TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Waldbach Braga, Tess A1 - Rackiewicz, Michal A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Design of Experiment (DoE) for Optimization of HPLC Conditions for the Simultaneous Fractionation of Seven α-Amylase/Trypsin Inhibitors from Wheat (Triticum aestivum L.) T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1260 KW - wheat KW - α-amylase/trypsin inhibitors KW - fractionation KW - purification KW - reversed-phase chromatography KW - ion-exchange chromatography KW - design of experiment KW - LC–MS/MS KW - MALDI-TOF-MS Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-559282 SN - 1866-8372 SP - 1 EP - 18 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - GEN A1 - Figueroa Campos, Gustavo A. A1 - Perez, Jeffrey Paulo H. A1 - Block, Inga A1 - Tchewonpi Sagu, Sorel A1 - Saravia Celis, Pedro A1 - Taubert, Andreas A1 - Rawel, Harshadrai Manilal T1 - Preparation of activated carbons from spent coffee and coffee parchment and assessment of their adsorbent efficiency T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The valorization of coffee wastes through modification to activated carbon has been considered as a low-cost adsorbent with prospective to compete with commercial carbons. So far, very few studies have referred to the valorization of coffee parchment into activated carbon. Moreover, low-cost and efficient activation methods need to be more investigated. The aim of this work was to prepare activated carbon from spent coffee grounds and parchment, and to assess their adsorption performance. The co-calcination processing with calcium carbonate was used to prepare the activated carbons, and their adsorption capacity for organic acids, phenolic compounds and proteins was evaluated. Both spent coffee grounds and parchment showed yields after the calcination and washing treatments of around 9.0%. The adsorption of lactic acid was found to be optimal at pH 2. The maximum adsorption capacity of lactic acid with standard commercial granular activated carbon was 73.78 mg/g, while the values of 32.33 and 14.73 mg/g were registered for the parchment and spent coffee grounds activated carbons, respectively. The Langmuir isotherm showed that lactic acid was adsorbed as a monolayer and distributed homogeneously on the surface. Around 50% of total phenols and protein content from coffee wastewater were adsorbed after treatment with the prepared activated carbons, while 44, 43, and up to 84% of hydrophobic compounds were removed using parchment, spent coffee grounds and commercial activated carbon, respectively; the adsorption efficiencies of hydrophilic compounds ranged between 13 and 48%. Finally, these results illustrate the potential valorization of coffee by-products parchment and spent coffee grounds into activated carbon and their use as low-cost adsorbent for the removal of organic compounds from aqueous solutions. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1158 KW - coffee by-products KW - spent coffee grounds KW - parchment KW - valorization KW - calcination KW - activated carbon KW - organic compounds adsorption Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-521914 SN - 1866-8372 IS - 8 ER - TY - GEN A1 - Tchewonpi Sagu, Sorel A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - Effect of sample preparation on the detection and quantification of selected nuts allergenic proteins by LC-MS/MS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1157 KW - nut allergenic proteins KW - sample preparation KW - protein extraction KW - tryptic digestion KW - microwave assisted digestion KW - SDS PAGE KW - LC-MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-521871 SN - 1866-8372 IS - 15 ER - TY - GEN A1 - Tchewonpi Sagu, Sorel A1 - Landgräber, Eva A1 - Henkel, Ina M. A1 - Huschek, Gerd A1 - Homann, Thomas A1 - Bußler, Sara A1 - Schlüter, Oliver K. A1 - Rawel, Harshadrai Manilal T1 - Effect of cereal α-amylase/trypsin inhibitors on developmental characteristics and abundance of digestive enzymes of mealworm larvae (Tenebrio molitor L.) T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1153 KW - growth behavior KW - Tenebrio molitor larvae KW - feeding KW - cereal meals KW - α-amylase/trypsin inhibitors KW - digestive enzymes quantification KW - LC-MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-520924 SN - 1866-8372 IS - 5 ER - TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Landgräber, Eva A1 - Rackiewicz, Michal A1 - Huschek, Gerd A1 - Rawel, Harshadrai Manilal T1 - Relative Abundance of Alpha-Amylase/Trypsin Inhibitors in Selected Sorghum Cultivars T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1068 KW - sorghum KW - α-amylase/trypsin inhibitors KW - reducing agents KW - cysteine alkylation KW - SDS PAGE KW - targeted proteomics KW - LC–MS/MS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-488096 SN - 1866-8372 IS - 1068 ER - TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Zimmermann, Lynn A1 - Landgräber, Eva A1 - Homann, Thomas A1 - Huschek, Gerd A1 - Özpinar, Haydar A1 - Schweigert, Florian J. A1 - Rawel, Harshadrai Manilal T1 - Comprehensive Characterization and Relative Quantification of α-Amylase/Trypsin Inhibitors from Wheat Cultivars by Targeted HPLC-MS/MS T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1028 KW - α-amylase/trypsin inhibitors KW - wheat cultivars KW - SDS-PAGE KW - peptides markers KW - relative quantification KW - mass spectrometry KW - LC-MRM-MS Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-486118 SN - 1866-8372 IS - 1028 ER - TY - GEN A1 - Figueroa Campos, Gustavo A. A1 - Sagu Tchewonpi, Sorel A1 - Saravia Celis, Pedro A1 - Rawel, Harshadrai Manilal T1 - Comparison of batch and continuous wet-processing of coffee BT - changes in the main compounds in beans, by-products and wastewater T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - Many technical challenges still need to be overcome to improve the quality of the green coffee beans. In this work, the wet Arabica coffee processing in batch and continuous modus were investigated. Coffee beans samples as well as by-products and wastewaters collected at different production steps were analyzed in terms of their content in total phenols, antioxidant capacity, caffeine content, organic acids, reducing sugars, free amino group and protein content. The results showed that 40% of caffeine was removed with pulp. Green coffee beans showed highest concentration of organic acids and sucrose (4.96 ± 0.25 and 5.07 ± 0.39 g/100 g DW for the batch and continuous processing). Batch green coffee beans contained higher amount of phenols. 