Optimization Of Fermentation Conditions And The Application Formulation Study For Metschnikowia Citriensis

Postharvest decays resulting from infection by pathogens cause a significant reduction in citrus industry.Using synthetic chemicals to control these pathogens not only harm for human health and environmental protection,but also cause the emergence of fungicide-resistant strains.Antagonistic yeast,as an effective biological control agent for postharvest diseases,is a promising alternative to chemical fungicides.Metschnikowia citriensis,a new species of Metschnikowia spp,isolated from citrus leaves and identified by our laboratory,was efficient to control postharvest green mold and sour rot caused by Penicillium digitatum and Geotrichum citri-aurantii on citrus fruit.Due to the unstable control effect of biological control agents and unclear biological mechanism,the success rate of commercial application of antagonistic yeast was low.Thus,the development of antagonistic yeast biocontrol agents with long shelf life and stable biocontrol effect is crucial to realize the commercialization of M.citriensis.In this study,firstly,the fermentation conditions of M.citriensis were screened and optimized,and expanded culture was conducted in fermenter to explore the possibility of realizing large-scale culture of M.citriensis.Secondly,in order to achieve commercialization,preparation of live agents of antagonistic yeast was studied,liquid bioformulation and freeze-dried formulation were prepared respectively to explore the effects of different dosage forms on the storage stability and biocontrol effect of biocontrol agents.The main results are as follows:（1）The fermentation conditions of M.citriensis were studied.Cultivated in different media,and the results showed that it grew better in yeast extract peptone dextrose medium.The single factor screening test of medium composition was conducted on YPD based medium,and four components of medium were screened out,included sucrose,soybean pepone,magnesium sulfate and potassium dihydrogen phosphate.By optimizing an appropriate mixed formula through orthogonal experiment,the medium composition was constituted with sucrose 20.00 g L-1,soybean pepone 18.46 g L-1,magnesium sulfate 0.52 g L-1 and potassium dihydrogen phosphate 1.16 g L-1.The growth condition of M.citriensis selected as follows:culture temperature was 28℃,inoculation amount was 3%,uncontrolled pH.The results showed that M.citriensis grew well in 15 L fermentation tank.After 18 h of fermentation,the growth of entered a stable period,and the maximum of biomass reached 12.23 g L-1.The kinetic model of M.citriensis growth was established according to Logistic equation,and the values predicted by the models were in good agreement with experiment values.X=0.8561e^0.4473t/12.16+0.07e^0.4473t（2）A novel liquid biocontrol agent using M.citriensis as the main active ingredient was developed.Through single factor screening test and response surface optimization test of liquid protectant,the optimal protective formula was determined to be trehalose 19.74%（w/v）combined with sodium glutamate 1.05%（w/v）,Tween 80 4.24%（w/v）and proline 1.11%（w/v）.The predicted viability could reach up to 53.75%,the validation experiment showed that viability of yeast in liquid bioformulation was 54.96%±0.52%,compared with the control group,the viability of yeast increased about ten times after 7 days of storage at 25℃.The result of evaluating the storage stability of liquid bioformulation showed that as the temperature decreased,the decrease of yeast survival rate was slowed down.But,the storage performance of liquid bioformulation should be improved,for example,the survival of yeast was 47.67%± 1.64%stored at-20℃ for 90 days.Compared with fresh M.citriensis,there was no significant change in the control effect of yeast in liquid bioformulation on postharvest pathogens in vitro.Also,there was no significant differences on the growth dynamics between fresh M.citriensis and bioformulation in citrus fruit wounds.The fresh M.citriensis was effective in controlling green and blue mold,sour rot and anthracnose.The biocontrol efficacy of liquid bioformulation was generally similar to the efficacy of fresh M.citriensis.The optimal protective formula had no effect on controlling diseases in postharvest citrus fruit.（3）A novel freeze-dried preparation using M.citriensis as the main active ingredient was developed.Through single factor screening test and mixture experiment,the optimal formula was determined to be soap powder 5.69%（w/w）,NNO 8.74%（w/w）,and high concentration dust 85.57%（w/w）.The predicted viable yeast in the lyophilized preparation was 4.74×108 CFU g-1,the validation experiment showed that viable yeast in the lyophilized preparation was 4.72×108 CFU g-1,which was close to the predicted result.The accelerated storage test demonstrated that the storage performance of freeze-dried bioformulation was better.In addition,the freeze-dried preparation results exhibited that the viable yeast in the preparation is 4.6×108 CFU g-1 after storage at 4℃for one year.There were no significant differences in biocontrol effect between fresh M.citriensis and freeze-dried preparation in vitro or in vivo.In conclusion,this article studied systematically the process of commercialization for M.citriensis,which expanded fermentation scale,provided some theoretical and technical support to realize the expansion of production.In addition,the study on the preparation of harvested cells,two formulations were obtained,which improved the storage stability of biocontrol agents.These results provided some reference basis for the development of biocontrol agents of M.citriensis.