Inhibition Activity And Potential Mechanism Of Limonin-loaded Nanoemulsion On Citrus Postharvest Pathogens

Limonoids are important bioactive substances in citrus seeds with various biological activities such as anticancer,antioxidant,hypocholesterolemic,antiviral,and antibacterial.Using citrus seeds,monomers of limonoids with high purity have been obtained.Nevertheless,data on systematic studies on limonoids in different citrus species are scarce,which is not conducive to the further utilization of limonoids.In addition,the poor water solubility and bioavailability of limonoids had limited their application in functional food,pharmaceutical,and insecticides.Nano-encapsulation is an effective approach to solve the above problems.A large number of studies have reported that nanotechnology will impart important properties such as slow-release and target-delivery to natural bioactive substances,so as to enhance their functional properties.The use of nanoemulsions as plant protection carriers or agrochemical nano-delivery systems is being extensively investigated in agricultural practices.The application can improve the solubility,bioavailability,stability and effectiveness of active ingredients during the application,thus achieving better control of pests and diseases or spoilage fungi.Postharvest decay of citrus caused by Penicillium spp.is a major problem to the citrus industry,seriously affecting the quality and economic value of citrus fruit.To investigate the application prospects of citrus limonin in citrus postharvest rot,this study firstly assessed the differences of limonin content in the seeds of 21 citrus accessions,and analyzed the correlation between the content of limonin,nomilin and obacunone,which can provide basic information for material selection and product development.Secondly,we isolated and purified limonin using an anti-solvent crystallization method,and designed a nano-delivery system for its encapsulation.In addition,we investigated the inhibition mechanism of the antifungal activity against Penicillium italicum and Penicillium digitatum.Furthermore,we make efforts to explore the antibacterial targets of the nanoemulsion and to provide a theoretical basis for further improvement of its inhibition activity.The main results are as follows.(1)Variation in limonoids content of different citrus seedsUsing UPLC-PDA,the main components of limonoids in the seeds of 21 citrus accessions were quantified,of which the highest content was 14.67 g/kg in‘Cupigoushigan',followed by 13.61 g/kg in‘Dahongpao',and the lowest content was0.14 g/kg in‘Chintan'.The highest content was 105 times the lowest.Among the Citrus reticulata accessions,except for‘Yanxiwanlu'and‘Nianju',all others contained limonoids higher than 8.50 g/kg.Among the Citrus grandis,the highest content of 8.04g/kg was found in‘Dianjiang Yu',which was significantly higher than that in‘Shatian Yu'and‘Liangpin Yu'.In other samples,the content of limonoids was higher in‘Jiwei'grapefruit and‘Jiangjin'sour orange,with 13.23 g/kg and 11.48 g/kg,respectively.While,‘Eureka'lemon,‘Zhishi',and‘Liuyang'kumquat seeds contained lower levels.In conclusion,limonoids in citrus seeds were related to the varieties and species.Among Citrus reticulata,the limonoids levels were relatively high and evenly distributed.‘Cupigoushigan'and‘Dahongpao'were potential resources with high limonoids content.The three major limonoid glycosides were present in most of the citrus samples,with limonin correlating well with nomilin and obacunone.And their contents declined in the order limonin>nomilin>obacunone.Furthermore,the crystallization efficiency of limonin could be improved using ultrasound-assisted antisolvent crystallization and we obtained 97.1%purity of limonin.(2)Limonin-loaded nanoemulsion and its antifungal activityStable limonin-eugenol nanoemulsions(EuLm)were obtained using eugenol as a carrier and paraffin oil as a ripening inhibitor.EuLm inhibited 100%of the spore germination of the pathogens within 24 h.After 7 days of incubation,EuLm inhibited59.2%and 60.3%of the mycelial growth of P.italicum and P.digitatum,respectively.In addition,EuLm significantly improved the resistance of citrus fruit to the fungus and reduced fruit morbidity and mildew in an artificial disease-induced citrus resistance test.Therefore,eugenol nanoemulsions loaded with limonin had good fungicidal activity against citrus Penicillium and can be used as an alternative fungicide for the control of citrus decay.