See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/318185030
An Effective Gluten Extraction Method Exploiting Pure Choline
Chloride-Based Deep Eutectic Solvents (ChCl-DESs)
Article in Food Analytical Methods · June 2017
DOI: 10.1007/s12161-017-0979-y CITATIONS 8 READS 166 6 authors, including:
Some of the authors of this publication are also working on these related projects:
Development of an electrochemical aptasensor for gliadin detection in food View project Rossella Svigelj University of Udine 13PUBLICATIONS 77CITATIONS SEE PROFILE Nicolò Dossi University of Udine 58PUBLICATIONS 1,150CITATIONS SEE PROFILE Agnese Giacomino
Università degli Studi di Torino
52PUBLICATIONS 1,579CITATIONS SEE PROFILE Gino Bontempelli University of Udine 174PUBLICATIONS 3,067CITATIONS SEE PROFILE
All content following this page was uploaded by Gino Bontempelli on 21 June 2018. The user has requested enhancement of the downloaded file.
An Effective Gluten Extraction Method Exploiting Pure Choline
Chloride-Based Deep Eutectic Solvents (ChCl-DESs)
Rossella Svigelj1&Renzo Bortolomeazzi1&Nicolò Dossi1&Agnese Giacomino2& Gino Bontempelli1&Rosanna Toniolo1
Received: 21 April 2017 / Accepted: 19 June 2017 / Published online: 30 June 2017 # Springer Science+Business Media, LLC 2017
Abstract Gluten analysis is still affected by the lack of an efficient and universal extraction solvent for gliadin, i.e., the prolamine fraction of gluten which is the analytical target used to quantify gluten in food. In this investigation, the possibility of adopting pure choline chloride deep eutectic solvents (ChCl-DESs) as gliadin extraction media was tested. As pro-totypes of ChCl-DESs, ethaline and reline were chosen in view of their good dipolar and hydrogen bond donor (HBD) properties, as well as of their moderate enough viscosity. Their ability to act as effective solvents for gliadin and their inability to affect the subsequent gliadin detection by a frequently adopted and commercially available ELISA kit were first assessed. Ethaline and reline extraction performance was then tested by evaluating gliadin recoveries achieved on gluten-free food real samples before and after their spiking with con-trolled gliadin amounts. Higher recoveries were found by using these DESs in comparison with those gained by the usually adopted 60% (v/v) ethanol-water solvent. In fact, glu-ten recoveries ranging from 78 to 113%, with a RSD better than 13%, were achieved by using ethaline. Less good recov-eries (from 67 to 132% with a RSD of 20%) were achieved with reline, even though they were however better than those found with 60%v/v ethanol-water solvent. These results make the proposed media profitable solvents for gliadin extraction and its subsequent detection by a commonly used immunoas-say kit, without any change of the usual detection procedure.
Keywords Deep eutectic solvents (DESs) . Gluten . ELISA kit . Celiac disease . Ethaline . Reline
Introduction
In the last decades, quantification of gluten in food is getting more and more important, since celiac disease, a gluten intol-erance, is a chronic inflammatory enteropathy that affects ap-proximately 1% of world peoples (Guandalini and Assiri 2014). In fact, ingestion of food containing gluten by geneti-cally predisposed people triggers an immune reaction leading to damages of the intestinal mucosa, thus causing serious side effects. Even though several treatments have been tested, the only effective therapy for celiac disease is up to now a lifelong
gluten-free diet (Akobeng and Thomas2008).
For this reason, the European Union has recently set that products containing less than 20 ppm (mg kg−1) of gluten alone can be labeledBgluten-free,^ while food labeled Bvery low gluten^ must contain less than 100 ppm of gluten
(Commission Implementing Regulation2014). In USA, the
corresponding FDA regulation (Food Labeling; Gluten-Free Labeling of Foods2013) sets instead that food with a gluten content below 20 ppm can be labeled gluten-free, while no provision is set for other gluten contents.
On the basis of these limits, the development of analytical methods for the detection of gluten in food appears to be an
important topic in food analysis (Haraszi et al. 2011).
