Name: using capillary electrophoresis to quantify organic acids from plant tissue: a test case examining coffea arabica seeds
Uploaded: 2016-11-12T14:00:00.000+00:00
Duration: 10 min 13 s
Description: Watch this Scientific Journal Video about Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds at JoVE.com

The authors would like to acknowledge the financial support of this project by The J.M. Smucker company.

1.样品制备<ol><li>组装为短链羧酸（SCCA）提取样品。在一个时间准备1.0克咖啡种子，以确保有足够的样本将保持处理之后。 <ol><li>如果样品的研磨前冷冻，保持整个处理冻结，以防止冻结/解冻损伤和氧化样组织。移除仅在需要用于研磨从冷冻室或零度存储的样本。 </li><li>在液氮冷冻的Flash新鲜的样本，以试样磨前夕。以通过将它们在研钵中预先填充有液氮减少样品处理，闪光灯冷冻样品。 </li><li>分析立即在液氮中并存储生成，或闪存冷冻在-20 C或-80ºC直至分析如下液体样品。在分析之前，从存储删除冷冻样本，并允许解冻。对液体样品进行到步骤（3.5）进行处理。 </li></ol></li><li> Appro公司磨损priate个人防护设备（包括安全眼镜，手套和白大褂）用液氮工作之前。 </li><li>使用陶瓷研钵和杵三重研磨组织样品到一个均匀细粉末（ 即，均匀的粒径）在液氮中。 注:实现有均匀的颗粒尺寸为最大化SCCA提取效率是至关重要的。 <ol><li>预冷却的研钵和与前加入样品液氮杵。保持填充有作为被加入样品液体氮的小体积的砂浆。 </li><li>用勺子，补充足够的液体氮砂浆完全淹没样品。 </li><li>加容易地样本，如叶或烘焙咖啡，以液氮，并使用圆磨运动粉碎。开始研磨样品时，液氮级别已经降低到它只是勉强覆盖采样点。 </li><li>加硬研磨样品，这样的š生咖啡种子，以液态氮，并允许他们在研磨之前冻结10-30秒（或直到液氮停止剧烈沸腾）。打破使用垂直破碎运动组织成更小的片段，并使用圆磨运动然后完整组织研磨。 </li><li>重复步骤1.3.2-1.3.4两次以上，使样品被研磨，共三次。通常情况下，研磨三个连续两轮将样本缩小与面粉般的一致性粉末。 </li><li>如果没有实现一个面粉状的稠度，重复步骤1.3.2-1.3.4直到样品被还原成均匀的小粒径的粉末（提取效率成反比粒径）。 </li></ol></li><li>转移粉的玻璃小瓶或1.5 ml离心管中。启动下游加工研磨后立即（推荐），或储存样品在-80ºC至样品准备提取。 <ol><li>如果样品必须在存储分析，分裂样品放入500毫克等分试样（或液体样品500μl的等分试样）和多管用于存储之间拆分。避免使样品反复冷冻/解冻循环，因为这些可能会改变样品的组成和负面地影响未来的采样测量。 </li></ol></li></ol> 2.有机酸标准溶液配制<ol><li>组装为感兴趣的SCCAs可信标准。这些将被用于在确定SCCA浓度来创建使用外部和内部标准溶液。对于咖啡样品包括柠檬酸，苹果酸，乙酸和乳酸为感兴趣和己二酸为内标酸。 <ol><li>确保确保所选择的内标不与样品中天然发现的，该标准没有共同洗脱与样品轮廓其他峰。 </li><li>运行的标准曲线为每个感兴趣的SCCA，并确定线性响应范围为每个SCCA（见第4节运行研究所ructions）。确保每个SCCA运行标准曲线样品中进行测量。 注意:标准曲线可以在缓冲液或待测定样品的背景中运行。在第二种情况下（"标准加入"），对于每一个标准曲线点的峰面积值将减去离开样品的背景来确定。 </li><li>预标签与SCCA酸名，浓度和测定的日期所需要的所有管和玻璃器皿。 </li></ol></li><li>在使用一个容量瓶，确保标准制备过程中精确度的已知浓度（10毫克/毫升）的每个标准制备储备溶液。 <ol><li>溶解在超纯水（18.2MΩ）固体SCCA标准（柠檬酸和苹果酸）以获得所需的储备溶液的浓度。 <ol><li>创建加入100毫克的标准为10毫升容量瓶中有10毫克/毫升储液。补液至10毫升线用超纯水到Dissolve酸。 </li><li>创建酸标准的较低浓度或轻轻加热烧瓶以驱动难以溶解SCCA标准，如己二酸，成溶液。 </li></ol></li><li>在超纯水稀释液相酸标准（醋酸和乳酸），以达到所需的浓度。 <ol><li>预填充容量瓶用5ml超纯水。添加足够的酸，以制备10mg / ml的溶液到烧瓶中（使用由供应商提供的酸密度计），然后加入足够的水使溶液的最终体积至10ml。 </li></ol></li></ol></li><li>传输各储备溶液到干净15毫升玻璃管，用聚四氟乙烯（PTFE）衬里帽，并用塑料石蜡膜密封。储备溶液可以在4ºC被储存在密封的管中1周。 <ol><li>确保SCCA标准没有在使用前溶液中沉淀出来，如果库存解决方案已经prepar之后被冷藏通货膨胀。驱动沉淀放回通过温和加热解决方案。 </li></ol></li><li>准备SCCA提取液。包括在浓度足以用于检测的样品中（0.05毫克/毫升）内标。准备足够的解决方案来提取所有样本。 <ol><li>通过稀释在超纯水中内标储备溶液到0.05毫克/毫升制备50毫升的SCCA的提取液。添加250微升内标储备溶液（10毫克/毫升），以49.75毫升水中。 </li><li>如果提取物需要稀释，调整在萃取溶液中的内标浓度，以使最终浓度将落在量化的限度内（给予可检测的，但不是过度饱和，峰值，参见下文5.2.2节）的稀释后CE系统（0.05毫克/毫升）。 </li><li>准备每个系列提取新鲜的提取液（ 即，每个实验系列）。 </li></ol></li><li>制备标准曲线样品对于感兴趣SCCAs（一系列适当稀释），使用至少5个点的最低限度。所使用的标准曲线的浓度将需要在一个给定的SCCA的线性响应范围内，并且跨越预期样品中的SCCA的浓度。 <ol><li>包括在标准曲线溶液选定内标，以允许样品中的内标定量。内标将被用于在峰值识别和计算提取效率（第6节），以帮助。 </li><li>稀释各SCCA上述确定的浓度（0.01，0.02，0.04，0.06，0.08毫克/毫升;见上文2.4.2）在新的离心管（每个浓度/标准曲线点），使用超纯水。制备1毫升溶液每个标准曲线的集中点。确保每个浓度点包含所有四个酸标准并在正确的浓度的内标。 </li><li>准备新的标准曲线的点为每套样品被毛细管电泳系统上运行。保持在4ºC标准曲线SCCA样品直至分析，这将发生SCCAs从靶组织（第3节）提取以下。 </li></ol></li></ol> 3.有机酸提取<ol><li>预标签样品管，准备至少一个管的每个样本被提取。 </li><li>删除要从存储中提取样本，将它们放在冰上，而称出材料的提取。 </li><li>称出100毫克的样品进行提取SCCA。 <ol><li>称量每个样品后，组织转移到一个干净的1.5 ml离心管。量出的样品作为接近目标质量尽可能减少可变性的量。 </li><li>跟踪测定各样品的质量的，作为检测SCCAs的量将使用样品质量被标准化（参见6.5.2，下文）。 </li></ol></li><li>权衡所有的样品后，加入1毫升提取液（在水:R +从步骤2.4内标混合物）向每个样品管中。保持的提取液，以组织块在所有提取相同的比率（在该壳体1毫升100±5毫克组织）。通过涡旋10秒拌匀。 </li><li>允许样品在室温下静置1小时。在这个时候，混合每根管子每15分钟如步骤3.4。 </li><li>提取1小时后，混合样品一个最终的时间和样品管转移到一个小离心。在4℃下离心样品10000×g离心10分钟以沉淀固体物质（细胞壁，颗粒物等 ）。 <ol><li>当处理液体样品，添加内部标准适当浓度，混合短暂离心。离心后，相同地对待液体样品从固体样品的提取物。 </li><li>检查样品的pH值，以确认它们与pH范围在检测试剂盒中的运行缓冲液相容（步骤4.6）或缓冲液系统被EMPloyed。作为样品的体积通常是相当小的，pH值可以使用pH试纸进行监测。 </li></ol></li><li>准备用注射器式盘式过滤器（3.8）来过滤样品。 注:确保每个过滤器正确连接到注射器转移上清之前，防止样品损失。制备装备有盘式过滤对于要分析的每个样品（包括标准曲线样品）一个针筒。 </li><li>样品的离心分离和制备的注射器过滤器后，从每个样品转移上清液至配备0.2微米针筒过滤器3毫升注射器。使用每个样品新的过滤器和注射器。筛选所有样品，包括标准曲线样品和缓冲控制，CE系统上运行之前。 </li><li>直接过滤样品到一个干净的离心管中。过滤后，关闭滤液含管和丢弃的注射器/过滤装置。 </li><li>立即将样品过滤入CE系统对SCCA检测;或在4℃存储样品过夜。