Screening And Study On Fermentation And Antagonistic Substances Of Antagonist Against Jujube Fruit's Postharvest Diseases

In this thesis, we set Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola as target, which are common pathogens in the research of'Dongzao'Jujube, screened antagonists from the soil of the forest and garden aimed at controlling the three pathogens. By means of measuring antagonistic ability of the different we successfully determined its antagonistic spectrum, and then screened antagonists of important value for future research and development. We identified taxonomic status of antagonist B26 which is antagonistically more effective and with wider antimicrobial spectrum; By conducting the orthogonal experiments and single factor experiments, the optimal conditions for the fermentation of B26 was studied; also, we further studied the antagonistic extract's stability under the treatment of temperature, pH, ultraviolet radiation, organic solvents, proteases and the results were as follows:Eleven strains of antagonists were isolated from the soil samples, which had better antagonistic effects on Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola. The duel cultural tests showed B26 had the strongest antagonism against the three selected fungal pathogens, the average radius of inhibition zone against Alternaria alternate was 15.8 mm and inhibition rate against Alternaria alternate was 78.8%.The duel culture tests also showed B26 antagonistic had effect on Fusarium decemcellulare Brick and Monilia fructicola, the average radius of inhibition zone were 14.3 mm and 12.8 mm, inhibition rates were 71.3% and 63.8%, so B26 is valuable for further study. Based on the observation of B26's morphological features on PDA and combined usage of 16s rDNA sequence analysis with physiological and biochemical experiments, we initially identified B26 as Bacillus subtilis.Through orthogonal experiments and single-factor experiments, the medium components and dosages were optimized and the optimum conditions of fermentation for producing antibacterial substances were selected. Microbial strains were inoculated into different fermentation mediums with different prescriptions after B26 was intermediate cultured in Seed culture medium, after that, the optimum medium components and dosages for producing antagonistic substances were obtained as follows: peptone 25 g, starch 40 g, sodium chloride 1.5 g, ammonium sulfate 0.8 g, potassium hydrogen phosphate 1.5 g. The optimum conditions of fermentation for producing antagonistic substances were:liquid seed age 24 h, inoculum sizes 5%, initial pH 7, aeration rates 20%, glycerol 0.5%, fermentation temperature 30℃, rotation speed 150 rpm, fermentation time 60 h.The average inhibition diameter of B26 fermented filtrates larger than other conditions against Fusarium decemcellulare Brick.The fermented filtrates of B26 from optimum culture conditions were centrifuged for 20 min at 8000rpm,4℃and then filtered through 0.45μm filter membrane before analysis. After that, Fermentation supernatant was precipitated with (NH4)2SO4 solution at 70% saturation degree. Aseptic crude extractions were dialyzed from precipitation solution, which still have antagonistic ability against Alternaria alternate, Fusarium decemcellulare Brick and Monilia fructicola. The antagonistic substance stability with different treatments of temperature, pH, ultraviolet radiation, organic solvents, proteases were obtained by further study as follows:Inhibition diameter was 78.2% control after crude antagonistic protein of B26 was treated by 100℃,20min,it means crude antagonistic protein can be resistant to high temperature, but lost the antagonistic activity completely after the treatment with 121℃,20min. Purification of antagonistic protein of B26 should be at neutral pH for antagonistic ability was the highest, regardless that the antagonistic protein have antagonistic activity at pH 3-pH 10, a wide range of pH. Crude antagonist protein solution was partially sensitive to Ultraviolet. Inhibition diameter was 78.1% of control after exposed for 80 min under UV light with distance 40 cm, Power 20 w. Organic solvents have little effect on crude antagonist protein, Inhibition diameters were 96.4%,85.7%,82.1%,81.5% respectively after treatment with Ether, chloroform, methanol, and acetone for 30 min.Crude antagonist protein was partially sensitive to the three protease, when treated with Proteinase K, trypsin,pepsin, inhibition diameter was 15.2 mm,16.2 mm,16.3 mm, which was76.7%,81.8%,83.3% control respectively.