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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

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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

Sorel Tchewonpi Sagu et al. Molecules.

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.

Keywords: Doehlert design; LC-MS/MS; MALDI-TOF/MS; Plackett–Burman design; SDS-PAGE; extraction; wheat; α-amylase/trypsin inhibitors.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Standardized Pareto graph showing effect of the type of solvent, X1; the composition of solvents mixture, X2; the ratio of sample/solvent, X3; the concentration of Urea, X4; the extraction temperature, X5; the extraction time, X6; stirring speed, X7; pH, X8; type of salt, X9; concentration of salt, X10; and centrifugation speed, X11 on: (a,b) concentration of protein and (c,d) inhibition of amylase activity for Julius and Ponticus, respectively. The blue vertical line indicates the limit of the standardized effect whose values are significantly different from zero at the 95.0% confidence level. The pink color shows that the parameter has a positive effect and the red color a negative effect on the experimental response.
Figure 2
Figure 2
Response surfaces of (a,b) protein concentration and (c,d) amylase activity inhibition as function of salt concentration and pH with Julius and Poncticus, respectively. Extraction time is fixed at central point (105 min).
Figure 3
Figure 3
HPLC fractionation (a), SDS-PAGE (b) and MALDI TOF MS (c) of extracted ATIs from Ponticus.
Figure 3
Figure 3
HPLC fractionation (a), SDS-PAGE (b) and MALDI TOF MS (c) of extracted ATIs from Ponticus.
Figure 4
Figure 4
HPLC fractionation (a), SDS-PAGE (b) and MALDI TOF MS (c) of extracted ATIs from Julius.
Figure 4
Figure 4
HPLC fractionation (a), SDS-PAGE (b) and MALDI TOF MS (c) of extracted ATIs from Julius.

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References

    1. Makela M.J., Eriksson C., Kotaniemi-Syrjanen A., Palosuo K., Marsh J., Borres M., Kuitunen M., Pelkonen A.S. Wheat allergy in children-new tools for diagnostics. Clin. Exp. Allergy. 2014;44:1420–1430. doi: 10.1111/cea.12393. - DOI - PubMed
    1. Young E., Stoneham M.D., Petruckevitch A., Barton J., Rona R. A population study of food intolerance. Lancet. 1994;343:1127–1130. doi: 10.1016/S0140-6736(94)90234-8. - DOI - PubMed
    1. Jin Y., Acharya H.G., Acharya D., Jorgensen R., Gao H., Secord J., Ng P.K.W., Gangur V. Advances in Molecular Mechanisms of Wheat Allergenicity in Animal Models: A Comprehensive Review. Molecules. 2019;24:1142 doi: 10.3390/molecules24061142. - DOI - PMC - PubMed
    1. Verhoeckx K.C.M., Vissers Y.M., Baumert J.L., Faludi R., Feys M., Flanagan S., Herouet-Guicheney C., Holzhauser T., Shimojo R., van der Bolt N., et al. Food processing and allergenicity. Food Chem. Toxicol. 2015;80:223–240. doi: 10.1016/j.fct.2015.03.005. - DOI - PubMed
    1. Berin M.C., Sampson H.A. Mucosal immunology of food allergy. Curr. Biol. 2013;23:R389–R400. doi: 10.1016/j.cub.2013.02.043. - DOI - PMC - PubMed

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