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

<ol>
	<li>Ara&#250;jo, W. L., Nunes-Nesi, A., Nikoloski, Z., Sweetlove, L. J., Fernie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Control+and+Regulation+of+the+Tricarboxylic+Acid+Cycle+in+Photosynthetic+and+Heterotrophic+Plant+Tissues:+TCA+Control+and+Regulation+in+Plant+Tissues.">Metabolic Control and Regulation of the Tricarboxylic Acid Cycle in Photosynthetic and Heterotrophic Plant Tissues: TCA Control and Regulation in Plant Tissues.</a> Plant Cell Environ. 35 (1), 1-21 (2012).</li><li>Finkemeier, I., Konig, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+Analysis+of+the+Role+of+Carboxylic+Acids+in+Metabolite+Signaling+in+Arabidopsis+Leaves.">Transcriptomic Analysis of the Role of Carboxylic Acids in Metabolite Signaling in Arabidopsis Leaves.</a> Plant Physiol. 162 (1), 239-253 (2013).</li><li>Doyle, M. P., Buchanan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Food Microbiology: Fundamentals and Frontiers. , (2013).</li><li>T&#367;ma, P., Samcov&#225;, E., &#352;tul&#236;k, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> Anal Chim Acta. 685 (1), 84-90 (2011).</li><li>L&#243;pez-Bucio, J., Nieto-Jacobo, M. F., Ram&#305;&#769;rez-Rodr&#305;&#769;guez, V., Herrera-Estrella, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+Acid+Metabolism+in+Plants:+From+Adaptive+Physiology+to+Transgenic+Varieties+for+Cultivation+in+Extreme+Soils.">Organic Acid Metabolism in Plants: From Adaptive Physiology to Transgenic Varieties for Cultivation in Extreme Soils.</a> Plant Sci. 160 (1), 1-13 (2000).</li><li>Cebolla-Cornejo, J., Valc&#225;rcel, M., Herrero-Mart&#236;nez, J. M., Rosell&#242;, S., Nuez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+Efficiency+Joint+CZE+Determination+of+Sugars+and+Acids+in+Vegetables+and+Fruits:+CE+and+CEC.">High Efficiency Joint CZE Determination of Sugars and Acids in Vegetables and Fruits: CE and CEC.</a> Electrophoresis. 33 (15), 2416-2423 (2012).</li><li>Rosello, S., Galiana-Balaguer, L., Herrero-Martinez, J. M., Maquieira, A., Nuez, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+the+Main+Organic+Acids+and+Carbohydrates+Involved+in+Tomato+Flavour+Using+Capillary+Zone+Electrophoresis.">Simultaneous Quantification of the Main Organic Acids and Carbohydrates Involved in Tomato Flavour Using Capillary Zone Electrophoresis.</a> J Sci Food Agr. 82 (10), 1101-1106 (2002).</li><li>Wasielewska, M., Banel, A., Zygmunt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+in+Determination+of+Low+Molecular+Mass+Organic+Acids.">Capillary Electrophoresis in Determination of Low Molecular Mass Organic Acids.</a> Int J Environ Sci Dev. 5 (4), 417-425 (2014).</li><li>Galli, V., Garc&#236;a, A., Saavedra, L., Barbas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+for+Short-Chain+Organic+Acids+and+Inorganic+Anions+in+Different+Samples.">Capillary Electrophoresis for Short-Chain Organic Acids and Inorganic Anions in Different Samples.</a> Electrophoresis. 24 (1213), 1951-1981 (2003).</li><li>Klampfl, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Organic+Acids+by+CE+and+CEC+Methods.">Determination of Organic Acids by CE and CEC Methods.</a> Electrophoresis. 28 (19), 3362-3378 (2007).</li><li>Kenney, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Organic+Acids+in+Food+Samples+by+Capillary+Electrophoresis.">Determination of Organic Acids in Food Samples by Capillary Electrophoresis.</a> J Chromatogr A. 546, 423-430 (1991).</li><li>Galli, V., Barbas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis+for+the+Analysis+of+Short-Chain+Organic+Acids+in+Coffee.">Capillary Electrophoresis for the Analysis of Short-Chain Organic Acids in Coffee.</a> J Chromatogr A. 1032 (1-2), 299-304 (2004).</li><li>Schmitt-Kopplin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+Electrophoresis:+Methods+and+Protocols.">Capillary Electrophoresis: Methods and Protocols.</a> Methods in Molecular Biology. , 384 (2008).</li><li>Nollet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatographic analysis of the environment 3rd ed. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ElixerOA Organic Acids/Anions Operating and Instruction Manual, MicroSolv Technology Corperation. , (2001).</li><li>Dahlen, J., Hagberg, J., Karlsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+low+molecular+weight+organic+acids+in+water+with+capillary+zone+electrophoresis+employing+indirect+photometric+detection.">Analysis of low molecular weight organic acids in water with capillary zone electrophoresis employing indirect photometric detection.</a> Fresenius J. Anal. Chem. 366 (5), 488-493 (2000).</li><li>Ibanez, A. B., Bauer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+method+for+the+determination+of+organic+acids+in+dilute+acid+pretreated+biomass+hydrolysate+by+liquid+chromatography-time-of-flight+mass+spectroscopy.">Analytical method for the determination of organic acids in dilute acid pretreated biomass hydrolysate by liquid chromatography-time-of-flight mass spectroscopy.</a> Biotech. For Biofuels. 7 (145), (2014).</li></ol>

The authors declare that they have no competing financial interests.