Quantification of this species is quite challenging since gluten is a complex mixture of storage proteins prolamines and glutelins, in a mass ratio of ca 1:1 (Wieser2007), found in wheat, rye, barley, oats, and their crossbred varieties. The prolamin fraction (gliadins) consists of monomeric proteins with a single polypeptide chain characterized by a globular
conformation and a molecular mass of 32–70 kDa (Barak
* Rosanna Toniolo rosanna.toniolo@uniud.it 1
Department of Agrifood, Environmental and Animal Sciences, University of Udine, Via Cotonificio 108, 33100 Udine, Italy
2 Department of Drug Science and Technology, University of Torino,
et al.2015), which is commonly used as analytical target to quantify gluten in food (Scherf and Poms2016). Thus, gluten mass in food is conventionally evaluated by doubling the mass of determined gliadin.
Currently, the CODEX official analytical method for gluten detection is an enzyme-linked immunosorbent assay (ELISA) using the R5 antibody (Slot et al.2016). Despite the availabil-ity of this reference immunosorbent assay, gluten detection in food still remains a problem (Diaz-Amigo and Popping2013). This is due to the possible occurrence of cross-reactivity pro-cesses, incomplete gluten extraction from heat-treated food, and the lack of a standardized reference material for calibra-tion procedures. This is the reason why many other enzyme-immunosorbent assays based on other anti-gliadin antibodies, extraction solvents, or calibration materials have been
devel-oped (Diaz-Amigo and Popping2013).
In any case, the main problem concerning gluten quantifi-cation remains its extraction from food matrices because this step turns out to be crucial in order to obtain a proper analyte quantification. At present, gluten extraction is usually per-formed by ethanol-water solutions 60–40% (v/v) or by patent-ed ethanol-water solutions containing rpatent-educing agents such as mercaptoethanol or tris(2-carboxyethyl)-phosphine (García et al.2005; Mena et al.2012). However, ethanol extraction is not always efficient and the presence of reducing agents can interfere at times with immunoassays (Doña et al.2007).
Recently, a new generation of ionic liquids (ILs), named deep eutectic solvents (DESs), obtained by mixing two safe components able to form an eutectic mixture with a melting point lower than that of starting components, are gaining a growing interest as green solvents. DESs exhibit physical and chemical properties similar to those of ILs, as well as improved biodegradability and lower toxicity (Zhang et al. 2012; Toniolo et al.2016). These solvents are generally ob-tained by mixing a hydrogen bond acceptor (HBA), such as quaternary ammonium salts, with a hydrogen bond donor (HBD), such as amines, carboxylic acids, alcohols, or carbo-hydrates (Abbott et al.2004). Up to now, explorative studies focused on their capacity to act as effective solvents have highlighted their ability to dissolve poorly soluble drugs and nucleic acids, while studies on their solvent ability toward organic macromolecules are still very scarce (Zeng et al. 2014; Zhang et al.2016). In addition, it was also hypothesized that the occurrence of a large number of biological processes could become possible in media such as DESs (Dai et al. 2013).
Thus, in this work we tested an alternative, green, and effective extraction procedure for gliadin, exploiting pure cho-line chloride-based deep eutectic solvents (ChCl-DESs) which are the most commonly employed DESs.
When this investigation was nearly completed, a paper dealing with a similar subject was published, in which water-diluted sugar-based natural deep eutectic solvents
(NADESs) in combination with an ultrasound-assisted extrac-tion was applied to gluten quantificaextrac-tion (Lores et al.2017). The results presented here prove that the use of pure ChCl-DESs as extraction media for gluten consents a simpler pro-cedure and makes possible the achievement of better recoveries.
The profitable capability of ChCl-DESs to extract gliadin from different food was also compared with that displayed by the ethanol-water medium usually adopted as gluten extrac-tion solvent, by resorting to a commercially available ELISA kit for the final gluten detection.
