对虾酚氧化酶微量测定体系的建立与评价
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摘要
快速跟踪对虾保藏过程中的酚氧化酶活力变化,对于改善对虾感官品质和提高产业经济价值具有重要意义。基于酶促反应动力学原理,通过考察反应时间及底物浓度对酶活的影响,建立微量测定体系,并在测定准确度、速度和试剂消耗方面与常量法作对比。结果表明,当反应时间2 min、底物与酶液体积比3∶2时可建立微量测定体系。与常量法相比,微量法在检测酶活性方面无显著性差异,且检测速度是常量法的2倍,试剂消耗量仅为常量法的1/20。微量法具有准确性高,试剂用量少,重复性好,方法简单、快捷等优点。
It is the great significance for improving the sensory quality and economic value of shrimp industry by real-time and accurately monitoring the profile of phenoloxidase activity during storage. Based on the enzyme kinetics,the effects of time course and substrate concentration on the velocity were investigated and the microdetermination system was thus established. And its assessment in accuracy, velocity and reagent consuming was made by comparing with the macrodetermination system. The results showed that microdetermination can be reliably established when the reaction time and the volume rate of substrate to crude enzyme sample were 2 min and 3 ∶ 2, respectively. Compared with the macrodetermination system, this mcirosystem had no significant difference in determination of phenoloxidase activity, but exhibited double speed in velocity and amounted to the volume of 1/20 in reagent consuming. With the advantages of high accuracy, little reagent consuming, excellent repeatability and simplicity, it can be conclude that this microdetermination system will found wide application in phenoloxidase activity monitoring.
It is the great significance for improving the sensory quality and economic value of shrimp industry by real-time and accurately monitoring the profile of phenoloxidase activity during storage. Based on the enzyme kinetics,the effects of time course and substrate concentration on the velocity were investigated and the microdetermination system was thus established. And its assessment in accuracy, velocity and reagent consuming was made by comparing with the macrodetermination system. The results showed that microdetermination can be reliably established when the reaction time and the volume rate of substrate to crude enzyme sample were 2 min and 3 ∶ 2, respectively. Compared with the macrodetermination system, this mcirosystem had no significant difference in determination of phenoloxidase activity, but exhibited double speed in velocity and amounted to the volume of 1/20 in reagent consuming. With the advantages of high accuracy, little reagent consuming, excellent repeatability and simplicity, it can be conclude that this microdetermination system will found wide application in phenoloxidase activity monitoring.
引文
[1]Encarnacion AB,Fagutao F,Jintasataporn O,et al.Application of ergothioneine-rich extract from an edible mushroom Flammulina velutipes for melanosis prevention in shrimp,Penaeus monodon and Litopenaeus vannamei[J].Food Research International,2012,45:232-237.
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[3]Nirmal NP,Benjakul S.Effect of catechin and ferulic acid on melanosis and quality of Pacific white shrimp subjected to prior freeze-thawing during refrigerated storage[J].Food Control,2010,21:1263-1271.
[4]徐丽敏.南美白对虾与鹰爪虾的保鲜及防黑变初步研究[D].青岛:中国海洋大学,2008:30-38.
[5]樊廷俊,任秉新,樊现远.甲壳动物酚氧化酶分子结构与生物化学性质的研究进展[J].山东大学学报:理学版,2012,47(1):1-7.
[6]陈闽榕.对虾微冻保鲜技术及多酚氧化酶的生化特性研究[D].福州:福建农林科技大学,2009:31-32.
[7]纪淑娟,尹竞男,李家政.蜜柚多酚氧化酶的酶学特征与活性测定研究[J].生物技术,2010(17):171-174
[8]刘凯.南美白对虾血细胞中酚氧化酶原系统的激活[J].动物医学进展,2009,30(1):27-32.
[9]肖婷,刘守柱,薛超彬,等.紫外分光光度计法与酶标仪微量法测定酚氧化酶蛋白含量[J].昆虫知识,2008,45(2):306-309.
[10]王建国,陆宏达.酚氧化酶活力测定方法中关于测定时间的研究[J].上海海洋大学学报,2009,18(6):765-771.
[11]李忠光,龚明.植物多酚氧化酶活性测定方法的改进[J].云南师范大学学报,2005,25(1):44-49.
[12]尹丽红,王堔柱,钦俊德.棉铃虫血淋巴酚氧化酶活性的微量测定[J].昆虫知识,2001,38(2):119-122.
[13]熊建华,李世传,罗秋水,等.金银花叶多酚氧化酶的酶学特性[J].中国食品学报,2012,12(11):59-64.
[2]曹荣.对虾生物保鲜与其熟制品保藏技术的研究[D].青岛:中国海洋大学,2009:10-45.
[3]Nirmal NP,Benjakul S.Effect of catechin and ferulic acid on melanosis and quality of Pacific white shrimp subjected to prior freeze-thawing during refrigerated storage[J].Food Control,2010,21:1263-1271.
[4]徐丽敏.南美白对虾与鹰爪虾的保鲜及防黑变初步研究[D].青岛:中国海洋大学,2008:30-38.
[5]樊廷俊,任秉新,樊现远.甲壳动物酚氧化酶分子结构与生物化学性质的研究进展[J].山东大学学报:理学版,2012,47(1):1-7.
[6]陈闽榕.对虾微冻保鲜技术及多酚氧化酶的生化特性研究[D].福州:福建农林科技大学,2009:31-32.
[7]纪淑娟,尹竞男,李家政.蜜柚多酚氧化酶的酶学特征与活性测定研究[J].生物技术,2010(17):171-174
[8]刘凯.南美白对虾血细胞中酚氧化酶原系统的激活[J].动物医学进展,2009,30(1):27-32.
[9]肖婷,刘守柱,薛超彬,等.紫外分光光度计法与酶标仪微量法测定酚氧化酶蛋白含量[J].昆虫知识,2008,45(2):306-309.
[10]王建国,陆宏达.酚氧化酶活力测定方法中关于测定时间的研究[J].上海海洋大学学报,2009,18(6):765-771.
[11]李忠光,龚明.植物多酚氧化酶活性测定方法的改进[J].云南师范大学学报,2005,25(1):44-49.
[12]尹丽红,王堔柱,钦俊德.棉铃虫血淋巴酚氧化酶活性的微量测定[J].昆虫知识,2001,38(2):119-122.
[13]熊建华,李世传,罗秋水,等.金银花叶多酚氧化酶的酶学特性[J].中国食品学报,2012,12(11):59-64.