Använda kapillärelektrofores att kvantifiera organiska syror från växtvävnad: ett pilotfall Undersöka<em> Coffea arabica</em> Frön
Summary
I denna artikel presenteras en metod för detektion och kvantifiering av organiska syror från växtmaterial med hjälp av gratis zoner kapillärelektrofores. Ett exempel på den potentiella tillämpningen av denna metod att bedöma effekterna av en andra jäsning på organiska syranivåer i kaffefrön, tillhandahålls.
Abstract
Karboxylsyror är organiska syror innehållande en eller flera terminala karboxylgruppen (COOH) funktionella grupper. Kortkedjiga karboxylsyror (SCCAs, karboxylsyror innehållande tre till sex kolatomer), såsom målat och citrat, är avgörande för en väl fungerande många biologiska system, där de fungerar i cellandningen och kan fungera som indikatorer på cell hälsa. I livsmedel, kan halten organisk syra ha en betydande inverkan på smak, med ökade SCCA nivåer resulterar i en sur eller "syra" smak. På grund av detta, metoder för snabb analys av organiska syranivåer är av särskilt intresse för livsmedels- och dryckesindustrin. Men tyvärr, de flesta metoder som används för SCCA kvantifiering är beroende av tidskrävande protokoll kräver derivatisering av prover med farliga reagens, följt av dyra kromatografiska och / eller masspektrometrisk analys. Denna metod detaljer en alternativ metod för detektion och kvantifiering av orgAnic syror från växtmaterial och livsmedelsprover med hjälp av gratis zoner kapillärelektrofores (CZE), ibland helt enkelt benämnd kapillärelektrofores (CE). CZE ger en kostnadseffektiv metod för att mäta SCCAs med en låg detektionsgräns (0,005 mg / ml). Den här artikeln detaljer utvinning och kvantifiering av SCCAs från växtprover. Medan den metod som fokuserar på mätning av SCCAs från kaffebönor, kan den metod som föreskrivs tillämpas på flera växtbaserade livsmedelsmaterial.
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
Som med alla analysteknik, finns det flera viktiga faktorer som väsentligt kan påverka kvaliteten och tillförlitligheten hos de data som genereras. För det första är det viktigt att bearbeta prover effektivt, med ett minimum av frysnings / upptiningscykler. Upprepad frysning och upptining kan äventyra den kemiska sammansättningen av provet före bearbetning eller analys. För det andra är det viktigt att tillämpa stegen i detta protokoll till alla prover konsekvent och jämnt. Tekniska fel till följd av inkon…
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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