如果样品将被保存在一夜之间，密封管道用塑料石蜡膜，防止气体交换和样品的蒸发。 <ol><li>如果样品必须比过滤后的第二天被保存更长的时间，在-20ºC液氮冷冻或冻结闪光提取。解冻后，但是，需要确保在必要的每个样本被检查的沉淀的形成和过滤器（如在步骤3.6）。 </li></ol></li></ol> 4.设置了SCCA检测运行<ol><li>请咨询编程 - 和控制软件的特定细节CE用户手册。 </li><li>准备SCCA检测毛细管电泳（CE）样品瓶。确保小瓶有适当的标签，清洁，无缺陷。 </li><li>洗净并在使用前干燥CE小瓶的瓶盖。 <ol><li>通过在超纯水中浸没他们并允许它们浸泡过夜洗帽。浸泡帽后，丢弃浸泡ING溶液并用超纯水冲洗帽两倍以上。 </li><li>传输冲洗帽带无绒内衬组织清洁干燥的表面，并让他们风干。确保帽使用前完全干透，避免压故障。 </li></ol></li><li>转移1ml样品到每个CE小瓶，小心翼翼地避免意外样品溅入瓶的颈部。转让完成后，将在每个小瓶CE帽。 <ol><li>使用超纯水直接100中的CE小瓶:如果需要稀释，稀释咖啡种子样品1:10或1。为稀释大于1:100，创建中间稀释，以避免移液误差。 </li></ol></li><li>通过从步骤2.5转印标准曲线溶液（1.0毫升）的CE小瓶准备为每个标准曲线点一个小瓶。转让后盖上各管。 </li><li>通过添加0.04重量克NaOH的含90毫升超纯水的烧杯中，并允许粒料吨准备0.1M的溶液氢氧化钠（NaOH）的Ø溶解。转移的0.1M NaOH溶液到100ml容量瓶中，使总体积至100ml。 </li><li>将1ml的0.1M NaOH溶液添加到一个干净的CE小瓶和帽。 </li><li>制备的CE缓冲器小瓶每批样品被CE系统上运行。分离试剂盒可或定制的缓冲区可以使用13。 <ol><li>制备1小瓶用1ml起始缓冲液溶液和帽。 </li><li>每运行缓冲液和帽1毫升准备三小瓶中。更换样品和标准曲线的点每批前的运行缓冲液，或35后的样品，以先到为准。 </li><li>填三个附加小瓶用1ml超纯水和帽每个小瓶。 </li><li>准备1空"浪费"帽小瓶。 </li></ol></li><li>制备步骤4.8中所述的小瓶后，装入含缓冲液和水的小瓶，以及废液瓶（从步骤4.8.2-5）到毛细管电泳SYS的缓冲盘透射电镜，按照制造商的说明15。仔细记下每小瓶自动进样器编程的位置。 </li><li>装载含有标准曲线点小瓶和含有样品的小瓶待测定到入口样品盘，如在制造商的说明。注意每个小瓶的自动进样器程序的位置（请参阅以下步骤4.11.1）。 </li><li>制备调理和样品分离的方法（在表1和2中 ，分别提供了用于这些程序），并写一个样本序列文件按照仪器操作的指令。使用表2中详述的分离方法:用清水冲洗剂（20磅）0.2分列;与加速器（20 psi）的0.5分钟冲洗，在20kV注入样品（0.5磅）到柱上为0.09分钟，单独的样品12分钟，用NaOH（20 psi）的0.5分钟冲洗，最后用水冲洗（ 20 psi）的0.5分钟。 <ol><li> USI纳克在CE控制软件，写样品运行序列（ 即，工作列表;列表文件详述在其中的样品进行分析的顺序，并且该方法也可以用来将每个样品中分离）使用"序列"电子表格界面。在电子表格上的每一行都会对应一个样品运行并产生一个数据文件。 <ol><li>确保写入顺序文件时，样品使用在采样托盘的正确位置标识。确保每个数据文件具有防止软件覆盖以前的文件的唯一名称。程序的CE毛细管空调运行如之前含有样品的分离方法的顺序运行的单独序列。 </li></ol></li><li>开始与标准曲线溶液运行采样序列，然后SCCA的样品，并与标准曲线溶液的第二次运行结束。这将允许发生期间样品分析任何信号损失的程度的计算。 </li> &lt;/ OL&gt; </li></ol><img alt="表格1" src="/files/ftp_upload/54611/54611table1.jpg" /> 表 1: 用于通过毛细制备用于短链羧酸的分离毛细管调理方法的程序电泳 。 <img alt="表2" src="/files/ftp_upload/54611/54611table2.jpg" /> 表 2:用于通过毛细管电泳一个以分析短链羧酸的分离方法程序。 5. SCCA检测运行执行和数据收集<ol><li>启动毛细管空调运行。期望以调节毛细管两次或三次前列准备样品分离。柱调理应当如表1中所述来进行。