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Abstract:

Wheat bread is an important staple food for a significant part of the world population. Its recipe contains the essential ingredients wheat flour, water, yeast and salt, and optionally also some non-essential ingredients. Straight-dough wheat bread making, the most commonly used bread making process, starts by mixing the ingredients to form visco-elastic dough. During dough mixing, the wheat gluten proteins, which consist of glutenins and gliadins, form a continuous network which relies on non-covalent interactions and covalent bonds. During dough mixing, air, and thus also molecular oxygen (O2), is incorporated. The latter is involved in several reactions including the oxidation of sulfhydryl (SH) groups into disulfide (SS) bonds. The gluten network allows wheat flour dough to retain gas during fermentation and the early phases of baking. During further baking, the gas cells are opened up which results in bread with a typical spongy crumb structure. Chemical redox agents are often part of the recipe as non-essential ingredients to optimize wheat flour bread making performance or to overcome deficiencies in wheat flour quality. Ascorbic acid (AH2) and azodicarbonamide (ADA) are two important components in this context. During dough mixing, AH2 is oxidized by O2 into dehydroascorbic acid (DHA) through action of AH2 oxidase. However, the O2 in dough necessary for DHA formation is rapidly consumed by yeast and in reactions catalyzed by enzymes present in wheat flour. DHA oxidizes glutathione (GSH) in the presence of GSH dehydrogenase to its dimer GSSG. Otherwise, GSH would react with SS bonds of the glutenin component of wheat gluten proteins through SH/SS exchange reactions which would depolymerize glutenin to some extent resulting in dough weakening and low bread quality. It has been suggested that ADA rapidly oxidizes free SH groups of proteins during dough mixing resulting in additional SS bonds between and within glutenin proteins. The bread volume enhancing effect of ADA has mainly been linked to a reduction in the amount of work input needed during mixing. A major drawback of the use of chemical agents in bread making is that, in most territories where its use is allowed, they need to be listed on labels. In order to develop clean label alternatives, it is essential to fully understand their mechanisms of action. Against this background, the main objective of this doctoral dissertation was to study the mechanisms of action of AH2 and ADA during wheat bread making. In the first part of this doctoral dissertation, the impact of AH2 and ADA on dough and bread properties on both molecular and macroscopic scale was examined using a multitude of (novel) techniques. The use of AH2 seemed to prevent the partial gluten depolymerization by oxidizing GSH which is only present in dough in small quantities. Furthermore, AH2 probably not only impacted the level of gluten SS bonds, but also (indirectly) the non-covalent interactions. ADA resulted in a higher degree of glutenin polymerization by oxidizing free SH groups and better stabilizing dough during fermentation. The higher degree of glutenin polymerization did not always result in a higher bread volume. When ADA was overdosed, an over-tenacious dough was formed leading to poor gas retention properties and a lower bread volume. As the amount of free SH groups in the glutenin fraction was lower when ADA was used, less heat-induced SH/SS exchange reactions took place during baking. As a consequence, the level of gliadin proteins incorporated in the glutenin network during baking was much lower. The effect of yeast and varying mixing times on the dough strengthening effects of AH2 and ADA was studied in the second part of this work. Under the experimental conditions, yeast did not have a detrimental effect on the dough strengthening effect of AH2. This was rather unexpected as yeast addition increases the GSH level in dough and consumes a considerable amount of the available O2. The very small levels of GSH associated with compressed yeast may swiftly have led to some gluten depolymerization. This may have resulted in conformational changes making previously buried, yet rheologically important SH groups available for oxidation and/or SH/SS exchange reactions at a later stage. These results show that yeast and the amount of GSH associated with it, are important determinants for the state of the redox couples present in wheat flour-based bread dough and, thus, influence the efficiency of the dough strengthening effect of AH2. The dough strengthening