باستخدام شعري الكهربائي لقياس الأحماض العضوية من الأنسجة النباتية: حالة اختبار فحص<em> كافيا ارابيكا</em> بذور
Summary
تقدم هذه المقالة طريقة للكشف وتقدير الأحماض العضوية من المواد النباتية باستخدام برنامج منطقتين الكهربائي الشعري. مثال على إمكانية تطبيق هذه الطريقة، وتحديد الآثار المترتبة على تخمير الثانوي على مستويات حمض عضوي في بذور القهوة، وتقدم.
Abstract
الأحماض الكربوكسيلية هي الأحماض العضوية التي تحتوي على واحد أو أكثر من محطة الكربوكسيل (COOH) مجموعات وظيفية. سلسلة قصيرة من الأحماض الكربوكسيلية (SCCAs، الأحماض الكربوكسيلية التي تحتوي على 3-6 الكربونات)، مثل مالات وسترات، حاسمة لحسن سير العمل في العديد من الأنظمة البيولوجية، حيث أنها تعمل في التنفس الخلوي، ويمكن أن تكون بمثابة مؤشرات الصحة الخلية. في الأطعمة، يمكن أن محتوى حمض عضوي يكون لها تأثير كبير على الذوق، مع زيادة مستويات سوروس مما أدى إلى الذوق "حامض" الحامض أو. وبسبب هذا، طرق تحليل السريع للمستويات حمض عضوي ذات أهمية خاصة بالنسبة لصناعات الأغذية والمشروبات. ولكن للأسف، فإن معظم الطرق المستخدمة لسوروس الكمي تعتمد على بروتوكولات تستغرق وقتا طويلا يتطلب اشتقاق من العينات مع الكواشف الخطرة، تليها الكروماتوغرافي مكلفة و / أو الطيفي الشامل يحلل. تفاصيل هذه الطريقة أسلوب بديل للكشف وتقدير من غزالهالأحماض القرآنية من المواد النباتية والعينات الغذائية باستخدام برنامج منطقتين الكهربائي الشعرية (تشيكيا)، وأحيانا مجرد يشار إلى الكهربائي الشعرية (CE). يوفر تشيكيا وسيلة فعالة من حيث التكلفة لقياس SCCAs مع الحد الأدنى للكشف عن (0.005 ملغ / مل). تفاصيل هذه المقالة استخراج وتقدير من SCCAs من العينات النباتية. بينما يركز طريقة المقدمة على قياس SCCAs من حبوب البن، يمكن تطبيق الطريقة التي قدمت إلى عدة مواد الغذائية ذات الأصل النباتي.
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
كما هو الحال مع أي تقنية تحليلية، وهناك العديد من العوامل الحاسمة التي يمكن أن تؤثر بشكل كبير على نوعية وموثوقية البيانات التي تم إنشاؤها. أولا، من المهم معالجة عينات بكفاءة، مع حد أدنى من دورات تجميد / الذوبان. تكرار التجميد والذوبان يمكن أن تعرض للخطر التركيب الكيم?…
Divulgazioni
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 |
Riferimenti
- Araújo, W. L., Nunes-Nesi, A., Nikoloski, Z., Sweetlove, L. J., Fernie, A. R. Metabolic Control and Regulation of the Tricarboxylic Acid Cycle in Photosynthetic and Heterotrophic Plant Tissues: TCA Control and Regulation in Plant Tissues. Plant Cell Environ. 35 (1), 1-21 (2012).
- Finkemeier, I., Konig, A. C., et al. Transcriptomic Analysis of the Role of Carboxylic Acids in Metabolite Signaling in Arabidopsis Leaves. Plant Physiol. 162 (1), 239-253 (2013).
- Doyle, M. P., Buchanan, R. . Food Microbiology: Fundamentals and Frontiers. , (2013).
- Tůma, P., Samcová, E., Štulìk, K. Determination of the Spectrum of Low Molecular Mass Organic Acids in Urine by Capillary Electrophoresis with Contactless Conductivity and Ultraviolet Photometric Detection-An Efficient Tool for Monitoring of Inborn Metabolic Disorders. Anal Chim Acta. 685 (1), 84-90 (2011).
- López-Bucio, J., Nieto-Jacobo, M. F., Ramı́rez-Rodrı́guez, V., Herrera-Estrella, L. Organic Acid Metabolism in Plants: From Adaptive Physiology to Transgenic Varieties for Cultivation in Extreme Soils. Plant Sci. 160 (1), 1-13 (2000).
- Cebolla-Cornejo, J., Valcárcel, M., Herrero-Martìnez, J. M., Rosellò, S., Nuez, F. High Efficiency Joint CZE Determination of Sugars and Acids in Vegetables and Fruits: CE and CEC. Electrophoresis. 33 (15), 2416-2423 (2012).
- Rosello, S., Galiana-Balaguer, L., Herrero-Martinez, J. M., Maquieira, A., Nuez, F. Simultaneous Quantification of the Main Organic Acids and Carbohydrates Involved in Tomato Flavour Using Capillary Zone Electrophoresis. J Sci Food Agr. 82 (10), 1101-1106 (2002).
- Wasielewska, M., Banel, A., Zygmunt, B. Capillary Electrophoresis in Determination of Low Molecular Mass Organic Acids. Int J Environ Sci Dev. 5 (4), 417-425 (2014).
- Galli, V., Garcìa, A., Saavedra, L., Barbas, C. Capillary Electrophoresis for Short-Chain Organic Acids and Inorganic Anions in Different Samples. Electrophoresis. 24 (1213), 1951-1981 (2003).
- Klampfl, C. W. Determination of Organic Acids by CE and CEC Methods. Electrophoresis. 28 (19), 3362-3378 (2007).
- Kenney, B. F. Determination of Organic Acids in Food Samples by Capillary Electrophoresis. J Chromatogr A. 546, 423-430 (1991).
- Galli, V., Barbas, C. Capillary Electrophoresis for the Analysis of Short-Chain Organic Acids in Coffee. J Chromatogr A. 1032 (1-2), 299-304 (2004).
- Schmitt-Kopplin, P. Capillary Electrophoresis: Methods and Protocols. Methods in Molecular Biology. , 384 (2008).
- Nollet, L. . Chromatographic analysis of the environment 3rd ed. , (2006).
- . . ElixerOA Organic Acids/Anions Operating and Instruction Manual, MicroSolv Technology Corperation. , (2001).
- Dahlen, J., Hagberg, J., Karlsson, S. Analysis of low molecular weight organic acids in water with capillary zone electrophoresis employing indirect photometric detection. Fresenius J. Anal. Chem. 366 (5), 488-493 (2000).
- Ibanez, A. B., Bauer, S. Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectroscopy. Biotech. For Biofuels. 7 (145), (2014).
