冷等离子体抑制鲜切苹果酶促褐变的分子酶学和miRNA调控机制研究

Cold plasma as a non-thermal sterilization technology has become a research hotspot in the field of food science. However, the studies on the physiological metabolism and quality safety control of fresh-cut fruits and vegetables are less, especially the inhibition mechanism of enzymatic browning is still obscure. In this project, the technologies and methods of biochemistry, molecular biology and transcriptomics will be used to study the effects of plasma activated water (PAW) treatment on phenoloxidase activity, polyphenol substrate synthesis and cell membrane permeability during the process of enzymatic browning of fresh-cut apple. And then the mechanism of PAW regulating enzymatic browning will be identified. In order to clarify the molecular enzymology mechanism of PAW inhibitiing enzymatic browning, the kinetic model of PAW inactivation phenoloxidase will be established, and the effect of PAW on gene expression and activity of enzymatic browning related enzyme will also be investigated. MiRNAs and their target genes, which have regulatory function for enzymatic browning related enzymes, will be identified and analyzed. The regulatory effect of PAW treatment on miRNAs will also be analyzed. The above researches will deeply reveal the molecular enzymology and miRNA regulation mechanism of PAW inhibiting enzymatic browning of fresh-cut apples. This study can provide a theoretical basis for the Postharvest biological research of horticultural products, and also can open up a new way for the quality and safety control of fresh-cut fruits and vegetables.

冷等离子体作为一种安全的非热杀菌保鲜技术在食品科学领域应用已成为研究热点，但在鲜切果蔬生理代谢调控和质量安全控制等方面的研究较少，尤其是对鲜切果蔬酶促褐变的抑制机制尚不清楚。本项目以鲜切苹果为研究对象，综合生物化学、分子生物学和转录组学等多学科方法，研究等离子活性水（PAW）处理对鲜切苹果酶促褐变发生过程中酚氧化酶活性、多酚类底物合成及细胞质膜透过性的影响，明确PAW调控酶促褐变的作用机制；建立PAW失活酚氧化酶的动力学模型，解明PAW对酶促褐变相关酶的基因表达及活性的影响，明确PAW抑制酶促褐变的分子酶学机制；分析鉴定出对酶促褐变相关酶具有调控功能的miRNAs及其靶基因，解析PAW对酶促褐变相关miRNAs的调控作用，系统揭示PAW抑制鲜切苹果酶促褐变的分子酶学及miRNA调控机制。本研究可为园艺产品采后生物学研究提供理论基础，还可为鲜切果蔬品质与安全控制开辟一个新途径。

酶促褐变是鲜切苹果加工与贮藏保鲜过程中面临的主要问题之一，有效控制鲜切苹果的褐变是当前研究的热点问题。等离子活性水（PAW）作为新型防腐保鲜技术可以有效抑制鲜切苹果的酶促褐变，但其调控机理尚不清楚。本项目从“PAW处理→调控酶、底物和膜→抑制褐变”的角度，通过动态监控鲜切苹果在贮藏期间酚氧化酶活性、底物含量、膜脂过氧化程度以及抗氧化活性变化，利用扫描电镜（SME）和透射电镜（TME）技术在亚细胞水平监测鲜切苹果细胞组织结构变化，并且利用miRNA-转录组-降解组联合测序分析技术，分析鉴定出对酶促褐变相关酶具有调控功能的miRNAs及其靶基因，深入分析H2S抑制鲜切苹果酶促褐变的miRNA调控机制。.结果显示PAW处理显著延缓了鲜切苹果在贮藏期间的褐变；抑制了多酚氧化酶（PPO）的活性，但在贮藏后期提高了过氧化物酶（POD）的活性；同时PAW减轻了总酚含量的降低和丙二醛（MDA）的积累；PAW处理虽然提高了其超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和POD等抗氧化酶的活性，但同时也提高了其超氧阴离子生成速率和H2O2的含量，从而对鲜切苹果的抗氧化能力无显著影响；亚细胞水平监测结果显示PAW处理能够维持鲜切苹果的表面细胞结构完整，防止其细胞结构松散和质壁分离，保持细胞壁和细胞膜完整连续；miRNA-转录组-降解组联合测序显示miR156靶向SPL、miR164靶向NAC、miR319靶向TCP4、GAMYB和acyl-CoA-binding protein 4、miR6478靶向patatin-like protein 2，可能在H2S通过调节ROS、苯丙酸和脂质代谢抑制鲜切苹果褐变中发挥重要作用。本研究为丰富鲜切果蔬生物学基础理论及开发鲜切果蔬贮藏保鲜技术提供理论和实验依据。