Preparation of plant essential oils and cyclodextrin inclusion complexes and its inhibition effects on citrus green mold

Green mold caused by Penicillium digitatum is one of the most destructive postharvest diseases in citrus fruit. Chemical fungicides (such as prochloraz) can be expected to effectively control this disease. But the widespread use of chemicals can easily lead to drug resistance of the pathogen, environmental pollution and food safety risks. Previous studies have shown that the plant essential oils and their bioactive components can be served as a type of potential biofungicide, due to the effective reduction of the postharvest disease in citrus fruit without impairing the fruit quality. However, the scale application can be largely limited by the volatility and easily oxidized property. Fortunately, the inclusion of cyclodextrins can be expected to improve the effectiveness of plant essential oils. In this study, the saturated aqueous solution was used to prepare the inclusion complexes of trans-2-hexenal, an active component in plant essential oils, particularly with four kinds of cyclodextrins including α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD), and hydroxypropyl-β- cyclodextrin (HP-β-CD). An effort was also made to improve the antifungal efficiency of trans-2-hexenal. At the same time, the antifungal activity of these inclusion complexes against P. digitatum was analyzed by in vitro assay, in order to determine the structure and the inclusion mode of the inclusion complex with the highest efficiency. As a result, four inclusion compounds designated as α-CDTH, β-CDTH, γ-CDTH, and HP-β-CDTH, respectively, were prepared by the saturated aqueous solution. The morphological results showed that the α-CDTH, β-CDTH, and γ-CDTH powders were fine and dense, while the HP-β-CDTH powder was rough with distinct particles. The in vitro antifungal data demonstrated that these inclusion compounds were used to effectively inhibit the growth of P. digitatum mycelium in a concentration-dependent manner. The minimum antifungal concentration (MFC) of β-CDTH and γ-CDTH to the mycelial growth of P. digitatum was estimated to be both 1.00 mg/mL, while the MFCs of α-CDTH and HP-β-CDTH were 2.00 and 4.00 mg/mL, respectively. The entrapment efficiency indicated that the highest entrapment efficiency (75.36%) was found in the γ-CDTH, whereas the HP-β-CDTH was the lowest entrapment efficiency (38.63%). The AL type was used to describe the phase solubility curves of the four cyclodextrin inclusion compounds. The solubility was also ranked in the descending order of HP-β-CD, γ-CD, β-CD, α-CD. The best overall performance was found in the γ-CDTH considering the in vitro antifungal efficiency, entrapment efficiency, and solubility. Thus, the γ-CDTH was selected as the material for the follow-up experiments. In vivo assay showed that the γ-CDTH at different concentrations reduced the incidence of green mold in citrus fruit at varying degrees (P<0.05), with 8.00 g/L γ-CDTH as the most effective concentration. Once the control fruits were totally rotten after 6 d storage, the disease incidence in the samples of 8.00 g/L γ-CDTH treatment was only 58.3%, which was comparable to that of prochloraz (56.7%). In addition, the γ-CDTH treatment effectively maintained the citrus fruit firmness without any adverse effects on the weight loss rate, color, vitamin C, and total soluble solids contents of citrus fruit. The Scanning Electron Microscopy (SEM) observation revealed that the shapes and sizes of γ-CDTH were quite different from those of γ-CD and physical mixtures, the varying sizes and irregular crystals were found in γ-CD. In contrast, the physical mixtures presented a rough surface, whereas the γ-CDTH was a smooth surface with a flake-like morphology. The nuclear magnetic resonance (NMR) analysis demonstrated that the hydrogen bonding interactions between H-3? and H-5? of γ-CD and H-6 of trans-2-hexenal were attributed to the formation of γ-CDTH. In summary, four inclusion compounds of trans-2-hexenal with cyclodextrins were prepared to further verify the antifungal efficiency and structural characteristics of γ-CDTH. The findings can provide a direct basis for the γ-CDTH to control citrus postharvest diseases in the development of plant-derived natural preservatives.