Study On The Antifungal Activity Of Litsea Cubeba Essential Oil And Its Inclusion Complex With ?-cyclodextrin Against Geotrichum. Citri-aurantii Causing Sour Rot In Citrus Fruit

Citrus fruit has sweet and juicy tastes and high nutrient components,and it is very popular among consumers.Citrus production in China is abundant,however,it is highly susceptible to fungal diseases,leading to serious decay and huge economic losses during storage.Sour rot is a common disease infected by Geotrichum citri-aurantii of citrus fruit,which is wet rotten.Unlike the blue and green molds,G.citri-aurantii cannot be controlled efficiently by commercial fungicides(Imazalil,Fludioxonil and Thiabendazole).Moreover,these synthetic chemicals can induce fungal resistance,and cause environmental problems and increase human health risks.Therefore,there is an urgent need to find natural alternative preservatives to control sour rot in citrus.Litsea cubeba essential oil(LCEO)is extracted from the litsea cubeba fruit.It consists of numerous bioactive compounds with broad-spectrum antibacterial properties,it is eco-friendly,safe and non-toxic.In this paper,chemical compositions of LCEO were analyzed,the antibacterial activity of LCEO against G.citri-aurantii was studied,and the antifungal mechanism was also investigated.In addition,the inclusion complex of LCEO and?-cyclodextrin(?-CD)was prepared,The preparation process was optimized by response surface methodology(RSM).The main results are as follows:(1)The chemical components of LCEO were characterized by GC-MS,twenty kind of compounds were identified,and citral was the main compound of LCEO,which accounted for about 67.00%.The MIC and MBC of LCEO against G.citri-aurantii were 0.78 mg·L^-1and1.56 mg·L^-1,respectively.LCEO has obvious inhibitory effects on the mycelial growth and spores germination.At the concentration of 800 mg·L^-1,the inhibition rates of hypha growth and spores germination reached 74.34%and 71%,respectively.vivo test showed that LCEO concentrations of 1600,3200 and 6400 mg·L^-1could significantly reduce the incidence of sour rot disease in fruits(p<0.05),and reduce the lesion diameter,which suggest that LCEO has a certain control effect on the occurrence of sour rot on citrus fruit.(2)LCEO exhibited good antibacterial activity against G.citri-aurantii,the antibacterial mechanism was investigated.After G.citri-aurantii in PDB were treated by LCEO at concentrations of 0.5MIC,MIC and 2MIC,the extracellular conductivity increased.The value of OD₂₆₀also significantly increased after treated for 30min.These results indicated that the permeability of cell membrane was altered,and a large amount of intracellular substances such as nucleic acid,DNA and so on were leaked out.In the laser confocal experiment,red fluorescence was observed in the groups treated with the concentration of MIC and 2MIC,while nearly no red fluorescence was observed in the control group,indicating that the integrity of the cell membrane was destroyed after LCEO treatment,the cells were deactivated,and PI dye could flow into the intracellular environment,so the fluorescence signal was detected.Images of scanning electron microscopy(SEM)showed that the surface of G.citri-aurantii treated with LCEO was rough,swelled,wrinkled and distorted.The change of fungal hyphae treated with 2MIC concentration were more obvious,which collapsed and distorted seriously.The morphology of control group presented smooth and normal.(3)LCEO/?CD-IC was prepared using saturated aqueous solution method.The optimal prepared condition was obtained by response surface methodology,and the?-CD/LCEO ratio was 4.2,temperature was 44°C,and time was 2 h.The inclusion yield was 71.07%under such condition.EE%and LC%were 33.60±0.08%and 9.07%±0.02%,respectively.The LCEO/?CD-IC prepared at optimum condition was characterized by FTIR,XRD,particle size analysis,SEM observation and TGA.The results showed that the infrared absorption peaks of LCEO/?CD-IC had obvious changes,and the crystal structure also changed.The particle size was smaller and more uniformly distributed than?-CD and physical mixture,it was normal distribution,which is consistent with that observed under SEM.Furthermore,the thermal stability was obviously improved as LCEO went into the inner cavity of?-CD.LCEO/?CD-IC was initially applied to the preservation test of citrus,and the results showed that it had a good antibacterial effect on G.citri-aurantii,which manifested that LCEO/?CD-IC has great potential as a preservative agent for citrus.