植物組織からの有機酸を定量化するためにキャピラリー電気泳動を使用した：テストケースを調べます<em>コーヒーノキアラビカ</em>種子
Summary
この記事では、無料の帯状キャピラリー電気泳動を用いて植物材料からの有機酸の検出および定量するための方法を提示しています。コーヒー種子中の有機酸濃度の二次発酵の効果を決定するこの方法の潜在的なアプリケーションの例が提供されます。
Abstract
カルボン酸は、一つ以上の末端カルボキシル（COOH）官能基を含む有機酸です。短鎖カルボン酸（SCCAs; 3〜6個の炭素を含有するカルボン酸）、そのようなリンゴ酸やクエン酸などは、それらが細胞呼吸で機能し、細胞の健康の指標として役立つことができる多くの生物学的システムの適切な機能に重要です。食品中に、有機酸の含有量は、酸味又は「酸」味をもたらす増加SCCAレベルと、味に大きな影響を与えることができます。このため、有機酸濃度の迅速な分析のための方法は、食品及び飲料産業に特に重要です。しかし、残念ながら、SCCAの定量化のために使用されるほとんどの方法は、高価で、クロマトグラフィーおよび/または質量分析解析に続いて、危険な試薬を用いたサンプルの誘導体化を必要とする時間のかかるプロトコルに依存しています。この方法は、組織の検出および定量化のための別の方法を詳述しますフリーゾーンキャピラリー電気泳動（CZE）を用いて植物材料と食品試料からANIC酸は、単に、キャピラリー電気泳動（CE）と呼ばれます。 CZEは、検出の下限（0.005ミリグラム/ ml）でSCCAsを測定するための費用対効果の高い方法を提供します。この記事では、植物試料からSCCAsの抽出と定量化について詳しく説明します。提供される方法は、コーヒー豆からSCCAsの測定に焦点を当てているが、提供される方法は、複数の植物をベースとする食品材料に適用することができます。
Introduction
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.
Protocol
Representative Results
Discussion
任意の分析技術と同様に、大幅に生成されたデータの品質と信頼性に影響を与えることができるいくつかの重要な要因があります。まず、凍結/融解サイクルの最小値と、効率的にサンプルを処理することが重要です。凍結融解の繰り返しは、処理や分析の前に、試料の化学組成を妥協することができます。第二に、一貫して均等にすべてのサンプルにこのプロトコルのステップを適用するこ…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the financial support of this project by The J.M. Smucker company.
Materials
| Ceramic Moarter and Pestle | Coorstek | 60310 | |
| Beckman Coulter P/ACE MDQ CE system | Beckman Coulter | Various | |
| Glass sample vials | Fisher Inc. | 033917D | |
| 1.5 ml microcentrifuge tubes | Fisher Inc. | 02-681-5 | |
| LC/MS grade water | Fisher Inc. | W6-1 | Milli-Q water (18.2 MΩ.cm) is also acceptable |
| 15 ml glass tube/ Teflon lined cap | Fisher Inc. | 14-93331A | |
| Parafilm M | Fisher Inc. | 13-374-12 | |
| CElixirOA detection Kit pH 5.4 | MicroSolv | 06100-5.4 | |
| BD Safety-Lok syringes | Fisher Inc. | 14-829-32 | |
| 17 mm Target Syringe filter, PTFE | Fisher Inc. | 3377154 | |
| 32 Karat, V. 8.0 control software | Beckman Coulter | 285512 | |
| capillary electrophoresis (CE) sample vials | Beckman Coulter | 144980 | |
| caps for CE vials | Beckman Coulter | 144648 | |
| Liquid Nitrogen | N/A | N/A | Liquid nitrogen is available from most facilities services |
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