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Open AccessArticle

A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett–Burman and Box–Behnken Designs

1
University of Potsdam, Institute of Nutritional Science, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Potsdam, Germany
2
IGV-Institut für Getreideverarbeitung GmbH, Arthur-Scheunert-Allee 40/41, D-14558 Nuthetal OT Bergholz-Rehbrücke, Germany
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(19), 3589; https://doi.org/10.3390/molecules24193589
Received: 20 August 2019 / Revised: 26 September 2019 / Accepted: 3 October 2019 / Published: 5 October 2019
(This article belongs to the Special Issue Methods for the Purification and Characterization of Proteins/Enzymes)
Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential. View Full-Text
Keywords: wheat; α-amylase/trypsin inhibitors; extraction; Plackett–Burman design; Doehlert design; SDS-PAGE; MALDI-TOF/MS; LC-MS/MS wheat; α-amylase/trypsin inhibitors; extraction; Plackett–Burman design; Doehlert design; SDS-PAGE; MALDI-TOF/MS; LC-MS/MS
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Tchewonpi Sagu, S.; Huschek, G.; Bönick, J.; Homann, T.; Rawel, H.M. A New Approach of Extraction of α-Amylase/trypsin Inhibitors from Wheat (Triticum aestivum L.), Based on Optimization Using Plackett–Burman and Box–Behnken Designs. Molecules 2019, 24, 3589.

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