简言之，将冲洗柱用0.1M的NaOH（20 psi）的1分钟，用水（20磅），用于冲洗1分钟，用引发剂（20 psi）的0.5分钟，用加速器（20 psi）的0.5分钟，独立加速器漂洗在30千伏10分钟冲洗，用0.1M NaOH水溶液（20 psi）的0.5分钟冲洗柱，最后0.5分钟用清水冲洗干净（20磅）的列。 <ol><li>条件之前，每个样品序列运行毛细管。通过保持分离电压达到适当的调节是在整个运行恒定并观察在光电二极管阵列跟踪的平坦的基线。 </li></ol></li><li>调理后，在软件中打开"控制"菜单，然后选择（ 即点击）启动样本分离"的运行顺序。"监控光电二极管阵列（PDA）跟踪，以确保适当的分离。 <ol><li>观察第一跟踪，并确保它有一个平坦的基线和完全溶解（基线分辨率），个别酸峰。超载样本显示长高峰的尾巴，或拖尾，并且将需要稀释（ 图1）。</li></ol></li></ol><img alt="图1" src="/files/ftp_upload/54611/54611fig1.jpg" /> 图1:PDA的比较突出的痕迹重载样本作为分析物浓度的增加，个别高峰几何可能开始变得不对称。在（ 一 ）0.05毫克/毫升，乙酸呈现良好定义的，左右对称的峰。作为乙酸增加到浓度（B）的0.07毫克/毫升和（c）0.10毫克/毫升，峰值尾形式（箭头）。这拖尾是一个很好的迹象表明，样品超载。 <a href="http://ecsource.jove.com/files/ftp_upload/54611/54611fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="3"><li>在序列运行的结论，打开数据文件的一个在所述的CE分析软件的时间和整合的峰值。 <ol><li>整合样品峰。 <ol><li>打开第一个文件并设置自动集成步骤排除运行（其中的电压，并且因此柱的流动方向，如在表1中描述的反相）的前3分钟。在同一个菜单中，将峰的选择标准，以50个单位的最小峰宽和100单位（或制造商的默认设置）的峰面积，以提供选择性的适度高的水平，从背景噪声中的微量分离酸的峰。 </li></ol></li><li>手动检查峰边界和基线每个PDA跟踪，以确保正确的峰积分。不当集成峰（ 即，已被集成为通过自动化软件两个分开的峰稍微不对称SSCA峰值）将需要手动重新结合。 </li></ol></li><li>整合后，查看％的面积报告，然后突出显示并复制面积百分比报告，并将其粘贴到一个单独的电子表格。重复每个样本构建完整的运行峰值不到原始吨，表示单峰的每行（ 即 ，每个峰应该包含单个化合物如果分离工作良好）。 </li></ol> 6.数据分析<ol><li>使用在（5.4）构成的电子表格分析准备数据。通过标记的酸标准，为每个标准曲线的点，其中包括内标开始分析。 </li><li>通过将保留时间供该样品中的内标物的保留时间与样品中每个峰计算每个峰的保留指数。排序的数据通过所得保留指数设定为确定的每个样品中感兴趣的峰。 </li><li>识别的峰值为每个感兴趣的酸后，构造为使用外部标准曲线点每种酸的标准曲线。 <ol><li>通过确定峰面积的SCCA标准各浓度，并标绘浓度对XA产生每个SCCA标准标准（浓度）的曲线XIS与在y轴峰面积。绘制每个SCCA标准曲线的线性回归和定义的斜率公式（表达式y = mx + B）。 </li><li>确保线性回归的R 2的值是0.90或更高的quantifications在标准曲线的线性范围最准确地进行。 </li></ol></li><li>计算使用峰面积与标准曲线（在上面6.3.2计）的线性回归线的斜率方程样品中对于给定的SCCA的酸浓度。简要地说，划分观测峰面积用于通过回归线对于酸的标准曲线的斜率的给定酸。 <ol><li>使用内部标准（步骤6.5），在加工过程中改正步骤6.4样品损失计算的原始浓度值。 </li></ol></li><li>计算使用内标各样品的校正因子。 <ol><li>划分内标物的实际浓度（即 KNO由样品中的内标（的观测值在实验开始时加入）WN量即使用内标标准曲线的斜率方程计算出的量）。乘以该修正系数的样本中的每个SCCA的原始浓度值。 </li><li>除以用于萃取以校正在样品质量的任何变化的样品的质量校正SCCA浓度的内标。这种计算得到每个样品的质量（毫克SCCA每毫克在本实施例研磨咖啡），然后可将其转化为针对给定的研究适当的单位（毫克/毫克，毫克/克，克/克， 等分析物的量）。 </li></ol></li><li>计算SCCA浓度后（归一化至任一质量[固体或液体样品]或体积[液体样品]），使用这些值进行统计分析按实验设计和感兴趣的问题的需求。 </li></ol>