Experimental
Reagents and Apparatus
Ethaline and reline (see Table 1) were the choline chloride-based deep eutectic solvents used as gluten extraction solvents throughout this investigation. They were supplied by Scionix Ltd. (London, UK) and employed as received, without further purification. Ethanol used to prepare 60% (v/v) ethanol-water extraction solutions was purchased from Sigma-Aldrich
(Milan, Italy). Reference gliadin (90.8% w/w purity) from
the Prolamin Working Group (PWG gliadin) was supplied by the Group on Prolamin Analysis and Toxicity (Germany) and used as the standard. This standard was obtained from a mixture of 28 wheat cultivars representative of the European
wheat-producing countries. A 1000 mg L−1 PWG gliadin
standard solution was prepared in 60% (v/v) ethanol-water. Gluten in food samples was quantified by the ELISA immu-noassay test performed by using the Gluten-Check™ kit sup-plied by Bio-Check (UK) (Gluten-Check™ ELISA kit, instruc-tion for use). Water used as solvent was ultrapure water, purified by an Elgastat UHQ PS system (ELGA LabWater, Siershahn, Germany). Both unheated (flour) and heated (biscuits and crack-ers) gluten-free foods were purchased from local supermarkets. Water content in ChCl-DESs (ethaline and reline) was de-termined by a Karl Fischer titrator DL32 (Mettler-Toledo, Greifensee, Switzerland). When necessary, a Model 1210 ul-trasonic bath from Bransonic Co. (USA) was used to prepare high-concentration PWG gliadin standard solutions. ELISA spectrophotometric measurements were carried out with a
PerkinElmer VICTOR31420 Multilabel Counter absorbance
microplate spectrophotometer (Waltham, MA, USA), while an Agilent Cary 50 UV-Vis spectrophotometer (Santa Clara, CA, USA) was used to record UV-Vis spectra. An analytical balance CP Sartorius (Sartorius, Goettingen, Germany) was used to weight samples. In agreement with the procedure
rec-ommended by the Gluten-Check™ELISA kit, all solid
sam-ples were grinded, when necessary, by a Sterilmixer homoge-nizer (International PBI, Milan, Italy) and shaken using a vor-tex (International PBI, Milan, Italy). An IKA magnetic stirrer
with a water bath (Werke GmbH & Co. KG, Staufen, Germany) and a Thermo Scientific Heraeus Labofuge 200 Centrifuge (Waltham, MA, USA) were used for sample ex-traction and centrifugation, respectively.
Sample Preparation and Extraction Procedure
Extraction of gluten from food was performed by using the abovementioned ChCl-DESs and, for the sake of comparison, by usual 60% (v/v) ethanol-water solutions.
About 50 g of each food sample was milled in a grinder to prepare a fine powder. Afterward, 0.35 g of the obtained pow-dered sample was extracted in Eppendorf vials with 3.5 mL of 60% (v/v) ethanol-water solution or pure ChCl-DESs. These vials were shaken by a vortex for 2 min, and then they were left in a water bath at 55 °C for 45 min. After this time, they were shaken again for 2 min and centrifuged for 10 min at 5000 rpm. Similar aliquots of powdered food were also spiked with known amounts of PWG gliadin (leading to spiked con-tents ranging from 5 to 36 mg kg−1) and subjected to the same treatment described above.
After extraction, 100μL of supernatant was diluted (10:1 or 20:1) with the dilution solution provided by the ELISA kit (phosphate-buffered ethanol-water). Sample preparation was carried out in a laboratory separated from that where analyses were performed, to avoid contamination, and all extractions were carried out in triplicate.
Gluten-Check™ELISA kit
Gluten content in food samples was determined by the
Gluten-Check™ELISA test mentioned above (http://www.biocheck.
uk.com/gluten/lab-test-kits). This test is a two-step method which profits from the use of the 401/21 antibody developed by Skerritt and Hill (1991), approved by the Association for Official Analytical Chemists (AOAC) for the quantification of gluten in food.
First, after suitable dilution with the kit solvent, extracted samples were incubated with the 401/21 monoclonal antibody immobilized onto the bottom of the wells, to form the corre-sponding complex. After incubation for 20 min, wells were emptied and washed three times with the washing solution supplied by the ELISA kit (phosphate-buffered ethanol-wa-ter). Afterward, a solution containing the antibody labeled with the enzyme horseradish peroxidase (HRP) was added, so that PWG gliadin formed an adduct sandwiched between the antibody attached to the well and antibody labeled with the enzyme. After further 20 min, the excess of non-immobilized labeled antibody was removed by using the kit washing solu-tion (four repeated washings). Then, a colorless solusolu-tion con-taining a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate spe-cific for the enzyme was added in the dark, so that the enzyme can promote the TMB oxidation, by giving rise to the corre-sponding blue-colored oxidized species.
