Peña-Farfal Carlos, Moreda-Piñeiro Antonio, Bermejo-Barrera Adela, Bermejo-Barrera Pilar, Pinochet-Cancino Hugo, de Gregori-Henríquez Ida
Faculty of Basic and Mathemathics Sciences, Institute of Chemistry, Pontificia Universidad Católica de Valparaiso, Avda. Brasil 2950, Valparaiso, Chile.
Talanta. 2004 Oct 20;64(3):671-81. doi: 10.1016/j.talanta.2004.03.042.
A systematic evaluation of different variables affecting the enzymatic hydrolysis of mussel soft tissue by five enzymes, three proteases (pepsin, pancreatin and trypsin), lipase and amylase, has been carried out for the determination of trace elements (As, Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn) by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Enzymatic hydrolysis methods offers advantages such as a less species alteration, safer laboratory conditions and a less contaminant wastes. The enzymatic hydrolysis was performed in an incubation camera Boxcult with orbital and horizontal shaker. Variables affecting the enzymatic hydrolysis process were simultaneously studied by applying a Plackett-Burman design (PBD). For a confidence interval of 95%, the significant factors for all enzymes and for most of the elements were the pH, the incubation temperature and the ionic strength. These significant factors were optimized later by using a central composite design (CCD), which gave optimum conditions at pH of 1, incubation temperature of 37 degrees C and ionic strength fixed by sodium chloride at 0.2M when using pepsin. For pancreatin, trypsin, lipase and amylase there were found two different optimum condition sets. The first one involves the use of a 0.5M phosphate buffer (ionic strength), at a pH of 6 and at an incubation temperature of 37 degrees C, which allows the quantitative extraction of Al, Cr, Mn, Pb and Zn. The second conditions set employees a 0.1M phosphate buffer (ionic strength), a pH of 9 and an incubation temperature at 37 degrees C, and it results adequate to extract As, Cd, Cu, Fe and Ni. Analytical performances, repeatability of the over-all procedure and accuracy, by analyzing DORM-1, DORM-2 and TORT-1 certified reference materials, were finally assessed for each enzyme. Good agreement with certified values has been assessed for most of the elements (As, Cd, Cr, Cu, Mn, Ni, Pb and Zn) when using trypsin, pepsin and/or pancreatin, except for Cd and Pb in DORM-1 and DORM-2 because of the certified contents in such certified reference materials are lower than the limit of detection (0.10 and 0.16mugg(-1) for Cd and Pb, respectively, for the use of trypsin).
通过电感耦合等离子体原子发射光谱法（ICP - AES）测定微量元素（砷、铝、镉、铬、铜、铁、锰、镍、铅和锌），对五种酶（三种蛋白酶：胃蛋白酶、胰酶和胰蛋白酶，脂肪酶和淀粉酶）作用下影响贻贝软组织酶解的不同变量进行了系统评估。酶解方法具有物种变化小、实验室条件更安全以及污染物废物更少等优点。酶解在配备轨道和水平振荡器的培养箱Boxcult中进行。通过应用Plackett - Burman设计（PBD）同时研究了影响酶解过程的变量。对于95%的置信区间，所有酶以及大多数元素的显著因素是pH值、孵育温度和离子强度。这些显著因素随后通过中心复合设计（CCD）进行了优化，使用胃蛋白酶时，在pH值为1、孵育温度为37℃且氯化钠固定离子强度为0.2M的条件下得到了最佳条件。对于胰酶、胰蛋白酶、脂肪酶和淀粉酶，发现了两组不同的最佳条件。第一组涉及使用0.5M磷酸盐缓冲液（离子强度），pH值为6，孵育温度为37℃，可实现铝、铬、锰、铅和锌的定量提取。第二组条件使用0.1M磷酸盐缓冲液（离子强度），pH值为9，孵育温度为37℃，适用于提取砷、镉、铜、铁和镍。最后通过分析DORM - 1、DORM - 2和TORT - 1认证参考物质评估了每种酶的分析性能、整个过程的重复性和准确性。使用胰蛋白酶、胃蛋白酶和/或胰酶时，大多数元素（砷、镉、铬、铜、锰、镍、铅和锌）与认证值具有良好的一致性，但DORM - 1和DORM - 2中的镉和铅除外，因为这些认证参考物质中的认证含量低于检测限（使用胰蛋白酶时，镉和铅的检测限分别为0.10和0.16μg g⁻¹）。