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The authors declare that they have no competing financial interests.

正如任何分析技术，存在可以显著影响生成的数据的质量和可靠性的几个关键因素。首先，为了有效地处理样品，用最少的冷冻/解冻循环是很重要的。反复冻融可以处理或分析之前破坏样品的化学组成。其次，它是至关重要的这个协议的步骤一致并均匀地施加到所有样品。从不一致的样品制备和处理产生的技术错误可以显著影响所产生的数据的质量，并导致在SCCA测量增加的"噪音"。例如，样品后...

Carboxylic acids are organic compounds containing one or more terminal carboxyl functional groups, each attached to an R-group containing one or more carbons (R-C[O]OH). Short chain, low molecular weight carboxylic acids (short chain carboxylic acids, SCCAs) containing between one and six carbons, are essential components of cellular respiration, and function in several biochemical pathways necessary for cell growth and development. SCCAs play critical roles in cellular metabolism1, cell signaling2, and organismal responses to the environment (such as antibiosis3). Because of this, SCCAs can serve as useful indicators of disruptions to cellular metabolism, plant stress responses4,5, and fruit quality6,7. To date, SCCAs have been quantified primarily through chromatographic techniques such as high performance liquid chromatography (HPLC) or gas chromatography-mass spectroscopy (GC-MS). While these methods, are capable of achieving very low limits of detection, they can be expensive, require the derivatization of target SCCAs using caustic and/or toxic reagents, and include lengthy separation runs on the GC or HPLC. Because of this, interest in the use of free zonal capillary electrophoresis (CZE), which does not require sample derivatization, to quantify organic acids has steadily increased8.
Free zonal capillary electrophoresis (CZE) is a chromatographic separation methodology that, due to its high number of theoretical plates, speed, and relative ease-of-use, is increasingly replacing both GC-MS and high-pressure liquid chromatography as an analytical method for the quantification (particularly for quality control purposes) of anions, cations, amino acids, carbohydrates, and short chain carboxylic acids (SCCAs)8,9,10. CZE-based separation of small molecules, including SCCAs, is based two primary principles: the electrophoretic movement of charged ions in an electrical field established across the buffer filling the capillary; and the electro-osmotic movement of the entire buffer system from one end of the capillary to the other, generally towards the negative electrode. In this system, small molecules move towards the negative electrode at varying speeds, with the speed of each molecule determined by the ratio of the net charge of the molecule to the molecular mass. As the movement of each individual molecule in this system is dependent on the charge state of the molecule and the overall rate of electro-osmotic flow (which is itself based on the ion content of the buffer used to fill the capillary), the buffer pH and ionic composition heavily impact the degree to which molecules can be efficiently separated using CZE. Because of this, SCCAs, with their relatively high charge-to-mass ratios, are ideal targets for CZE-based separation. Metabolites separated using CZE can be detected using a variety of methods, including UV absorbance, spectral absorbance (which is generally performed using a photo-diode array [PDA]), and/or mass spectroscopy (CE-MS or CE-MS/MS)8. The diversity of separation and detection methods provided by CZE makes it an extremely flexible and adaptable technique. Because of this, CZE has been increasingly applied as a standard method of analysis in the areas of food safety and quality11,12, pharmaceutical research13, and environmental monitoring13,14. 
Capillary electrophoresis has been used to detect and quantify short chain carboxylic acids for nearly two decades13. The resolving power (particularly for small, charged molecules), short run time, and low per sample cost of CZE analyses make CZE an ideal technique for the separation and quantification of SCCAs13. This method presents a protocol to utilize CZE to measure the concentration of organic acids from plant tissues. Example data was generated through the successful implementation of this protocol to measure the change in organic acid levels in coffee seeds following a secondary fermentation treatment. The protocol details the critical steps and common errors of CZE-based separation of SCCAs, and discusses the means by which this protocol can be successfully applied to quantify SCCAs in additional plant tissues.