(3)Antifungal mechanism of EuLm against P.italicum and P.digitatumThe mechanism of antimicrobial action of the nanoemulsions was initially analyzed based on the microbial physiological index,then GC-MS-EI metabolomics was used to analyze the influence of the emulsions on fungal metabolites.The results showed that after EuLm treatment:the structure of Penicillium mycelium was disrupted,and SEM showed wrinkles,shrinkage and a large number of fragments on the surface of treated mycelium.The mitochondrial membrane potential(MMP)of P.italicum and P.digitatum decreased by 43.0%and 48.3%,respectively.The integrity,permeability and fluidity of fungal cell membranes were disturbed,and PI staining showed that EuLm caused severe damage to the cell membranes,which in turn led to increased leakage of Ca²⁺,K⁺and Mg²⁺.EuLm also increased the extracellular conductivity,and reduced contents of intracellular proteins,lipids and sugars.In addition,the nanoemulsion interfered with ergosterol synthesis,which is closely related to the structural and functional properties of cell membranes.EuLm treatment decreased ergosterol by 62.3%and 53.2%in P.italicum and P.digitatum,respectively,and stimulated severe lipid peroxidation in fungal cell membranes.Also,the accumulation of malondialdehyde(MDA)increased gradually with exposure time.Metabolite thermograms showed that EuLm treatment caused a decrease in the abundance of 34 metabolites in P.italicum,such as L-glutamine,L-leucine,L-threonine,L-aspartate,serine,D-fructose,and sorbitol.In contrast,EuLm had decreased the expression of 40 metabolites in P.digitatum,including sorbitol,arabitol,proline,malate,and glucosamine.The analysis of the screened metabolites in combination with the KEGG metabolic database showed that EuLm treatment on the metabolic pathways of Penicillium focused on amino acid metabolism,energy metabolism,and carbohydrate metabolism,such as valine,leucine and isoleucine biosynthetic pathway,glycine,serine and threonine biosynthetic pathway,lysine biosynthetic pathway,citric acid cycle pathway,glycolytic pathway,and fatty acid synthesis pathway,etc.In summary,the potential antifungal mechanisms of EuLm are summarized in the following:interfering with the integrity,permeability and fluidity of cell membranes,causing a decrease in the content of cellular vital substances,and disordering the metabolic pathways of Penicillium spp.(4)Preservation of citrus fruit by EuLm combined with chitosan(CS)A composite coating solution(CSNE)was prepared by combining EuLm and natural film-forming agent chitosan.Then we investigate CSNE's efficacy on the control of postharvest rot of citrus and evaluate its effect on the quality index during storage.The results showed that CSNE with 2%chitosan significantly reduced citrus rotting rates,which were 26.7%,15.6%and 5.6%in the control,CS and CSNE groups after 27 days of storage,respectively.Compared with the control,CSNE could delay fruit senescence and maintain good quality indicators of citrus fruits.For instance,the nanoemulsion can reduce the weight loss rate,slow down the increase of soluble solids(TSS)and decrease of titratable acid(TS),maintain good fruit hardness and color,and reduce the respiration rate and ethylene release rate of fruits.Moreover,CSNE could protect the antioxidant components such as vitamin C,phenolic compounds and total flavonoids.The physical isolation of the coated film also reduced the dispersion of volatile components from the peel,which could maintain the resistance of the peel to pathogens.In addition,CSNE treatment had a significant effect on the activities of defense enzymes(PAL,CAT,SOD,and GR)and defense-related genes(PAL,chitinase,c APX,MDAR,MYC,and WARK6)in citrus fruits,the expression of which may be important for improving fruit storage tolerance.Taking the physiological and biochemical index and molecular studies into consideration,CSNE treatment is considered to have important effect in citrus postharvest application.