5-caffeoylquinic Acid (5-CQA) was the main constituent (67.1 and 66.0% for the batch and continuous processing, respectively). Protein content was 15 and 13% in the green coffee bean in batch and continuous processing, respectively. A decrease of 50 to 64% for free amino groups during processing was observed resulting in final amounts of 0.8 to 1.4% in the processed beans. Finally, the batch processing still revealed by-products and wastewater with high nutrient content encouraging a better concept for valorization. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1010 KW - Arabica coffee beans KW - coffee by-products KW - batch process KW - continuous process KW - nutritional characteristics Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-481691 SN - 1866-8372 IS - 1010 ER - TY - GEN A1 - Eggert, Kai A1 - Rawel, Harshadrai Manilal A1 - Pawelzik, Elke T1 - In vitro degradation of wheat gluten fractions by Fusarium graminearum proteases T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - Fusarium spp. infection of cereal grain is a common problem, which leads to a dramatic loss of grain quality. The aim of the present study was to investigate the effect of Fusarium infection on the wheat storage protein gluten and its fractions, the gliadins and glutenins, in an in vitro model system. Gluten proteins were digested by F. graminearum proteases for 2, 4, 8 and 24 h, separated by Osborne fractionation and characterised by chromatographic (RP-HPLC) and electrophoretic analysis (SDS-Page). Gluten digestion by F. graminearum proteases showed in comparison with gliadins a preference for the glutenins whereas the HMW subfraction was at most affected. In comparison with a untreated control, the HMW subfraction was degraded of about 97% after 4 h incubation with Fusarium proteases. Separate digestion of gliadin and glutenin underlined the preference for HMW-GS. Analogue to the observed change in the gluten composition, the yield of the proteins extracted changed. A higher amount of glutenin fragments was found in the gliadin extraction solution after digestion and could mask a gliadin destruction at the same time. This observation can contribute to explain the frequently reported reduced glutenin amount parallel to an increase in gliadin quantity after Fusarium infection in grains. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 877 KW - gluten KW - gliadin and glutenin fractions KW - peptides KW - serine and trypsin protease Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-435102 SN - 1866-8372 IS - 877 SP - 697 EP - 705 ER - TY - GEN A1 - Sagu Tchewonpi, Sorel A1 - Huschek, Gerd A1 - Bönick, Josephine A1 - Homann, Thomas A1 - Rawel, Harshadrai Manilal T1 - 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 T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 805 KW - wheat KW - α-amylase/trypsin inhibitors KW - extraction KW - Plackett–Burman design KW - Doehlert design KW - SDS-PAGE KW - MALDI-TOF/MS KW - LC-MS/MS Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-442229 SN - 1866-8372 IS - 805 ER - TY - GEN A1 - Rawel, Harshadrai Manilal A1 - Huschek, Gerd A1 - Sagu Tchewonpi, Sorel A1 - Homann, Thomas T1 - Cocoa Bean Proteins BT - Characterization, Changes and Modifications due to Ripening and Post-Harvest Processing T2 - Postprints der Universität Potsdam: Mathematisch-Naturwissenschaftliche Reihe N2 - The protein fractions of cocoa have been implicated influencing both the bioactive potential and sensory properties of cocoa and cocoa products. The objective of the present review is to show the impact of different stages of cultivation and processing with regard to the changes induced in the protein fractions. Special focus has been laid on the major seed storage proteins throughout the different stages of processing. The study starts with classical introduction of the extraction and the characterization methods used, while addressing classification approaches of cocoa proteins evolved during the timeline. The changes in protein composition during ripening and maturation of cocoa seeds, together with the possible modifications during the post-harvest processing (fermentation, drying, and roasting), have been documented. Finally, the bioactive potential arising directly or indirectly from cocoa proteins has been elucidated. The “state of the art” suggests that exploration of other potentially bioactive components in cocoa needs to be undertaken, while considering the complexity of reaction products occurring during the roasting phase of the post-harvest processing. Finally, the utilization of partially processed cocoa beans (e.g., fermented, conciliatory thermal treatment) can be recommended, providing a large reservoir of bioactive potentials arising from the protein components that could be instrumented in functionalizing foods. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 681 KW - cocoa processing KW - cocoa proteins KW - classification KW - extraction and characterization methods KW - fermentation-related enzymes KW - bioactive peptides KW - heath potentials KW - protein–phenol interactions Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-425953 SN - 1866-8372 IS - 681 ER -