<table><tbody><tr><td>Ceramic Moarter and Pestle</td><td>Coorstek</td><td>60310</td><td></td></tr><tr><td>Beckman Coulter P/ACE MDQ CE system</td><td>Beckman Coulter</td><td>Various</td><td></td></tr><tr><td>Glass sample vials</td><td>Fisher Inc.</td><td>033917D</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes&amp;nbsp;</td><td>Fisher Inc.</td><td>02-681-5</td><td></td></tr><tr><td>LC/MS grade water</td><td>Fisher Inc.</td><td>W6-1</td><td>Milli-Q water (18.2&amp;nbsp;M&amp;Omega;&amp;#183;cm) is also acceptable</td></tr><tr><td>15 ml glass tube/ Teflon lined cap&amp;nbsp;</td><td>Fisher Inc.</td><td>14-93331A</td><td></td></tr><tr><td>Parafilm M</td><td>Fisher Inc.</td><td>13-374-12</td><td></td></tr><tr><td>CElixirOA detection Kit pH 5.4&amp;nbsp;</td><td>MicroSolv</td><td>06100-5.4</td><td></td></tr><tr><td>BD Safety-Lok syringes</td><td>Fisher Inc.</td><td>14-829-32</td><td></td></tr><tr><td>17 mm Target Syringe filter, PTFE</td><td>Fisher Inc.</td><td>3377154</td><td></td></tr><tr><td>32 Karat, V. 8.0 control software</td><td>Beckman Coulter</td><td>285512</td><td></td></tr><tr><td>capillary electrophoresis (CE) sample vials&amp;nbsp;</td><td>Beckman Coulter</td><td>144980</td><td></td></tr><tr><td>caps for CE vials&amp;nbsp;</td><td>Beckman Coulter</td><td>144648</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>N/A</td><td>N/A</td><td>Liquid nitrogen is available from most facilities services</td></tr></tbody></table>

using capillary electrophoresis to quantify organic acids from plant tissue: a test case examining coffea arabica seeds

羧酸是含有一个或多个末端羧基（COOH）官能团的有机酸。短链羧酸（SCCAs;含有三至六个碳原子的羧酸），如苹果酸和柠檬酸，对许多生物系统，在那里它们在细胞呼吸功能，可以作为细胞的健康指标的适当运作的关键。在食品中，有机酸含量会对味道显著影响，导致酸或"酸"味增加SCCA水平。正因为如此，对于有机酸水平的快速分析方法特别适用于食品和饮料工业。然而不幸的是，用于SCCA量化大多数方法都依赖于需要具有危险的试剂样品的衍生费时协议，其次是昂贵的色谱和/或质谱分析。此方法详细介绍了组织的检测和定量替代方法从植物材料和使用自由纬向毛细管电泳（CZE）食物样本齐尼奇酸，有时简称为毛细管电泳（CE）。 CZE提供一种用于测量SCCAs与检测的下限（0.005毫克/毫升）的成本效益的方法。本文将详细介绍SCCAs从植物样品提取和量化。虽然所提供的方法主要针对从咖啡豆SCCAs的测定中，所提供的方法可应用于多个植物为基础的食品原料。

