Effect Of Zinc Oxide Nanocomplexes On Aspergillus Flavus In Stored Corn And Its Quality

Maize is one of the world’s major food crops and an important cash crop in China.Maize kernels are large and hygroscopic,and microorganisms multiply rapidly and in large numbers under the right temperature and humidity conditions,causing problems such as heat mould and excessive fungal toxins,which are a serious threat to maize storage safety.Aspergillus flavus is one of the main fungi that cause huge losses in maize after production and during storage,and its metabolism produces aflatoxin,which is also a major carcinogen.The detection and control of aflatoxins not only helps to mitigate the effects of fungal deterioration on grain quality,but also helps to control the metabolism of mycotoxins in grain and helps to ensure safe grain storage.Zinc oxide nanoparticles have been widely used because of their broader inhibition spectrum,longer inhibition period and stronger inhibition activity.In this study,a method based on SYBR Green I qPCR was firstly developed for the detection of Aspergillus flavus,and the sensitivity and specificity of the method were evaluated.Secondly,ZnO nanoparticles were prepared by hydrothermal method,and mesoporous silica was modified on their surface to prepare ZnO@Si O₂ which were applied to inhibit the growth of Aspergillus flavus in maize.Subsequently,the inhibitory effect of ZnO nanocomplexes on the growth of Aspergillus flavus was investigated in terms of several aspects,such as Aspergillus flavus spore germination,mycelial growth,fungal ultrastructure and changes in ROS levels.Finally,ZnO nanoparticles were added to maize storage process,and the inhibition effect of ZnO-NPs and ZnO@Si O₂ non bacteria and fungi in maize storage process and the effect of ZnO@Si O₂ on maize quality were tested,laying a theoretical foundation for their future application in maize storage process.The main research contents and results are as follows:（1）Construction of a real-time fluorescence PCR assay for Aspergillus flavus.A SYBR Green I-based Aspergillus flavus qPCR system was constructed based on the specific primers designed for the OMT-1 gene.The standard curve y=-3.5192x+55.905（R²=0.9973）and the amplification efficiency was 107.0%.The method was highly specific and sensitive,with a lower limit of detection of 0.015 ug/m L.Meanwhile,the copy number of OMT-1 gene correlated well with the total number of colonies obtained from the same set of samples（R²=0.97）.It indicated that the qPCR absolute quantification method developed in this paper could be used to monitor the expression of the aflatoxin gene in maize.（2）Preparation and characterization of ZnO nanocomplexes.ZnO nanoparticles were firstly prepared,and then mesoporous silica was modified on their surface to obtain ZnO@Si O₂.The prepared ZnO@Si O₂ showed a particle size of 47±3 nm under transmission electron microscopy（TEM）with good dispersion and an obvious bilayer structure,indicating that Si O₂ was well modified on the surface of the ZnO nanoparticles.The XRD and FT-IR spectra also showed distinctive peaks of Si O₂,indicating that Si O₂ was successfully encapsulated on the surface of ZnO nanoparticles while maintaining the crystal structure of ZnO;the nitrogen uptake/desorption curves indicated that Si O₂ was mesoporous with an average pore size of 7.94 nm.The inhibition effect of ZnO-NPs and ZnO@Si O₂ on the dominant bacteria（Fusarium oxysporum,Penicillium oryzae,Aspergillus oryzae,Blueform,Escherichia coli and Staphylococcus aureus）in maize was good and increased with the addition concentration.（3）Study on the effect of ZnO nanocomplexes on the growth of Aspergillus flavus in stored maize.The anti-Aspergillus flavus activity of the prepared ZnO nanocomplexes was further investigated using Aspergillus flavus,the dominant fungus in maize storage,as the model fungus.The results showed that the ZnO nanocomplex effectively inhibited the germination of Aspergillus flavus spores,and the majority of Aspergillus flavus conidia lost their germination ability under the effect of 1 mg/m L ZnO nanocomplex.The mycelium appeared broken and deformed;significantly reduced the content of Aspergillus flavus in maize storage process.The intracellular ROS levels of ZnO-NPs were significantly increased after treatment with ZnO nanocomplexes,indicating that the formation of ROS is the main mechanism for the antibacterial activity of ZnO-NPs,and the production of reactive oxygen species（ROS）such as H₂O₂ and O₂.^-can damage cellular components such as lipids,DNA and proteins,thus affecting the growth rate of Aspergillus flavus colonies,reducing mycelial biomass,inhibiting spore germination,destroying the normal morphology of Aspergillus flavus,and destroying superficial morphology.Mycelia normal morphology and ultrastructure,resulting in serious damage to cell morphology,leading to cell damage and death.（4）Effect of ZnO nanocomplex on the quality of maize during storage.600 mg/kg of ZnO nanocomplex was effective in reducing the electrical conductivity,malondialdehyde and H₂O₂content and O₂.^-production rate of the kernels during storage.It also had high SOD,POD and antioxidant capacity（FRAP and DPPH free radical scavenging capacity）.The addition of ZnO nanocomplex at 3000 mg/kg exacerbated the degree of membrane lipid peroxidation,reduced enzyme activity and antioxidant capacity of maize,thus causing some harm to the quality of maize and detrimental to maize storage.Therefore,choosing the right amount of ZnO nanocomplex can extend the storage time of maize and maintain the stability of grain storage.