常温酶解法测定基于南极磷虾中虾青素酯的方法研究  

A method for the determination of astaxanthin ester in Antarctic krill(Euphausia superba)by enzymatic hydrolysis at room temperature

作　　者：高岩 邢丽红[1] 孙伟红[1] 孙晓杰[1] 祖露 李兆新[1] GAO Yan;XING Lihong;SUN Weihong;SUN Xiaojie;ZU Lu;LI Zhaoxin(Key Laboratory of Sustainable Development of Polar Fishery,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;Shanghai WEIPU Chemical Technology Service Co.,Ltd,Shanghai 200082,China;Shanghai Fisheries Research Institute,Shanghai 200433,China)

机构地区：[1]中国水产科学研究院黄海水产研究所,农业部极地渔业开发重点实验室,山东青岛266071 [2]上海微谱化工技术服务有限公司,上海200082 [3]上海市水产研究所,上海200433

出　　处：《水产学报》2022年第3期430-438,共9页Journal of Fisheries of China

基　　金：国家重点研发计划(2018YFC1406805);中国水产科学研究院基本科研业务费资助(2020TD71)。

摘　　要：由于虾青素酯具有多种存在形态,其准确定量还存在难点,将其有效转化成可定量的游离态是解决问题的关键。本实验将南极磷虾作为不同形态虾青素研究的代表性品种,采用柱层析法从南极磷虾中分别制备了虾青素的单酯和双酯,明确了其组成和含量,作为典型特征样品,进一步通过单因素和正交实验优化确立最佳酶解条件,并对方法的准确性和适用性进行了评价。结果显示,①从南极磷虾中制备并鉴定出8种虾青素单酯和13种双酯作为典型特征样品;②单酯在底物浓度为0.5μg/mL,反应体系酶浓度1.14 U/mL,反应温度25℃,反应时间75 min时,游离虾青素回收率达94.56%±1.24%;双酯在底物浓度为1.0μg/mL,反应体系酶浓度0.92 U/mL,反应温度25℃,反应时间75min时,游离虾青素回收率为98.28%±0.84%。③将常温酶解法应用于实际样品的测定中,南极磷虾油中虾青素的含量为(265.09±20.35)mg/kg,雨生红球藻中虾青素的含量为(21759.36±90.19)mg/kg。为了验证方法的准确度,分别采用标准SC/T 3053-2019和GB/T 31520—2015与酶解法进行比较,结果分别为(260.42±11.57)和(21752.54±100.00)mg/kg,偏差均小于10%;进一步通过在样品基质中添加全反式虾青素标准溶液的方式进行了验证,南极磷虾中游离虾青素回收率为95.24%,RSD为2.03%;雨生红球藻中游离虾青素回收率为98.56%,RSD为0.75%,说明常温酶解法的准确度和精密度可以满足虾青素酯的准确定量。研究表明,常温酶解法的反应条件温和,减少了温度引起的虾青素氧化和异构化,最大化的将酯态转化成游离态,适用于水产品中虾青素酯的准确测定。It’s difficult for accurate quantification of astaxanthin esters because of their multiple forms,and hydrolysis of astaxanthin esters to free astaxanthin is the key to solve the problem.In this work,Antarctic krill(Euphausia superba)was used as a representative specie for the study of astaxanthins.The mono-and di-esters of astaxanthin were prepared from Antarctic krill by column chromatography,and the composition and content of them were clarified using high-resolution mass spectrometry with ultraviolet detection.The astaxanthin monoesters and the astaxanthin diesters were then taken as the typical research samples,the enzymatic hydrolysis conditions of which were optimized through single factor and orthogonal experiments,and the accuracy and applicability of the method were evaluated.Results showed that:(1)8 astaxanthin monoesters and 13 astaxanthin diesters were prepared and identified as typical characteristic compounds from krill.(2)For the astaxanthin monoesters,as the substrate concentration was 0.5μg/mL,the enzyme concentration of the reaction system was 1.14 U/mL,the reaction temperature was 25℃,and the reaction time was 75 mins,the yield of free astaxanthin could reach(94.56±1.24)%;And for the astaxanthin diesters,as the substrate concentration was 1.0μg/mL,the enzyme concentration of the reaction system was 0.92 U/mL,the reaction temperature was 25℃,and the reaction time was 75 minutes,the free astaxanthin yield reached(98.28±0.84)%.(3)Enzymatic hydrolysis at room temperature was applied to the analysis of actual samples,the astaxanthin content was(265.09±20.35)mg/kg in Antarctic krill oil,and the content was(21759.36±90.19)mg/kg in Haematococcus pluvialis.In order to verify the accuracy,the enzymatic hydrolysis method was compared with the standard methods from SC/T 3053—2019 and GB/T 31520—2015.The determination results were 260.42±11.57 mg/kg and 21752.54±100.00 mg/kg respectively,and the deviations of the results were all less than 10%.Then the results were further verified by addi

关 键 词：南极磷虾 虾青素酯 常温酶解法 定量测定 

分 类 号：TS254[轻工技术与工程—水产品加工及贮藏工程]