemical and synthetic fungicides. Some conclusions were drawn as following:(1) Screening of some medicinal plants for antimicrobial activityNinety six medicinal plants with antiphlogistic and antimicrobial activities were screened for potential antifungal activity against P. italicum and P. digitatum, two main postharvest pathongens of citrus fruit. The acetone extracts of cinnamon, clove buds and I. balsamina L. showed the best antifungal activity against both moulds. The antifungal activity of extracts was determined as the diameter of inhibitory zones and minimum inhibitory concentration (MIC). The MICs of cinnamon, clove buds and I. balsamina L. extracts against P. italicum were3.125mg mL-1、6.25mg mL-1、12.5mg mL-1respectively, while they were3.125mg mL-1、6.25mg mL-1、25mg mL-1L against P. digitatum. The I. balsamina extract was selected for deep and comprehensive study concerning about inhibitory efficiency and economic costs.(2) Extraction, isolation and identification of antifugal components from stems of I. balsaminaThe regression model of inhibitory zone (Y) and ethanol concentration (X1), extracting temperature (X2), solvent times (X3) and extracting time (X4) was established by quadric regression orthogonal rotational experiments. The regression equation is as following:Y=35.24570+1.12726X1-0.87843X12-0.56129X22-0.85810X32-0.67513X42-1.22737X,X2. The optimum extracting parameters were obtained by using nonlinear optimization method. The best extracting process parameters were that the ethanol concentration was96.8%, solvent times were10, extraction temperature was50℃, extraction time was3h. The diameter of inhibitory zone against P. italicum was36.95mm when inhibited by the tea extracted with above mentioned process according to the regression equation. The verification test results showed that resulting model calculation agreed well with experimental value.By using bioassay-guided fractionation, an antifungal compound from I. balsamina L. was isolated and identified as2-methoxy-1,4-naphthoquinone by IR, UV, LC-MS and (13)C-and (1)H-NMR data. This is the first report demonstrating that2-methoxy-1,4-naphthoquinone (MeONQ) has intensive in vitro antifugal activity against P. italicum and P. digitatum.(3) Evaluation of antimicrobial spectrum and inhibitory stability of I. balsamina extract and MeONQExtract of I. balsamina and MeONQ showed same antimicrobial spectrum and inhibitory stability, that proved MeONQ was the major antimicrobial agent of I. balsamina extract. The antimicrobial activities of the extract and MeONQ on4moulds,2yeasts,2Gram-positive bacterias and2Gram-negative bacterias were observed. All tested strains, except E. coli, were highly sensitive to MeONQ. The extract and MeONQ showed good thermal stability and storability property, and inhibited the microorganism strongly under the pH range2-8. The UV exposure time and concentration of Fe3+impacted the inhibitory activities significantly.(4) Preliminary study on inhibitory mechanism of MeONQ against P. italicum1) Through the mycelia growth rate test, toxicity regression equations and EC50of MeONQ against P. italicum were got. The toxicity regression equation was Y=6.2316X+2.3121, and the EC50was2.6998μg mL-1. MeONQ could inhibit the mycelia growth and conidia yield significantly.2) Scanning electron and transmission electron micrographs revealed that MeONQ could inhibit the mycelia growth. After affected by MeONQ, the meristematic cells of the mycilia were atrophying and collapsed, mycelia cells appeared serious plasmolysis. The cells structure was disordered and the subarea disappeared with electron density increasing and a large amount of material extravasation, hence, resulting in cell death.3) MeONQ could significantly inhibit SDH, MDH, CHT and GUN activities in the hyphae of P. italicum.4) Increase of cell membrane electrolytes permeability, measured by change of electrical conductivity of P. italicum hyphae, was found upon treatment with MeONQ. However, the macromolecular substances such as protein and nucleic acid selectively transporting through the cell membrane was not affected by MeONQ.In summary, MeONQ impacts the P. italicum through multiple targets on it’s growth and development, such as cell wall, cell membrane and mitochondria, which results in it’s desintigration and death.(5) Effects of composit preservating film on citrus fruitTo develop new preservative technology of citrus fruit, the effects of composite edible coating (based on15g L-1CMC,10μg mL-1MeONQ,0.5g L-1citric acid,5g L-1sucrose esters,5g L-1glycerin,20g L-1calcium propionate) on postharvest fruit quality such as weight loss, total soluble solids content (SSC), titratable acidity (TA), ascorbic acid content (AsA) were evaluated during cold storage at4℃. The physiological indicators such as activities of superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), chitinase (CHT), β-1,3-glucanase (GUN) and phenylalanine ammonia-lyase (PAL) were also periodically assayed. The data suggested that the edible coating exhibited a beneficial impact on the overall quality of’97-2’Nanfeng mandarin and ’Newhall’navel orange by reducing the moisture loss and fruit spoilage, maintaining titratable acidity and ascorbic acid content by comparing with the uncoated fruits. Further, the coating could effectively enhance the ability to scavenge reactive oxygen species and induce fruit desease resitance. The commosite coating showed better influence on Nanfeng mandarin than navel orange.