The enzymatic reaction was hence stopped by acidification with 0.5 M H2SO4which caused simultaneously inhibition of the enzyme and conversion of the oxidized form of TMB to the corresponding yellow-colored protonated form, whose ab-sorption band (remaining unaltered for about 1 h) showed a maximum at 450 nm. A calibration curve was constructed by plotting the absorbance at 450 nm against the gluten concen-tration of five standard solutions, in the range 5–110 mg L−1, provided with the ELISA kit.
Results and Discussion
Solubilization Tests of PWG Gliadin in ChCl-DESs DESs consisting of choline chloride combined in a 1:2 M ratio with 1,2-ethanediol(ethaline), glycerol (glyceline), urea (re-line), and malonic acid (maline) were recently reported to display dipolar properties, i.e., they behave as compounds exhibiting electron density shared unequally between atoms
Table 1 Formula and component molar ratios of ChCl-DESs adopted
Name
Salt-HBA
HBD
Molar
ratio
Ethaline
HO N+ Cl-choline chloride
OH
OH
ethylene glycol
1:2
Reline
HO N+ Cl-choline chloride
O NH2 NH2urea
1:2
because they have different electronegativity (Pandey et al. 2014). Moreover, also their HBD ability higher enough than those proper for short-chain alcohols was highlighted (Pandey et al.2014). On the other hand, current literature emphasizes that conventional gliadin solvents, such as water-ethanol, do not allow always its total extraction from some foods (García et al.2005). Consequently, it has to be expected that the dis-solution ability of these ChCl-DESs toward PWG gliadin should be profitable and worthy of investigation.
Thus, we have tested the ability of two choline chloride DESs, ethaline and reline, to extract gliadin from food, as well as their compatibility with a commercially available enzyme immunoassay. Ethaline and reline were chosen for these tests because they display a moderate viscosity and do not present handling difficulties at room temperature. Table2reports their viscosity at 25 and 55 °C from literature data (D’Agostino et al.2011), together with their water content determined by us.
Data reported in Table2show that ethaline and reline dis-play at 25 °C a viscosity which is quite higher than that displayed by common gliadin solvents (from 30 to 1000 times higher than water), but it becomes considerably lower at 55 °C which is the temperature usually recommended for gluten quantification. In fact, it is generally accepted that no gluten protein denaturation occurs up to 58 °C (Leon et al.2003). This temperature-induced viscosity decrease of DESs is ex-pected to improve their extraction efficiency since analyte diffusion becomes quicker, thus making these solvents quite promising.
Preliminary tests were performed to verify the ability of ethaline and reline to dissolve PWG gliadin. With this purpose, a series of solutions were first prepared by dissolving controlled amounts of PWG gliadin in each DES at 25 °C, so as to achieve concentrations ranging from 50 to 300 mg L−1. Higher concen-trations were not considered in view of the fact that they are not usually encountered after food extraction. Concomitantly, for the sake of comparison, PWG gliadin solutions with the same concentrations were also prepared in 60% (v/v) ethanol-water. All these prepared standard samples turned out to be clear so-lutions, without precipitates, which were subjected to absor-bance measurements in the UV-Vis range 250–450 nm, where proteins display an absorption band with a maximum at about
280 nm (Schmid2001). Recorded UV-Vis spectra, whose
typ-ical trends are summarized inFig. 1, showed in all cases a linear increase with the PWG gliadin concentration. In particular, spectra recorded in DES media did not show appreciable dif-ferences from those recorded in 60% (v/v) ethanol-water and also the corresponding calibration plots (insets inFig. 1) turned out to be similar enough. In fact, regression equations found in 60% (v/v) ethanol-water, ethaline, and reline, respectively, were the following: A = 7.042 × 10−4xCPWG (mg L−1)− 0.008; A = 7 . 5 3 5 × 1 0− 4x CP W G ( m g L− 1) + 0 . 0 1 6 ; a n d A = 8.017 × 10−4xCPWG(mg L−1)− 0.009.
Table 2 Viscosity and water content of DESs
ChCl-DESs Viscositya(25 °C) (mPa s−1) Viscositya(55 °C) (mPa s−1) Water contentb(%) Ethaline 37 24 2.7% Reline 750 95 3.1% a (D’Agostino et al.2011) b
Experimental data (Karl-Fischer titration)
Fig. 1 Absorption spectra, recorded at 25 °C, for PWG gliadin at
different concentrations (from 50 to 300 mg L−1) in 60% (v/v)
ethanol-water, ethaline, and reline.Insets: PWG gliadin absorbance at 278 nm
versus its concentration
When these tests were repeated in DESs at 55 °C, very similar UV-Vis spectra were once again recorded. In addition, when absorbance measurements in DES media were repeated every hour, the relevant spectra turned out to be repeatable for 1 day at least. These findings pointed out that DESs adopted by us are effective solvents for PWG gliadin and that they do not cause analyte degradation.
