Digestibility of gluten proteins is reduced by baking and enhanced by starch digestion

Frances Smith, Xiaoyan Pan, Vincent Bellido, Geraldine A Toole, Fred K Gates, Martin S J Wickham, Peter R Shewry, Serafim Bakalis, Philip Padfield, E N Clare Mills, Frances Smith, Xiaoyan Pan, Vincent Bellido, Geraldine A Toole, Fred K Gates, Martin S J Wickham, Peter R Shewry, Serafim Bakalis, Philip Padfield, E N Clare Mills

Abstract

Scope: Resistance of proteins to gastrointestinal digestion may play a role in determining immune-mediated adverse reactions to foods. However, digestion studies have largely been restricted to purified proteins and the impact of food processing and food matrices on protein digestibility is poorly understood.

Methods and results: Digestibility of a total gliadin fraction (TGF), flour (cv Hereward), and bread was assessed using in vitro batch digestion with simulated oral, gastric, and duodenal phases. Protein digestion was monitored by SDS-PAGE and immunoblotting using monoclonal antibodies specific for celiac-toxic sequences (QQSF, QPFP) and starch digestion by measuring undigested starch. Whereas the TGF was rapidly digested during the gastric phase the gluten proteins in bread were virtually undigested and digested rapidly during the duodenal phase only if amylase was included. Duodenal starch digestion was also slower in the absence of duodenal proteases.

Conclusion: The baking process reduces the digestibility of wheat gluten proteins, including those containing sequences active in celiac disease. Starch digestion affects the extent of protein digestion, probably because of gluten-starch complex formation during baking. Digestion studies using purified protein fractions alone are therefore not predictive of digestion in complex food matrices.

Keywords: Allergen; Baking; Celiac; Digestion; Gluten.

© 2015 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1
Figure 1
Simulated gastric digestion of a total gliadin fraction from wheat undertaken using a low pepsin protocol. Soluble (A, C, E) and insoluble (B, D, F) fractions of digests analysed by SDS‐PAGE (A, B), immunoblotting (C–F) using mAbs IFRN 0610 (C, D; brightened by + 40%) and 0065 (E, F). Selected bands (arrowed) were subjected to densitometric analysis (Supporting Information Table 3) and used for kinetic analysis. Soluble protein volumes loaded accounted for dilution factor between digestive phases whereas extracted insoluble protein samples were loaded in equivalent volumes. Pepsin was identified on the basis of its Mr defined by SDS PAGE analysis of the digestion enzymes (data not shown).
Figure 2
Figure 2
Simulated oral‐gastric digestion of wheat flour protein undertaken using a low pepsin protocol. Soluble (A, C) and insoluble (B, D) fractions of digests were analyzed by SDS‐PAGE (A, B) and immunoblotting (C, D) using mAb IFRN 0610 (C, D; brightened by + 40%). Selected bands (arrowed) were subjected to densitometric analysis (Supporting Information Table 3) and used for kinetic analysis. Soluble protein volumes loaded accounted for dilution factor between digestive phases whereas extracted insoluble protein samples were loaded in equivalent volumes. Pepsin was identified on the basis of its Mr defined by SDS PAGE analysis of the digestion enzymes (data not shown).
Figure 3
Figure 3
Simulated oral‐gastric digestion of bread protein undertaken using a low pepsin protocol. Soluble (A, C) and insoluble (B, D) fractions of digests were analyzed by SDS‐PAGE (A, B) and immunoblotting (C, D) using mAb IFRN 0610 (C, D brightened by + 40%). Selected bands (arrowed) were subjected to densitometric analysis (Supporting Information Table 3) and used for kinetic analysis. Soluble protein volumes loaded accounted for dilution factor between digestive phases whereas extracted insoluble protein samples were loaded in equivalent volumes.
Figure 4
Figure 4
Kinetics of selected prolamins under different digestion conditions. Kinetics (rate constant k (min−1) estimated from the fitting of an exponential curve) of the disappearance of substrate protein or appearance of product at “high” or “low” pepsin: protein ratios. Prolamins were reactive with mAb 0610 or 0065, present in digest immunoblots of matrices total gliadin fraction (TGF), flour or bread. (A) Kinetic analysis example from flour digestion, Mr 44 kDa insoluble substrate, k = 25.66 × 10−3min−1. (B) Kinetic analysis example from bread digestion (low pepsin), Mr 20 kDa soluble product, k = 37.09 × 10−3min−1. (C) Scatter plot of combined kinetic analysis for digests.
Figure 5
Figure 5
Simulated gastro‐duodenal digestion of protein from bread. SDS‐PAGE (A–D) and immunoblots (E–H) from oral, gastric, and duodenal/intestinal digestion of bread after 11 min low‐pepsin gastric digestion. Performed without (A, B, E, F) or with (C, D, G, H) HSA and PMSF‐inhibited pancreatic amylase present. Soluble (A, C, E, G) and insoluble (B, D, F, H) fractions were recovered by centrifugation. Immunoblots developed using mAb 0610 and brightened up to + 40%. Soluble protein volumes loaded accounted for dilution factor between digestive phases whereas extracted insoluble protein samples were loaded in equivalent volumes. Pancreatic α‐amylase, chymotrypsin, and trypsin were identified on the basis of its Mr defined by SDS PAGE analysis of the digestion enzymes (data not shown).
Figure 6
Figure 6
Breakdown of starch in bread samples during simulated gastro‐duodenal digestion. Insoluble starch as % dry matter at different time points of digestion. “0” refers to “chew” sample, with time points onwards as 0.3, 11 min gastric digestion and duodenal time points. Two digestion conditions plotted: with amylases only with amylases and proteases present. *Statistically significant data points (G11D0.3 p < 0.001, G11D5 p < 0.01). Bars show standard error of the mean.

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