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Modern high-speed mechanical dough development (MDD) alters the gluten macropolymer's (GMP) structure. Changes to both the protein and food matrix structure can influence protein digestibility and immunogenicity. This study investigated the relationship between protein structural changes imparted by MDD and gluten's digestibility plus celiac reactivity. Dough was prepared at three mixing speeds (63 rpm, 120 rpm and 200 rpm) to different degrees of development (between 10 and 180% wh.kg -1 ). Protein structural changes were characterised by confocal microscopy, free thiol determination and protein extractability assays. MDD altered the structure of gluten within bread, changing the protein's surface area and macrostructure. Breads were digested using the INFOGEST in vitro protocol. Gluten's antigenicity and digestibility were monitored using ELISA and mass spectrometry, by monitoring the concentration of six immunogenic peptides causative of celiac disease. The structural changes imparted by mixing did not affect bread's digestibility or celiac reactivity., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Microbial transglutaminase (mTG) catalyses the formation of protein crosslinks, deamidating glutamine in a side-reaction. Gluten deamidation by human tissue transglutaminase is critical to activate celiac disease pathogenesis making the addition of mTG to wheat-based products controversial. The ability of mTG (0-2000 U.kg -1 ) to alter gluten's structure, digestibility and the deamidation state of six immunogenic gluten peptides within bread was investigated. Gluten's structure was altered when mTG exceeded 100 U.kg -1 , determined by confocal microscopy, extractability and free sulfhydryl assays. The effect of mTG on six immunogenic peptides was investigated by in vitro digestion (INFOGEST) and mass spectrometry. The addition of mTG to bread (0-2000 U.kg -1 ) did not alter the deamidation state or digestibility of the immunogenic peptides investigated. Overall, this investigation indicated that the addition of mTG to bread does not create activated gluten peptides. This analysis provides evidence for risk assessments of mTG as a food processing aid., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Previous work has shown that baking induces structural changes within the gluten macropolymer (GMP) that reduce gluten protein digestibility. The precise nature of these structural changes within dough/bread, and how they alter the in vitro release profile of immunogenic gluten peptides that activate celiac disease is unknown. This work examined the effect of dough baking temperature and duration on the GMP's structure and the release profile of immunogenic gluten peptides. Dough was baked at either 150 °C or 230 °C for 25, 35 or 45 min. The structure of the GMP within the resulting loaves was defined and compared using confocal microscopy, quantitative protein network analysis, gliadin protein extractability (HPLC) and determination of the free thiol content. Both bread and dough were digested in vitro (INFOGEST) and the release profile of six immunogenic gluten peptides (including the immunodominant 33mer) defined using quantitative mass spectrometry. Higher baking temperatures and longer durations increased the degree of intermolecular disulfide bonds between the sulfur-rich gliadins and GMP backbone. The thermal load did not alter the GMP macrostructure, but significant differences between bread and dough were observed. Baking altered the concentration and release profile of the immunogenic gluten peptides throughout in vitro digestion causing the digestion of immunogenic gluten peptides differed between raw and heat-treated bread., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Celiac disease is an autoimmune illness activated by gluten peptides produced during gastrointestinal digestion. A simulated in vitro digestion of gluten was conducted to define the profile and kinetic release pattern of immunogenic gluten peptides in a physiologically relevant food matrix. White bread was digested using the INFOGEST in vitro standardised digestion protocol from 0 to 240 min and subsequently analysed by SDS-PAGE, quantitative LC-MS/MS, untargeted LC-MS/MS and ELISA. The release profile of six gluten peptides was defined by quantitative LC-MS/MS; none were detected in the gastric phase, but rapidly peaked in the intestinal phase. These results were corroborated by the ELISA analysis. Untargeted proteomics identified 83 immunogenic peptides. Their qualitative concentrations were defined throughout digestion, demonstrating complex relationships through proteolysis. This analysis suggests immunogenic gluten may peak within the intestinal duodenum and gives new insights into the complexity of gluten digestion from a physiologically relevant food matrix., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)