Endurance of the ELISA (Gluten-Check™) Assay
for ChCl-DES Media
In principle, the performance of the Gluten-Check™ELISA
immunoassay employed by us for gluten determination could be interfered by the presence of DESs used in the extraction step. In fact, these media could change to some extent the gliadin environment, thus causing its conformational alter-ation and making the ELISA immunoassay no longer suitable for its determination. Consequently, some preliminary tests were performed to verify whether ethaline and reline were able to affect the detection procedure.
With this purpose, standard solutions of PWG gliadin in both ethaline and reline were prepared by adding controlled amounts of the analyte to the pure solvents, so as to achieve
concentrations ranging from 0 to 50 mg L−1. One hundred
microliters of these solutions was then subjected to the detec-tion procedure reported in theBExperimental^ section.
The results of seven replicate measurements performed on each sample turned out to be satisfactory, in that PWG gliadin concentrations found turned out to be practically coincident with the expected contents, with a RSD of ±9%. This finding points out that the replacement of ethanol-water by ethaline or reline as extraction media is fully compatible with the subse-quent gluten detection procedure adopted by us.
Determination of Gluten in Food Samples
The ability of DESs adopted by us to act as effective extraction media for gluten from food was tested on real food samples. Both heat-untreated (flour) and heat-treated (crackers and bis-cuits) gluten-free food samples were assayed, in view of the fact that significant decreases of gluten contents were found on passing from flour to the corresponding bread when the usual 60% (v/v) ethanol-water extraction solvent was used (Gessendorfer et al.2010). For the sake of comparison, all real samples were also subjected to extraction with 60% (v/v) eth-anol-water. After extraction, these samples in both DESs and ethanol-water were subjected to analysis for their natural glu-ten conglu-tent.
Moreover, four spiked samples were prepared by adding controlled amounts of PWG gliadin standard solutions to 0.35 g of both heat-treated and heat-untreated gluten-free food samples. Three replicates of each sample and of each spike level were tested. Also, these spiked samples were extracted with each DES adopted and with ethanol-water, following the procedure described in theBExperimental^ section. After cen-trifugation, 100μL aliquots of supernatants were analyzed by the ELISA kit.
The results obtained are summarized in Table 3, where
recoveries achieved with the different solvents are compared with one another. Even though gliadin found in spiked sam-ples did not come from gluten, since equivalent amounts of glutelins were of course absent, all data reported in this table are expressed as milligrams per kilogram of the corresponding gluten (double of gliadin mass), as inferred from the calibra-tion curve provided by the ELISA kit.
Data in Table3show that when food samples characterized by small gluten concentrations were analyzed, such as unspiked gluten-free samples, all extraction solvents
Table 3 Recovery % ± relative standard deviation (RSD) for gluten-free and PWG gliadin spiked food samples
Extraction medium PWG gliadin
spike (mg kg−1)
Gluten found (recovery% ± SD) (mg kg−1)
Flour Crackers Biscuits
Ethaline – 2.3 ± 0.1 2.4 ± 0.1 2.1 ± 0.2 4.5 10.6 (94 ± 11) 12.9 (113 ± 12) 8.9 (80 ± 13) 9.0 19.0 (94 ± 7) 19.0 (93 ± 7) 15.8 (78 ± 3) 8 41.9 (109 ± 8) – – 36 76.0 (102 ± 12) – – Reline – 2.0 ± 0.2 2.5 ± 0.1 2.1 ± 0.2 9 13.5 (67 ± 15) 17.3 (84 ± 8) 26.5 (132 ± 20) 18 28.1 (74 ± 8) – – 60% (v/v) EtOH–H2O – 2.3 ± 0.1 1.9 ± 0.2 1.9 ± 0.1 9 5.1 (25 ± 20) 12.0 (60 ± 15) 7.1 (36 ± 18) 18 22.3 (58 ± 27) – –
employed led to comparable enough gluten contents (a little more than 2 mg kg−1).
