LC-MS/MS quantitation of α-amylase/trypsin inhibitor CM3 and glutathione during wheat sourdough breadmaking

Abstract

Aims: This study aimed to quantify α-amylase/trypsin inhibitor (ATI) CM3 and glutathione (GSH) during wheat sourdough breadmaking.

Methods and results: Breads were made with two wheat cultivars and fermented with Fructilactobacillus sanfranciscensis, F. sanfranciscensis ΔgshR or Latilactobacillus sakei; chemically acidified and straight doughs served as controls. Samples were analysed after mixing, after proofing and after baking. GSH and CM3 were quantified by multi-reaction-monitoring-based methods on an LC-QTRAP mass spectrometer. Undigested ATI extracts were further examined by SDS-PAGE.

Conclusions: GSH abundance was similar after mixing and after proofing but increased after baking (p < 0.001), regardless of fermentation. In breads baked with cv. Brennan, the samples fermented with lactobacilli had higher GSH abundance (p < 0.001) than in the controls. CM3 relative abundance remained similar after mixing and after proofing but decreased after baking (p < 0.001) across all treatments. This trend was supported by the SDS-PAGE analysis in which ATI band intensities decreased after baking (p < 0.001) in all experimental conditions. The overall effect of baking exerted a greater effect on the abundances of GSH and CM3 than fermentation conditions.

Significance and impact of the study: This is the first report to quantify ATI over the course of breadmaking by LC-MS/MS in sourdough and straight dough processes.

Keywords: Fructilactobacillus sanfranciscensis; LC-MS/MS; alpha-amylase/trypsin inhibitor; glutathione; lactic acid bacteria; non-celiac wheat sensitivity; sourdough; wheat allergens.