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

Abstract

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.

Synthesis and accumulation of amylase-trypsin inhibitors and FODMAPs in bread wheat (Triticum aestivum L.) during grain development

“…Besides gluten type proteins, the PWG spectrum revealed the presence of ATIs and avenin-like proteins, which was recently con rmed by Lexhaller et al [15]. As ATIs are rather small proteins that range from 13-18 kDa [13,16,17], sample peaks in this area were assigned predominately to ATIs and to other proteins from the prolamine superfamily (e.g. avenin-like proteins) in a minor extent [7].…”

Synthesis and accumulation of amylase-trypsin inhibitors and changes in carbohydrate profile during grain development of bread wheat (Triticum aestivum L.)

“…Besides gluten type proteins, the PWG spectrum revealed the presence of ATIs and avenin-like proteins, which was recently confirmed by Lexhaller et al [ 22 ]. As ATIs are rather small proteins that range from 13 to 18 kDa [ 20 , 23 ], sample peaks in this area were assigned predominately to ATIs and to other proteins from the albumin/globulin fraction (e.g. avenin-like proteins) in a minor extent [ 24 ].…”

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS

“…The sample preparation for SDS PAGE was performed as previously described [ 52 ]. Briefly, samples from different extraction methods were mixed in a ratio of 1:1 ( v/v ) with the sample buffer (consisting of 125 mM Tris buffer, pH = 6.8, 20% ( v / v ) glycerol, 2% ( v / v ) 2-mercaptoethanol, 2% ( w / v ) SDS, 0.1% ( w / v ) bromphenol blue) and the mixtures were heated at 95 °C for 5 min.…”