On the contrary, when spiked samples displaying higher gliadin concentrations (added amount plus natural content) were tested, the usual 60% (v/v) ethanol-water extraction sol-vent turned out to provide a quite modest performance. Better results were found when the extraction was conducted with the DES reline, but the best performance in terms of recovery was provided by the DES ethaline. This ability of ethaline to act as a more effective extraction medium with respect to reline can be conceivably accounted for considering the lower viscosity displayed by this DES, which is expected to increase the analyte mass transfer rate from the sample to the extraction medium.
In particular, it is worth noting that gluten recoveries achieved by ethaline turned out to be totally satisfactory just for samples spiked with gliadin amounts nearly 10 mg kg−1 (corresponding to about 20 mg kg−1of gluten), which is the gluten content typical of the cutoff region between gluten-free and gluten-containing food samples.
The better performance offered by ethaline with respect to
the 60%v/v ethanol-water solvent is conceivably closely
bound up with the gliadin solubility in these different media. In fact, solubility tests performed by us for gliadin in ethaline at room temperature showed that PWG solubility was largely
higher than 5000 mg L−1, while a solubility of ca.
1600 mg L−1 was reported for gliadin in ethanol at room
temperature (Lores et al.2017). No literature report is so far available about gliadin solubility in 60%v/v ethanol-water at higher temperatures, but it is expected that a temperature in-crease causes a solubility inin-crease in both ethanol-water and ethaline. Consequently, ethaline should remain conceivably a more effective solvent for PWG even at 55 °C.
Finally, it is the case of comparing our findings with those gained carrying out the gluten extraction from food by using sugar-based DESs which were recently reported (Lores et al. 2017). Recoveries found by us by using ChCl-DESs were almost comparable with those achieved in spiked real samples extracted by sugar-based DESs. However, extractions con-ducted with these last DESs required their preliminary dilution with water in order to reduce their quite high viscosity. On the contrary, the low enough viscosity displayed at 55 °C by ChCl-DESs (in particular by ethaline) allowed their direct use as solvent for gluten extraction.
Conclusions
The results achieved in this investigation point out that DESs such as ethaline and reline are very promising as extraction solvents of gluten from both unprocessed and processed food, because they provide reliable results with simple sample treat-ment. The use of these DESs is fully compatible with the
subsequent gluten detection with a usual sandwich immuno-assay exploiting the 401/21 antibody, without any change of the procedure recommended by the ELISA kit adopted.
Both DESs provided performance comparable enough with that offered by the usual 60% (v/v) ethanol-water solvent for samples containing small gluten contents, while they proved to be better extraction solvents when food samples character-ized by higher gluten concentrations were analyzed. As a mat-ter of fact, ethaline acted as a more effective extraction medi-um with respect to reline and this can be explained on consid-ering the higher viscosity of reline which causes conceivably the analyte mass transfer from the sample to the extraction solvent to be slowed down.
In particular, recoveries provided by ethaline from samples containing nearly 20 mg kg−1of gluten (range where it is crucial to distinguish between free and gluten-containing samples) turned out to be more accurate than those provided by other extraction solvents so far adopted.
Compliance with Ethical Standards
Conflict of Interest Rossella Svigelj declares that she has no conflict of
interest. Renzo Bortolomeazzi declares that he has no conflict of interest. Nicolò Dossi declares that he has no conflict of interest. Agnese Giacomino declares that she has no conflict of interest. Gino Bontempelli declares that he has no conflict of interest. Rosanna Toniolo declares that he has no conflict of interest.
Ethical Approval This article does not contain any studies with human
or animal subjects.
Informed Consent Informed consent was not applicable.
