Preparation Of Plant Essential Oil + Cyclodextrin Inclusion Complex And Its Application In Citrus Preservation

Green mildew is one of the main postharvest diseases of citrus caused by Penicillium digitatum.Chemical fungicides such as imazole and imidamide are commonly used to control postharvest green mildew in citrus.However,long-term use of chemical fungicides can easily lead to drug resistance of pathogenic bacteria and environmental and food safety problems^[1-3].Therefore,it is of great significance to develop green antiseptic fungicides to control citrus green mildew effectively.Plant essential oil has been proved to have strong bacteriostatic activity,and the inclusion compound formed after inclusion with cyclodextrin has good water solubility,and can effectively maintain the bacteriostatic property of essential oil.In this paper,38 kinds of cyclodextrin inclusion compounds of plant essential oils were prepared from 10typical components andα,β,γand HP-β-cyclodextrin by saturated aqueous solution method.Thymol/γ-cyclodextrin inclusion complex（γ-CDTL）,trans-2-hexenal/γ-cyclodextrin inclusion complex（γ-CDTH）and cinnamaldehyde/γ-cyclodextrin inclusion complex（γ-CDCL）with better comprehensive properties were screened out for subsequent experiments to explore the mechanism of action of the three inclusion complexes on Penicillium digitatum.The control effect on postharvest green mildew of citrus was also investigated.The main results are as follows:（1）Preparation and characterization of inclusion complexesWith 10 typical components of plant essential oils（trans-2-hexeneal,cinnamaldehyde,thyme,citronellal,trans-2-hepteneal,cuminaldehyde,eugenol,carvacrol,α-terpineol,β-linalool）and 4 kinds of cyclodextrins（α-cyclodextrin,β-cyclodextrin,γ-cyclodextrin,HP-β-cyclodextrin）as raw materials,prepared by saturated aqueous solution method 38 kinds of plant essential oil cyclodextrin inclusion complexes were obtained,and their inclusion rates,phase solubility experiments and in vitro antibacterial effects were compared.The results showed that the inclusion compounds prepared with thymol,trans-2-hexenal and cinnamaldehyde as guest molecules had good inhibitory effect on Penicillium digitatum,and the inclusion rate was generally high.The inclusion rate ofγ-CDTL prepared from thymol andγ-cyclodextrin was 86.10%,and the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MFC）were 1.00 mg/m L and 2.00mg/m L,respectively.The inclusion rate ofγ-CDTH prepared by trans-2-hexenal andγ-cyclodextrin was 75.36%,and 1.00 mg/m Lγ-CDTH could completely inhibit the growth of Penicillium digitatum;The inclusion rate of the refinedγ-CDCL was75.63%,and its MIC and MFC were 2.00 mg/m L and 4.00 mg/m L,respectively.Scanning electron microscopy（SEM）and nuclear magnetic resonance（NMR）analyses were performed on the above-mentioned inclusion complexes of thymols,trans-2-hexenals and cinnamaldehydes.SEM results showed that the surfaces of the inclusion complexes of thymol,trans-2-hexenal and cinnamaldehyde all became smooth and regular.α-CDTL is larger and thicker in shape,α-CDCL is narrower in block,α-CDTH is in flakes;β-CDCL is in slightly larger and thicker blocks,β-CDTL is in larger and thicker blocks,β-CDTH is a block with a smaller shape;γ-CDTL,γ-CDCL andγ-CDTH are all platelets with a smaller shape,of whichγ-CDTL has the smallest size;HP-β-CDTL is a triangular sheet with a smaller shape,HP-β-CDCL is an irregular shape,and HP-β-CDTH is a larger triangular block.NMR analysis shows that the methyl end of thymol enters the cyclodextrin cavity from the wide mouth end ofα-CD to formα-CDTL,while HP-β-CDTL andγ-CDTL are the phenolic hydroxyl groups of thymol entering HP-β-The narrow ends of CD andγ-CD form inclusion complexes.α-CDTH andγ-CDTH are formed by trans-2-hexenal aldehyde groups entering the narrow end ofα-CD and the wide end ofγ-CD,respectively,while HP-β-CD in HP-β-CDTH The narrow end of the cyclodextrin is completely embedded in the cyclodextrin.α-CDCL and HP-β-CDCL are formed by the benzene ring of cinnamaldehyde entering the cyclodextrin from the wide end ofα-CD and HP-β-CD,whileγ-CDCL is formed by the cinnamaldehyde benzene ring fromγ-CD narrow mouth end is embedded and formed.（2）Effects of threeγ-cyclodextrins inclusion complexes on storage and preservation of citrusThe effects ofγ-CDTL,γ-CDTH andγ-CDCL on the decay of citrus fruits inoculated with Penicillium digitatum were studied.The results showed thatγ-CDTL,γ-CDTH andγ-CDCL treatments could reduce the occurrence of green mildew in citrus fruits to different degrees.When the control group was completely rotten,the rot rate of citrus fruits treated by 8×MFCγ-CDTL was only 31.7%.In addition,8×MFCγ-CDTL and 8×MFCγ-CDTL had 58.3%and 61.7%fruit decay rates,respectively.4×MFCγ-CDTL,4×MFCγ-CDTH and 2×MFCγ-CDCL were selected to treat naturally occurring citrus fruits because the concentration was too high to be left on fruit surface.The results showed that after 30 days of storage,the fruit decay rates ofγ-CDTL,γ-CDTH andγ-CDCL treatments were 15.33%,13.33%and 6.67%,respectively,which were significantly lower than that of the control group（22.67%）（P<0.05）.The results of fruit quality determination showed thatγ-CDTL,γ-CDTH andγ-CDCL treatments had no adverse effects on the water loss rate,colour,p H and Vc content of citrus fruits.Among them,γ-CDCL treatment maintained the fruit hardness well,andγ-CDTL treatment could effectively maintain the titratable acid content of citrus fruits.（3）Mechanism ofγ-cyclodextrin inclusion Complex on Penicillium digitatumAfter treatment withγ-cyclodextrin inclusion complexes,the filaments of Penicillium digitatum were gradually distorted from smooth and round.When1/2MICγ-CDTL was treated for 30 min,the activity of extracellular alkaline phosphatase of Penicillium digitatum was significantly increased.Compared with the control group,the fluorescence intensity of the mycelium cell wall was significantly weaker,the integrity of the cell wall was destroyed,the chitinase activity increased,and the chitin content decreased.γ-CDTL may damage the mycelium by disrupting cell wall integrity by affecting chitin content.After treatment with 1/2MICγ-CDTH for 30 min,the content of total lipid and ergosterol in the mycelium decreased,and the integrity of the cell membrane of Penicillium digitatum.The structure inhibits the growth of Penicillium digitatum.When treated with 1/2MICγ-CDCL for 60 min,the total lipid content of Penicillium digitatum was significantly reduced,the integrity of the cell membrane was destroyed,and the mitochondrial membrane potential（MMP）of the mycelium was significantly reduced,suggesting thatγ-CDCL had a significant effect on the mitochondrial membrane potential（MMP）.The mechanism of action of Penicillium digitatum may be related to the disruption of mycelial membrane integrity and normal mitochondrial morphology and function.In conclusion,a variety of plant essential oil cyclodextrin inclusion compounds with better antibacterial effects were prepared and screened in this study,and the mechanism of action ofγ-cyclodextrin inclusion compounds on the growth of Penicillium digitatum was preliminarily revealed.Provide theoretical and technical basis for the study of cyclodextrin inclusion complexes of plant essential oils and the preservation of citrus.