中国农业科学 ›› 2020, Vol. 53 ›› Issue (16): 3356-3371.doi: 10.3864/j.issn.0578-1752.2020.16.013

• 食品科学与工程 • 上一篇    下一篇

基于线粒体能量代谢途径的金针菇采后纳米包装保鲜机制

马宁1(),王鹤潼1,方东路2,赵立艳2,杨文建1,裴斐1,胡秋辉1   

  1. 1南京财经大学食品科学与工程学院/江苏省现代粮食流通与安全协同创新中心/江苏省粮油品质控制及深加工技术重点实验室,南京 210023
    2南京农业大学食品科技学院,南京 210095
  • 收稿日期:2020-03-21 接受日期:2020-05-27 出版日期:2020-08-16 发布日期:2020-08-27
  • 作者简介:马宁,Tel:13951729396;E-mail:maning@nufe.edu.cn
  • 基金资助:
    国家自然科学基金(31801592);江苏省自然科学基金(BK20180526);江苏省研究生培养创新工程(KYCX19_1418)

Nano-Packaging Preservative Mechanism of Flammulina filiformis After Harvest Based on Mitochondrial Energy Status Pathways

MA Ning1(),WANG HeTong1,FANG DongLu2,ZHAO LiYan2,YANG WenJian1,PEI Fei1,HU QiuHui1   

  1. 1College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety/Key Laboratory of Grains and Oils Control and Processing of Jiangsu Province, Nanjing 210023
    2College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095
  • Received:2020-03-21 Accepted:2020-05-27 Online:2020-08-16 Published:2020-08-27

摘要:

【目的】从线粒体水平上揭示不同包装对金针菇能量代谢的影响,为进一步揭示纳米包装保鲜机制提供新思路。【方法】以金针菇为材料,对比不连续密度梯度离心法(DDGCM)、酵母线粒体提取试剂盒方法(YMEKM)和改进普利莱法(IPKM)3种提取方法对金针菇子实体与菌丝体中线粒体功能活性的影响,确定最佳提取方法。通过测定不同包装及不同贮藏期金针菇线粒体中ATP代谢系统物质含量(ATP、ADP、AMP及能荷)和线粒体主要复合体活性,揭示不同包装金针菇采后能量代谢规律。【结果】通过标志性污染物酶和线粒体呼吸速率指标测定,3种方法以IPKM提取的线粒体内乙醇脱氢酶活性及胞液内细胞色素C氧化酶活性最低,呼吸速率最高,证明IPKM所提的线粒体结构完整性最好;IPKM提取的线粒体超氧化物歧化酶活性最高,达到17.82 U·mg-1 pro,比DDGCM-1和YMEKM分别高7.31%和25.59%。结合透射电镜和健那绿染色结果可知,IPKM提取的线粒体结构相对完整,活性线粒体数量最多。在金针菇冷藏期间,纳米包装金针菇的ATP水平、能荷含量以及线粒体主要复合体活性都显著高于普通PE包装。纳米包装材料通过维持高水平的ATP含量,延缓能荷值的下降,促进了线粒体的氧化磷酸化过程。纳米包装组在第21天的ATP含量仍然比普通PE包装组高113.83 μg·g-1FW,从而避免金针菇线粒体复合体I、Ⅲ活性降低,减缓线粒体复合体Ⅳ的活性降低。普通PE包装金针菇的线粒体复合体Ⅳ活性在第9天达到第一个峰值,而纳米包装金针菇线粒体复合体IV活性在第15天达到第一个峰值(5.14 U·mg-1 pro),显著高于普通PE包装(1.12 U·mg-1 pro),相差达到78.23%。证明纳米包装能有效控制线粒体呼吸链中的电子传递不受阻碍,保证细胞能量供应,从而更好地保持金针菇的能量状态。【结论】改进普利莱法对线粒体损伤最小,并维持线粒体活性,适合金针菇采后线粒体能量代谢研究。同时,纳米包装通过维持较高水平的ATP含量与线粒体复合体I、Ⅲ活性并延缓能荷与线粒体复合体Ⅳ活性的下降,进而保持金针菇的能量状态并延缓衰老,延长贮藏期。

关键词: 金针菇, 线粒体, 改进普利莱法, 贮藏, 能量代谢

Abstract:

【Objective】 The purposes of this study were to illuminate the effects of different packaging on energy metabolism of Flammulina filiformis from mitochondrial level, providing a new idea for further revealing the preservation mechanism of nano-package. 【Method】 In this experiment, F. filiformis was used as the research object. By using three common methods, including discontinuous density gradient centrifugation method (DDGCM), yeast mitochondrial extraction kit method (YMEKM) and improved Pulilai kit method (IPKM), the optimal way to extract F. filiformis mitochondria was determined. The regularity of post-harvest energy metabolism in packaged F. filiformis was expounded via measuring the content of ATP metabolism system substances (ATP, ADP, AMP and energy charge) and mitochondrial complex activity. 【Result】 The alcohol dehydrogenase activity in mitochondria and the cytochrome C oxidase activity in cytosol were the lowest, and mitochondrial respiration rates were the highest in IPKM extracted samples compared to other two methods, demonstrating the IPKM obtained mitochondria had the best membrane integrity. The mitochondrial superoxide dismutase activity extracted by IPKM was the highest, reaching 17.82 U·mg-1 pro, which was 7.31% and 25.59% higher than that extracted by DDGCM-1 and YMEKM, respectively. Combined with the results of transmission electron microscopy and Janus green B staining test showed that the IPKM treatment had the largest number of active mitochondria, and their mitochondrial structure was relatively complete. During the cold storage period of F. filiformis, the sample in nano-packaging (NP) groups showed that not only the ATP and energy content but also the activity of the main mitochondrial complex was significantly higher than those of polyethylene (PE) packaged bags (control). NP materials promoted the process of oxidative phosphorylation of mitochondria by maintaining a high level of ATP content and delaying the decline of energy charge. At the 21st day, the ATP content of NP groups was still 113.83 μg·g-1FW, which was higher than that of control, thus avoiding the reduction of the activity of F. filiformis mitochondrial complex I and Ⅲ, and slowing down the activity of mitochondrial complex Ⅳ. The mitochondrial complex Ⅳ activity of F. filiformis in the PE packaging groups reached the first peak at the 9th day, while NP groups reached the first peak at the 15th day, and the activity of the mitochondrial complex IV (5.14 U·mg-1 pro) in the NP materials reached the first peak on the 15th day, which was significantly higher than that in the PE packaging groups (1.12 U·mg-1 pro), with a difference of 78.23%. It proved that NP could effectively control the electron transfer in the mitochondrial respiratory chain without hindrance, ensure the energy supply of cells, thus better maintain the energy state of F. filiformis . 【Conclusion】 Compared with other methods, the mitochondria proposed by IPKM was the best approach for leading to the minimal damage on mitochondria structure and bioactivity, which laid the foundation of mitochondrial energy metabolism research on F. filiformis in the future. Meanwhile, the energy state of F. filiformis was maintained and its shelf-life was prolonged by NP treatment via improving ATP content and mitochondrial complex I, Ⅲ activities, and delaying the decline of energy charge and mitochondrial complex Ⅳ activity.

Key words: Flammulina filiformis, mitochondria, improved Pulilai method, storage, energy metabolism