@misc{SaguTchewonpiHuschekBoenicketal.2019, author = {Sagu Tchewonpi, Sorel and Huschek, Gerd and B{\"o}nick, Josephine and Homann, Thomas and Rawel, Harshadrai Manilal}, title = {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}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {805}, issn = {1866-8372}, doi = {10.25932/publishup-44222}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-442229}, pages = {20}, year = {2019}, 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.}, language = {en} } @misc{SaguTchewonpiZimmermannLandgraeberetal.2020, author = {Sagu Tchewonpi, Sorel and Zimmermann, Lynn and Landgr{\"a}ber, Eva and Homann, Thomas and Huschek, Gerd and {\"O}zpinar, Haydar and Schweigert, Florian J. and Rawel, Harshadrai Manilal}, title = {Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1028}, issn = {1866-8372}, doi = {10.25932/publishup-48611}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-486118}, pages = {27}, year = {2020}, abstract = {The α-amylase/trypsin inhibitors (ATIs) are discussed as being responsible for non-celiac wheat sensitivity (NCWS), besides being known as allergenic components for baker's asthma. Different approaches for characterization and quantification including proteomics-based methods for wheat ATIs have been documented. In these studies generally the major ATIs have been addressed. The challenge of current study was then to develop a more comprehensive workflow encompassing all reviewed wheat-ATI entries in UniProt database. To substantially test proof of concept, 46 German and Turkish wheat samples were used. Two extractions systems based on chloroform/methanol mixture (CM) and under buffered denaturing conditions were evaluated. Three aspects were optimized, tryptic digestion, chromatographic separation, and targeted tandem mass spectrometric analysis (HPLC-MS/MS). Preliminary characterization with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) documented the purity of the extracted ATIs with CM mixture and the amylase (60-80\%)/trypsin (10-20\%) inhibition demonstrated the bifunctional activity of ATIs. Thirteen (individual/common) biomarkers were established. Major ATIs (7-34\%) were differently represented in samples. Finally, to our knowledge, the proposed HPLC-MS/MS method allowed for the first time so far the analysis of all 14 reviewed wheat ATI entries reported.}, language = {en} } @misc{SaguTchewonpiLandgraeberRackiewiczetal.2020, author = {Sagu Tchewonpi, Sorel and Landgr{\"a}ber, Eva and Rackiewicz, Michal and Huschek, Gerd and Rawel, Harshadrai Manilal}, title = {Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1068}, issn = {1866-8372}, doi = {10.25932/publishup-48809}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-488096}, pages = {21}, year = {2020}, abstract = {Sorghum is of growing interest and considered as a safe food for wheat related disorders. Besides the gluten, α-amylase/trypsin-inhibitors (ATIs) have been identified as probable candidates for these disorders. Several studies focused on wheat-ATIs although there is still a lack of data referring to the relative abundance of sorghum-ATIs. The objective of this work was therefore to contribute to the characterization of sorghum ATI profiles by targeted proteomics tools. Fifteen sorghum cultivars from different regions were investigated with raw proteins ranging from 7.9 to 17.0 g/100 g. Ammonium bicarbonate buffer in combination with urea was applied for protein extraction, with concentration from 0.588 ± 0.047 to 4.140 ± 0.066 mg/mL. Corresponding electrophoresis data showed different protein profiles. UniProtKB data base research reveals two sorghum ATIs, P81367 and P81368; both reviewed and a targeted LC-MS/MS method was developed to analyze these. Quantifier peptides ELAAVPSR (P81367) and TYMVR (P81368) were identified and retained as biomarkers for relative quantification. Different reducing and alkylating agents were assessed and combination of tris (2 carboxyethyl) phosphine/iodoacetamide gave the best response. Linearity was demonstrated for the quantifier peptides with standard recovery between 92.2 and 107.6\%. Nine sorghum cultivars presented up to 60 times lower ATI contents as compared to wheat samples. This data suggests that sorghum can effectively be considered as a good alternative to wheat.}, language = {en} } @misc{TchewonpiSaguLandgraeberHenkeletal.2021, author = {Tchewonpi Sagu, Sorel and Landgr{\"a}ber, Eva and Henkel, Ina M. and Huschek, Gerd and Homann, Thomas and Bußler, Sara and Schl{\"u}ter, Oliver K. and Rawel, Harshadrai Manilal}, title = {Effect of cereal α-amylase/trypsin inhibitors on developmental characteristics and abundance of digestive enzymes of mealworm larvae (Tenebrio molitor L.)}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {5}, issn = {1866-8372}, doi = {10.25932/publishup-52092}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-520924}, pages = {18}, year = {2021}, abstract = {The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21\% to 42\% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25\% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8\% and 14\% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.}, language = {en} } @misc{SaguTchewonpiHuschekWaldbachBragaetal.2022, author = {Sagu Tchewonpi, Sorel and Huschek, Gerd and Waldbach Braga, Tess and Rackiewicz, Michal and Homann, Thomas and Rawel, Harshadrai Manilal}, title = {Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.)}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, publisher = {Universit{\"a}tsverlag Potsdam}, address = {Potsdam}, issn = {1866-8372}, doi = {10.25932/publishup-55928}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-559282}, pages = {1 -- 18}, year = {2022}, abstract = {Wheat alpha-amylase/trypsin inhibitors remain a subject of interest considering the latest findings showing their implication in wheat-related non-celiac sensitivity (NCWS). Understanding their functions in such a disorder is still unclear and for further study, the need for pure ATI molecules is one of the limiting problems. In this work, a simplified approach based on the successive fractionation of ATI extracts by reverse phase and ion exchange chromatography was developed. ATIs were first extracted from wheat flour using a combination of Tris buffer and chloroform/methanol methods. The separation of the extracts on a C18 column generated two main fractions of interest F1 and F2. The response surface methodology with the Doehlert design allowed optimizing the operating parameters of the strong anion exchange chromatography. Finally, the seven major wheat ATIs namely P01083, P17314, P16850, P01085, P16851, P16159, and P83207 were recovered with purity levels (according to the targeted LC-MS/MS analysis) of 98.2 ± 0.7; 98.1 ± 0.8; 97.9 ± 0.5; 95.1 ± 0.8; 98.3 ± 0.4; 96.9 ± 0.5, and 96.2 ± 0.4\%, respectively. MALDI-TOF-MS analysis revealed single peaks in each of the pure fractions and the mass analysis yielded deviations of 0.4, 1.9, 0.1, 0.2, 0.2, 0.9, and 0.1\% between the theoretical and the determined masses of P01083, P17314, P16850, P01085, P16851, P16159, and P83207, respectively. Overall, the study allowed establishing an efficient purification process of the most important wheat ATIs. This paves the way for further in-depth investigation of the ATIs to gain more knowledge related to their involvement in NCWS disease and to allow the absolute quantification in wheat samples.}, language = {en} }