| Some losses of postharvest fruits are caused by molds,which are pathogenic and the source of mycotoxins,threatening human and animal health.The use of chemical fungicides is currently used as an effective control method,but it has disadvantages of environmental pollution and pesticide residues,requiring sustainable and environmentally friendly methods.Compared with chemical synthetic fungicides,natural antagonistic microorganisms for biological control have become a promising alternative.Bacillus can produce a variety of antifungal substances,and its metabolite antifungal protein is the main antifungal component,but the stability of antifungal protein is low.Sodium alginate and chitosan can be used as carriers for loading antifungal proteins due to their good antifungal properties and biocompatibility,which is conducive to improving the stability of antifungal proteins.In this study,we firstly screened the strain XZ38-3,which has antifungal effect on Aspergillus flavus,extracted the antifungal protein from the fermentation broth of XZ38-3,and performed mass spectrometry identification and analysis.Through encapsulation technology,sodium alginate and chitosan were used as wall materials to encapsulate antifungal protein to prepare microspheres.The preparation process,particle characteristics and stability of microspheres were studied.Finally,the control effect of antifungal protein microspheres on Aspergillus flavus infection in cherry tomatoes was determined.The main research results are as follows:（1）Screening of antagonistic bacteria and purification and identification of antifungal protein.In this study,a strain XZ38-3 with antagonistic effect on Aspergillus flavus was isolated and screened.This strain was identified as Bacillus velezensis XZ38-3 by morphological observation,physiological and biochemical identification,and molecular biological identification.The antifungal protein was extracted from the fermentation broth of Bacillus velezensis XZ38-3 by（NH4）2SO4fractional precipitation.When the saturation of（NH4）2SO4 was 80%,the inhibition rate of the antifungal protein against Aspergillus flavus was the highest at 58.95%.After purification and mass spectrometry identification analysis,the intensity based absolute quantification（iBAQ）of the chitin-binding protein in antifungal protein was 80.81%,and the relative molecular weight was 22.45 k Da,indicating that the main component of antifungal protein was chitin-binding protein,which was named CBP1.CBP1 has a strong inhibitory effect on a variety of pathogenic fungi,indicating that it has broad-spectrum antifungal activity.（2）Preparation process optimization and particle characterization of microspheres.The single-layer microspheres（CBP1-SA）and bilayer microspheres（CBP1-SA-CS）were prepared by using sodium alginate and chitosan as wall materials,and Ca Cl2 as cross-linking agents to encapsulate the antimicrobial protein.By orthogonal test,the preparation parameters of CBP1-SA were determined as follows:time 1 h,1.0%sodium alginate,2.0%Ca Cl2 and p H 6.5.Under these conditions,the encapsulation rate was 93.88±2.09%,the water content was 95.89±0.55%and the swelling rate was 126.05±1.81%.Chitosan was selected for secondary encapsulation of monolayer microspheres,and the results showed that when the chitosan concentration was 1%and p H=3,the maximum encapsulation rate of CBP1-SA-CS was 95.27±1.08%,the water content was 94.48±5.32%,and the swelling rate was 154.07±2.05%;when the mass ratio of CBP1:SA was 3:5 for encapsulation experiments,the encapsulation rates of CBP1-SA and CBP1-SA-CS were 94.58±1.01%and 93.10±2.24%.respectively,SEM observation showed wrinkles and depressions on the surface of CBP1-SA and CBP1-SA-CS;FTIR and XRD analysis showed that there was an interaction between CBP1,SA and CS,and CBP1 was encapsulated successfully.（3）Study on the stability of microspheres.The growth rate method was used to determine the temperature,p H and storage stability of CBP1,CBP1-SA and CBP1-SA-CS.The results showed that the temperature and p H stability of CBP1 were enhanced after CBP1was encapsulated by SA and CS.After being stored at 25°C,4°C and-20°C for 50 days,the antifungal effects of CBP1-SA and CBP1-SA-CS were higher than that of CBP1.（4）Antifungal effect of microspheres.After treatment with CBP1,CBP1-SA and CBP1-SA-CS solutions,the infection rate of cherry tomatoes was reduced by 70.00%,65.84%and 66.67%,respectively,compared with the control group with an infection rate of95.00%.It slowed that such method slowed down the decay of cherry tomato fruits caused by Aspergillus flavus and reduced the weight loss of these fruits.In summary,the antifungal protein encapsulated by sodium alginate and chitosan will be taken as an effective agent to prevent the cherry tomatoes rot caused by A.flavus. |