该协议已成功用于测量的绿色咖啡种子的SCCA含量种子处理的影响。在这个实验中，6个处理为: 明串珠菌属的饱和微生物悬浮液在其生长培养基pseudomesenteroides应变GCP674（1），在水中GCP674微生物的含水悬浮液（2），乙酸和乳酸（0.15和0.4的水溶液毫克分别/ ml）的（3），一个废M1生长培养基处理（4），卫生署2 O水（5），和未治疗的对照（没有加入到种子...

Watch this Scientific Journal Video about Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds at JoVE.com

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

本文介绍了检测和使用免费的纬向毛细管电泳从植物材料有机酸定量的方法。该方法的潜在应用中，确定在咖啡种子有机酸水平二次发酵的效果的一个例子中，提供了。

利用毛细管电泳从植物组织量化有机酸:一个测试用例检查<em&gt;小粒种咖啡</em&gt;种子

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

using-capillary-electrophoresis-to-quantify-organic-acids-from-plant

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

9.8K 次观看.  The Ohio State University, OARDC. 本文介绍了检测和使用免费的纬向毛细管电泳从植物材料有机酸定量的方法。该方法的潜在应用中，确定在咖啡种子有机酸水平二次发酵的效果的一个例子中，提供了。 

视频: Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

PTR-ToF-MS Coupled with an Automated Sampling System and Tailored Data Analysis for Food Studies: Bioprocess Monitoring, Screening and Nose-space Analysis

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that belongs to the Direct-Injection Mass Spectrometric (DIMS) technologies. These techniques allow the rapid determination of volatile organic compounds (VOCs), assuring high sensitivity and accuracy. In general, PTR-MS requires neither sample preparation nor sample destruction, allowing real time and non-invasive analysis of samples. PTR-MS are exploited in many fields, from environmental and atmospheric chemistry to medical and biological sciences. More recently, we developed a methodology based on coupling PTR-ToF-MS with an automated sampler and tailored data analysis tools, to increase the degree of automation and, consequently, to enhance the potential of the technique. This approach allowed us to monitor bioprocesses (e.g. enzymatic oxidation, alcoholic fermentation), to screen large sample sets (e.g. different origins, entire germoplasms) and to analyze several experimental modes (e.g. different concentrations of a given ingredient, different intensities of a specific technological parameter) in terms of VOC content. Here, we report the experimental protocols exemplifying different possible applications of our methodology: i.e. the detection of VOCs released during lactic acid fermentation of yogurt (on-line bioprocess monitoring), the monitoring of VOCs associated with different apple cultivars (large-scale screening), and the in vivo study of retronasal VOC release during coffee drinking (nosespace analysis).

Proton Transfer Reaction Time of Flight Mass Spectrometry allows high-sensitivity, rapid and non-invasive analysis of volatile organic compounds. To demonstrate its potential, we give three examples: lactic acid fermentation of yogurt (on-line bioprocess monitoring), different apple genotypes (large-scale screening), and retronasal space after drinking coffee (nosespace analysis).

PTR-ToF-MS结合自动采样系统和食品研究定制数据分析:生物过程监测，筛选和鼻空间分析

Proton Transfer Reaction (PTR), combined with a Time-of-Flight (ToF) Mass Spectrometer (MS) is an analytical approach based on chemical ionization that ...

科研

化学

Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a ...

生物化学

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

环境科学

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized.
Samples were collected to represent the entire plant, frozen at -80 &#176;C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

We present a fast and reliable method to quantify vitamin C in Lactuca spp. using UPLC-UV, potentially transferable to other plants. The key steps are the sample preparation and vitamin C extraction under stable conditions, the reduction of dehydroascorbic acid to ascorbic acid and the optimization of the chromatographic procedure.