כמו בכל שיטה אנליטית, ישנם מספר גורמים קריטיים שיכולים להשפיע על איכות ואמינות משמעותית של נתונים שנוצר. ראשית, חשוב לעבד דגימות ביעילות, עם מינימום של מחזורי הקפאה / פשרה. חוזרות הקפאה להפשרה יכולים לסכן את ההרכב הכימי של המדגם לפני עיבוד או ניתוח. שנית, חשוב ליישם את ...

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

חומצות קרבוקסיליות הן חומצות אורגניות המכילות carboxyl מסוף אחד או יותר (COOH) קבוצות פונקציונליות. קצר שרשרת חומצות קרבוקסיליות (SCCAs; חומצות קרבוקסיליות המכילות שלושה עד שישה פחמנים), כגון malate ציטראט, הן קריטיות לתפקוד התקין של מערכות ביולוגיות רבות, שבו הם מתפקדים נשימה תאית והוא יכול לשמש אינדיקטורים של בריאות תא. במזונות, תכולת חומצות אורגניות יכולה להיות השפעה משמעותית על טעם, עם רמות SCCA מוגברת וכתוצאה מכך טעם חמוץ או &quot;חומצה&quot;. בגלל זה, שיטות לניתוח מהיר של רמות חומצה אורגנית הם בעלי עניין מיוחד לתעשיות המזון והמשקאות. למרבה הצער, עם זאת, רוב שיטות כימות SCCA תלויות פרוטוקולי זמן רבים המחייבים את derivatization של דגימות עם ריאגנטים מסוכנים, ואחריו chromatographic היקר ו / או ספקטרומטריה מנתח. שיטה זו מפרט שיטה חלופית עבור איתור וכימות של orgחומצות anic מחומר צמח דגימות מזון באמצעות אלקטרופורזה נימי אזורים חינם (CZE), לפעמים פשוט המכונות אלקטרופורזה נימים (CE). CZE מספק שיטה חסכונית למדידת SCCAs עם הגבלה נמוכה של גילוי (0.005 מ&quot;ג / מ&quot;ל). מאמר זה מפרט את המיצוי וכימות SCCAs ממדגמים צמח. בעוד השיטה ספקה מתמקדת מדידת SCCAs מפולי קפה, השיטה הניתנת ניתן ליישם חומרי מזון על בסיס צמחי מרובים.

פרוטוקול זה נוצל בהצלחה למדוד את ההשפעות של טיפולים זרע על תוכן SCCA של זרעי קפה ירוקים. בניסוי זה, ששת הטיפולים היו: השעית חיידקים רוויה של Leuconostoc pseudomesenteroides GCP674 זן במדיום הגידול שלה (1), השעיה מימית של GCP674 חיידקים במים (2), בתמיסה מימית של אצטית וחומ?...

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

מאמר זה מציג שיטה איתור וכימות של חומצות אורגניות מחומר הצמח באמצעות אלקטרופורזה נימי אזורי חינם. דוגמא של יישום הפוטנציאל של שיטה זו, הקובע את ההשפעות של תסיסה משנית על רמות חומצה אורגניות זרעי קפה, מסופקת.

באמצעות אלקטרופורזה נימים לכמת חומצות אורגניות מרקמות צמח: מקרה מבחן בחינה<em&gt; קפה ערביקה</em&gt; זרעים

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

Research

JoVE Journal

Chemistry

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

9.8K Views.  The Ohio State University, OARDC.  מאמר זה מציג שיטה איתור וכימות של חומצות אורגניות מחומר הצמח באמצעות אלקטרופורזה נימי אזורי חינם. דוגמא של יישום הפוטנציאל של שיטה זו, הקובע את ההשפעות של תסיסה משנית על רמות חומצה אורגניות זרעי קפה, מסופקת. 

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

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

תמליל והמטבוליט Profiling להערכת של טבק ועץ צפצפה כחומר גלם לתעשייה המבוססת על ביו

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Environment

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 יחד עם מערכת הדגימה אוטומטית וניתוח נתונים מותאמים למחקרים על מזון: ניטור Bioprocess, הקרנה וניתוח שטח האף

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

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.

מיצוי קפאין, פעילות אנזימטיות, ביטוי גנים של קפאין סינתאז מ המתלים תא צמח

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 ...

Biochemistry

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.

לכימות הצמח חלבון מסיס והתוכן לעיכול פחמימות, באמצעות תירס (זיה מייז) כמו שיוצרו

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

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

קוונפיקציה של קואנזים A בתאים ורקמות

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

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.

שיטה כללית לקביעת הרכב חומצה פנולית מסיסה באופן חופשי ויכולת נוגדת חמצון של דגנים וקטניות

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 ...

<meta charset="utf8"></head><body>הפרדה איזומרית משופרת של אלקלואידים ארגוט באמצעות LC-IMS-MS

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules).
Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence.
Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

<meta charset="utf8"></head><body>זיהוי אלקלואידים ארגוטיים מוסדרים במטריצות מזון על ידי ספקטרומטריית ניידות יונים לכודים בכרומטוגרפיה נוזלית-ספקטרומטריית מסה בזמן טיסה

This protocol presents a validated liquid chromatography-ion mobility-high resolution mass spectrometry method to determine the presence of ergot alkaloids in food in compliance with the recently released Commission Regulation (EU) 2023/915.

זיהוי אלקלואידים ארגוטיים מוסדרים במטריצות מזון על ידי ספקטרומטריית ניידות יונים לכודים בכרומטוגרפיה נוזלית-ספקטרומטריית מסה בזמן טיסה

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) ...

הערכת מיני בוטנים פראיים לעמידות להצטברות אפלטוקסין

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.

Author Spotlight: Quantification of aflatoxins and phytoalexins in peanut seeds to identify genetic resistance against Aspergillus

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.

באמצעות אלקטרופורזה נימים לכמת חומצות אורגניות מרקמות צמח: מקרה מבחן בחינה

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