Gliadin extraction method exploiting pure choline chloridebased deep eutectic solvents (ChCl-DESs) Rossella Svigelj (
[email protected]) Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Italy Tutor: Prof. Rosanna Toniolo; Co-tutor: Prof. Renzo Bortolomeazzi Abstract The application of green solvents for the extraction of gliadin (which represents about 50% in mass of gluten) is here described. To evaluate the capability of two choline chloride DESs, Ethaline and Reline (see Table 1), to extract gliadin, we tested their performance on a set of heated and unheated gluten-free food. Gluten-free unspiked and spiked samples such as white flour, crackers and biscuits were assayed. The suitability of Ethaline and Reline to extract gliadin was evaluated by comparison with the usual 60% (v/v) ethanol-water gliadin extractant employing a commercially available ELISA kit.
Background
Results The compatibility of ChCl-DESs with a commercially available immunoassay was investigated. Then, extraction efficiency was evaluated on food samples such as white flour, crackers and biscuits. The overall experimental assay was conducted as sketched in Figure 1.
Deep eutectic solvents (DESs) are a new class of ionic liquids, obtained by mixing two safe components able to form an eutectic mixture with a melting point lower than that of starting components (Zhang et al. 2012). Salt-‐HBA
HBD
Molar ratio
Ethaline Reline
HO
N
+
Cl
choline c hloride HO
N+
-
Cl-
choline c hloride
OH
OH
ethilen g licole
1:2
O
NH2
NH2
urea
1:2
Table 1.
Gluten extraction is usually performed by ethanol (60%) or patented cocktail solution containing mercaptoethanol and guanidine hydrochloride (García et al. 2005). Since ethanol extraction in not always efficient and the presence of reducing agents in the cocktail solution may lead to interferences with immunoassays we decided to explore the use of pure choline chloride DESs.
Figure 1.
About 50 g of each food sample were milled in a grinder to prepare a fine powder. Afterward, 0.35 g of the obtained powdered sample were 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 they were then 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. Samples were also spiked with known amounts of gliadin, and subjected to the same treatment described above. Recovery % and reproducibility (RSD %) of gliadin extraction using ethaline, reline and 60% v/v ethanolwater solution are shown in Table 2.
Extrac'on medium
Gluten found (recovery% ± SD) ppm
PWG Gliadin spike ppm -‐
Ethaline
Reline
60% (v/v) EtOH–H2O
Flour
Crackers
Biscuits
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)
18
41.9 (109 ± 8)
-‐
-‐
36
76.0 (102 ± 12)
-‐
-‐
-‐
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)
-‐
-‐
-‐
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)
-‐
-‐
Table 2.
Conclusions These results seem to be quite promising, since the use of these green medias is fully compatible with the immunoassay here employed, which is one of the most common ELISA test used for gliadin detection. In addition Ethaline proved to be a better extraction solvent than ethanol solutions for samples with higher gluten concentrations.
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XXII WORKSHOP ON THE DEVELOPMENTS IN THE ITALIAN PHD RESEARCH ON FOOD SCIENCE, TECHNOLOGY AND BIOTECHNOLOGY BOLZANO, SEPTEMBER 20th-22nd, 2017