References
Abbott AP, Boothby D, Capper G, Davies DL, Rasheed RK (2004) Deep eutectic solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic liquids. J Am Chem Soc 126: 9142–9147
Akobeng AK, Thomas AG (2008) Systematic review: tolerable amount of gluten for people with coeliac disease. Aliment Pharmacol Ther
27:1044–1052
Barak S, Mudgil D, Khatkar BS (2015) Biochemical and functional prop-erties of wheat gliadins: a review. Crit Rev Food Sci Nutr 55:357– 368
Commission Implementing Regulation (2014) (EU) no 828/2014 on the requirements for the provision of information to consumers on the absence or reduced presence of gluten in food. Off J Eur Union
L228:5–8
D’Agostino C, Robert C, Harris RC, Abbott AP, Gladdena LF, Mantle MD (2011) Molecular motion and ion diffusion in choline chloride
based deep eutectic solvents studied by1H pulsed field gradient
NMR spectroscopy. Phys Chem Chem Phys 13:21383–21391 Dai YT, van Spronsen J, Witkamp GJ, Verpoorte R, Choi YH (2013)
Natural deep eutectic solvents as new potential media for green
technology. Anal Chim Acta 766:61–68
Diaz-Amigo C, Popping B (2013) Accuracy of ELISA detection methods for gluten and reference materials: a realistic assessment. J Agric
Food Chem 61:5681–5688
Doña VV, Fossati CA, Chirdo F (2007) Interference of denaturing and reducing agents on the performance of quantitative immunoassays
in gliadin analysis. Eur Food Res Technol 226:591–602
Food Labeling; Gluten-Free Labeling of Foods (2013) Federal Register
78: 47154–47179
García E, Llorente M, Hernando A, Kieffer R, Wieser H, Mendez E (2005) Development of a general procedure for complete extraction of gliadins from heat processed and unheated foods. Eur J
Gastroenterol Hepatol 17:529–539
Gessendorfer B, Wieser H, Koehler P (2010) Optimisation of a solvent for the complete extraction of prolamins from heated foods. J Cereal
Sci 52:331–332
Gluten-Check™ ELISA kit (n.d.), Instruction for use. (http://www.
biocheck.uk.com/gluten/lab-test-kits)
Guandalini S, Assiri A (2014) Celiac disease: a review. JAMA Pediatr
168:272–278
Haraszi R, Chassaigne H, Maquet A, Ulberth F (2011) Analytical methods for detection of gluten in food-method developments in support of food labeling legislation. J AOAC Int 94:1006–1025 Leon A, Rosell CM, de Barber CB (2003) A differential scanning
calo-rimetry study of wheat proteins. Eur Food Res Technol 217:13–16 Lores H, Romero V, Costas I, Bendicho C, Lavilla I (2017) Natural deep
eutectic solvents in combination with ultrasonic energy as a green approach for solubilisation of proteins: application to gluten deter-mination by immunoassay. Talanta 162:453–459
Mena MC, Lombardia M, Hernando A, Méndez E, Albaret JP (2012) Comprehensive analysis of gluten in processed foods using a new
extraction method and a competitive ELISA based on the R5
anti-body. Talanta 91:33–40
Pandey A, Rai R, Pal M, Pandey S (2014) How polar are choline chloride-based deep eutectic solvents? Phys Chem Chem Phys 16:
1559–1568
Scherf KA, Poms RE (2016) Recent developments in analytical methods
for tracing gluten. J Cereal Sci 67:112–122
Schmid FX (2001) Biological macromolecules: UV-visible spectropho-tometry. In: Encyclopedia of life sciences. Wiley, Chichester Skerritt JH, Hill AS (1991) Enzyme immunoassay for determination of
gluten in foods: collaborative study. JAOAC 74:257–264
Slot IB, Van der Fels-Klerx HJ, Bremer MG, Hamer RJ (2016) Immunochemical detection methods for gluten in food products:
where do we go from here? Crit Rev Food Sci Nutr 56:2455–2466
Toniolo R, Dossi N, Svigelj R, Pigani L, Terzi F, Abollino O, Bontempelli G (2016) A deep eutectic solvent-based amperometric sensor for the detection of low oxygen contents in gaseous atmospheres. Electroanalysis 28:757–763
Wieser H (2007) Chemistry of gluten proteins. Food Microbiol 24:115–
119
Zeng Q, Wang Y, Huang Y, Ding X, Chen J, Xu K (2014) Deep eutectic solvents as novel extraction media for protein partitioning. Analyst 139:2565–2573
Zhang Q, Vigier KDO, Royer S, Jérôme F (2012) Deep eutectic solvents:
syntheses, properties and applications. Chem Soc Rev 41:7108–
7146
Zhang HM, Wang YZ, Xu KJ, Li N, Wen Q, Zhou YG (2016) Ternary and binary deep eutectic solvents as a novel extraction medium for protein partitioning. Anal Methods 8:8196–8207
View publication stats View publication stats