改进的 UPLC-UV 方法，用于在莱图斯品种（Lactuca sativa L.）和作物野生亲亲（Lactuca spp.） 中对维生素 C 进行定量化

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

使用顶空固相微萃取与气相色谱-质谱联用分析黑醋栗果实中的挥发性化合物

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the bran of cereals or seed coat of legumes. They possess antioxidant properties that have generated much research interest in recent years, about their potential antioxidant protective health functions. This work presents a generalized method for the extraction of free soluble phenolic acids from whole grains and analysis of their antioxidant capacity. Five whole grain samples comprising two cereals (wheat and yellow corn) and three legumes (cowpea bean, kidney bean, and soybean), were used. The grains were milled into flour and their free soluble phenolic acids extracted using aqueous methanol. The compounds were then identified using a high-pressure liquid chromatograph (HPLC). The Folin-Ciocalteu method was used to determine their total phenolic content while their antioxidant capacities were determined using the DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The phenolic acids identified included vanillic, caffeic, p-coumaric and ferulic acids. Vanillic acid was identified only in cowpea while caffeic acid was identified only in kidney bean. p-Coumaric acid was identified in yellow corn, cowpea, and soybean, while ferulic acid was identified in all the samples. Ferulic acid was the predominant phenolic acid identified. The total concentration of phenolic acids in the samples decreased in the following order: soybean &gt; cowpea bean &gt; yellow corn = kidney bean &gt; wheat. The total antioxidant capacity (sum of values of DPPH, TEAC and ORAC assays) decreased as follows: soybean &gt; kidney bean &gt; yellow corn = cowpea bean &gt; wheat. This study concluded that HPLC analysis as well as DPPH, TEAC, and ORAC assays provide useful information about the phenolic acid composition and antioxidant properties of whole grains.

Phenolic acids are important phytochemicals that are present in whole grains. They possess bioactive properties such as antioxidant protective functions. This work aimed at reporting on a generalized method for the HPLC identification, total phenolic content estimation, and determination of the antioxidant capacity of phenolic acids in cereals and legumes.

一种测定谷物和豆类游离可溶性酚酸组成和抗氧化能力的广义方法

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the ...

野生花生物种对黄曲霉毒素积累的抗性评价

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance.
Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes&#160;(stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins.
We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

作者聚焦：定量分析花生种子中的黄曲霉毒素和植物抗毒素，以确定对曲霉菌的遗传抗性

We demonstrate a medium-throughput method for quantification of aflatoxins and stilbenoid phytoalexins in single peanut seeds using ultra performance liquid chromatography. This method was specifically developed for the analyses of wild Arachis species challenged by the aflatoxigenic Aspergillus species.

利用毛细管电泳从植物组织量化有机酸:一个测试用例检查

摘要

探索更多视频

此视频中的章节

PTR-ToF-MS结合自动采样系统和食品研究定制数据分析:生物过程监测，筛选和鼻空间分析

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

改进的 UPLC-UV 方法，用于在莱图斯品种（Lactuca sativa L.）和作物野生亲亲（Lactuca spp.）中对维生素 C 进行定量化

使用顶空固相微萃取与气相色谱-质谱联用分析黑醋栗果实中的挥发性化合物

一种测定谷物和豆类游离可溶性酚酸组成和抗氧化能力的广义方法

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

利用毛细管电泳从植物组织量化有机酸:一个测试用例检查

摘要

探索更多视频

此视频中的章节

PTR-ToF-MS结合自动采样系统和食品研究定制数据分析:生物过程监测，筛选和鼻空间分析

植物细胞悬浮液中咖啡因的提取、酶活性及咖啡因合成酶基因的表达

以玉米 (玉蜀黍) 为例量化植物可溶性蛋白和可消化碳水化合物含量

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

改进的 UPLC-UV 方法，用于在莱图斯品种（Lactuca sativa L.）和作物野生亲亲（Lactuca spp.） 中对维生素 C 进行定量化

使用顶空固相微萃取与气相色谱-质谱联用分析黑醋栗果实中的挥发性化合物

一种测定谷物和豆类游离可溶性酚酸组成和抗氧化能力的广义方法

基于 SPE-UPLC 的单花生 （Arachis spp.） 中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生 （Arachis spp.） 中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生 （Arachis spp.） 中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

改进的 UPLC-UV 方法，用于在莱图斯品种（Lactuca sativa L.）和作物野生亲亲（Lactuca spp.）中对维生素 C 进行定量化

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子