植物精油与环糊精包合物的制备及对柑橘绿霉病的抑制作用

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

植物精油及其活性组分能降低柑橘采后病害且不影响果实品质,是一种潜在的生物杀菌剂,但存在易挥发和不稳定等问题,限制了其实际应用。将植物精油与环糊精包合能有效克服上述缺陷,提高植物精油的应用效果。为了提升植物精油组分反式-2-己烯醛对柑橘绿霉病菌的控制效果,该研究拟采用饱和水溶液法制备反式-2-己烯醛与α-环糊精(α-CD)、β-环糊精(β-CD)、γ-环糊精(γ-CD)和羟丙基-β-环糊精(HP-β-CD)包合物,并考察包合物对指状青霉的离体和活体控制效果,在此基础上解析效果较优包合物的结构特点和包合模式。结果显示,通过饱和水溶液法成功制备了反式-2-己烯醛与α-CD、β-CD、γ-CD和HP-β-CD的4种包合物(α-CDTH、β-CDTH、γ-CDTH和HP-β-CDTH)。外观形态结果显示,α-CDTH、β-CDTH和γ-CDTH粉末细腻绵密,HP-β-CDTH粉末粗糙,颗粒分明。4种环糊精包合物均能有效抑制P.digitatum菌丝体的生长且具有浓度依赖性,β-CDTH和γ-CDTH抑制P.digitatum菌丝体生长的最小杀菌浓度(Minimum Fugicide Concentration,MFC)为1.00 mg/mL。包合率结果显示,γ-CDTH包合率最高,为75.36%。相溶解度结果表明,4种环糊精包合物的溶解性大小依次为HP-β-CD、γ-CD、β-CD、α-CD。综合离体抑菌效果、包合率和溶解性可知,γ-CDTH综合表现最佳。活体接种试验结果表明,不同浓度γ-CDTH处理能不同程度降低柑橘果实绿霉病发病率(P<0.05),以8.00 mg/mLγ-CDTH处理效果最佳。此外,γ-CDTH处理能有效维持柑橘果实硬度,且对柑橘果实失重率、色泽、维生素C含量、可溶性固形物无不良影响。微观结构观察表明,γ-CDTH与γ-CD、γ-CD与反式-2-己烯醛物理混合物的形状和大小有明显差异,表现在γ-CD形态大小不一,大部分呈无规则晶体,而物理混合物表面粗糙,γ-CDTH表面光滑,有类似片状的形态;γ-CD的H-3′和H-5′与反式-2-己烯醛的H-6之间的氢键相互作用是γ-CDTH的形成基础。研究结果可为γ-CDTH用于降低柑橘采后病害提供直接的依据,也为植物源天然防腐剂开发提供理论基础。

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.