The thermo-active serine peptidase aqualysin 1 (Aq1) of Thermus aquaticus was applied in bread making to study the relative contribution of thermoset gluten to bread crumb texture. Aq1 is active between 30 °C and 90 °C with an optimum activity temperature of around 65 °C. It is inhibited by wheat endogenous serine peptidase inhibitors during dough mixing and fermentation and starts hydrolyzing gluten proteins during baking above 80 °C when the enzyme is no longer inhibited and most of the starch is gelatinized and contributes to structure formation. Aq1 activity reduced the molecular weight of gluten proteins and significantly increased their extractability in sodium dodecyl sulfate containing medium. While it had no impact on the specific bread volume and only limited impact on hardness, cohesiveness, springiness, resilience and chewiness, it impacted bread crumb coherence. We conclude that starch has a greater impact on crumb texture than thermoset gluten.

Although the impact of heat on molecular properties of wheat gluten is well understood, changes in its microstructure have rarely been studied. Here, formation of the thermoset gluten network in a model system relevant for bread baking was studied with confocal laser scanning microscopy and protein network analysis. From 65 °C onwards, gluten converts from thick aligned protein strands in a highly branched and homogeneous network of small thin protein threads. Neither gliadin incorporation in the network nor application of aqualysin 1, the thermo-active serine peptidase from Thermus aquaticus which recently has been reported to hydrolyze gluten proteins in dough only at temperatures exceeding 80 °C, impacts on the gluten microstructure. As starch causes structure setting itself and thereby decreases protein mobility, molecular scale changes in the gluten network at temperatures exceeding 80 °C brought about by aqualysin 1 do not impact its microstructure.

The impact of Aqualysin 1 (Aq1), the thermo-active peptidase of Thermus aquaticus, on wheat albumin, globulin, gliadin and glutenin proteins during heat treatment of wheat dough and bread baking was examined. The level of protein extractable in sodium dodecyl sulfate containing medium under non-reducing conditions (SDS-EP-NR) from wheat dough decreases upon heating to a lesser extent when Aq1 is used than in control experiments. The higher SDS-EP-NR level is caused by the release by Aq1 of peptides from the repetitive gluten protein domains during baking. These peptides are also extractable from bread crumb with salt solution. The resultant thermoset gluten network in bread crumb is mainly made up by protein from non-repetitive gluten domains.

Background and objectives Wheat flour proteins inhibit the thermo‐active serine peptidase aqualysin 1 (Aq1) from Thermus aquaticus in a temperature‐dependent way. The impact of aqueous flour extracts (FEs) from maize, barley, or rice on Aq1 activity both in an assay and in a gluten‐starch model system (GSMS) for bread making was investigated. Findings The degree of Aq1 inhibition by maize and barley FEs decreased at temperatures exceeding 50°C. With higher levels of FEs, higher temperatures were required to undo inhibition. Rice FEs did not inhibit Aq1. Gluten hydrolysis started at lower temperatures in GSMS containing maize FE rather than wheat FE. Incubating Aq1 with maize FE prior to inclusion in GSMS containing wheat FE decreased the onset temperature of Aq1 activity. Conclusions In presence of their FEs, the onset and optimal temperatures for Aq1 activity increased in the order maize, wheat, and barley. When Aq1 was incubated with maize inhibitors prior to dough making, wheat inhibitors still impacted Aq1 activity. Significance and novelty That maize and barley contain inhibitors inactivating Aq1 in a heat‐sensitive way different from that exerted by those in wheat is novel and relevant in the context of the production of multigrain bread products.