effect of ADA was smaller than that of AH2, which is partly due to the formation of ADA's reaction product biurea. Varying mixing times also impacted the dough strengthening effect of ADA. Higher levels of ADA were needed when shorter mixing times were applied. This may be related to the amount of O2 incorporated during dough mixing. To study the influence of varying levels of O2 on the functionality of AH2 and ADA, the quantity of incorporated O2 was altered by: (i) mixing during a fixed time with gas mixtures containing different percentages of O2 and (ii) mixing for different timings under normal atmospheric conditions. AH2 first needs to be oxidized to DHA before the latter can exhibit the actual improving effect. However, under reduced O2 conditions, the use of AH2 still resulted in an increased bread volume. When AH2 was added to a dough prepared under O2 enriched conditions, no additional impact was observed. These findings suggest that AH2 oxidase is very effective in using O2 to form DHA. Under normal conditions, ADA use resulted in a bread volume enhancing effect up to a certain dosage. When the dosage was further increased, bread specific volume became lower due to the formation of an over-tenacious dough. When more O2 was incorporated into the dough, lower levels of ADA were needed to obtain its maximal bread volume enhancing effect. Conversely, to obtain maximal bread volume, higher ADA levels were needed when dough was prepared under O2 limited conditions. It is concluded that O2 and ADA at least partly oxidize the same substrates and that higher O2 incorporation results in lower needs of ADA and vice versa. Next, the impact of simultaneous use of AH2 and ADA on dough and bread properties was studied using regression analyses predicting the optimal conditions of AH2 level, ADA level and mixing time based on response surface models. No positive interaction effects were observed in any of the obtained models, indicating that AH2 and ADA do not act synergistically when used simultaneously in a dough recipe. However, to reach the maximal dough strength and bread volume, both agents should be included according to the estimate response surface models. They thus exhibit their dough strengthening and bread volume enhancing effect independently from each other. While AH2 prevents partial depolymerization of glutenin by oxidizing GSH, ADA directly oxidizes free SH groups of glutenin resulting in a higher degree of SS bonds between glutenin chains. The models predicted that, when simultaneously used, both agents in the tested concentration ranges counteract each other as evidenced by the dough stability parameters. Lastly, the conversion and degradation of AH2 during wheat bread making were studied. The chemical and enzymatic interconversion of AH2 and bicyclic hydrated DHA (DHA*), and the hydrolysis of the latter into 2,3-diketogulonic acid (DKGA) were monitored over time using 13C nuclear magnetic resonance (NMR) spectroscopy, thereby simultaneously detecting all three components. Aqueous model systems of pure water, tap water and (heat-treated) wheat flour extract to which 13C-labeled AH2 was added, were analyzed. AH2 oxidation by O2 is accelerated by metal ions and wheat endogenous enzymes. GSH stabilizes AH2 added to a wheat flour extract by: (i) limiting the extent to which it is oxidized and (ii) (partly) regenerating AH2 from DHA*. The latter is also partly hydrolyzed to DKGA. The degradation of AH2 was further investigated during dough fermentation and bread baking using radioactive 14C1-labeled AH2 in a bread dough recipe. The loss of C1-atoms as carbon dioxide (CO2) was measured. While there was no loss of C1-atoms from AH2 during dough mixing and fermentation, 14C-labeled CO2 was formed from AH2 during baking suggesting that its degradation is mainly thermally driven. The extent of CO2 loss by AH2 degradation was found to depend on the levels added with higher dosages of AH2 resulting in lower relative levels losing their C1-atom. It is concluded that the level of AH2 added in a bread making recipe has a large impact on the degree of degradation, possibly due to the limited quantity of O2 available to convert AH2 into DHA. In general, it is concluded that a correct redox state of wheat dough systems is important for obtaining optimal dough and bread properties. This redox state depends on the amount of O2 incorporated into the dough, the amount and type of yeast, and the dough GSH content. These variables evidently also influence the efficiency of the dough strengthening and bread volume enhancing capacity of the chemical agents AH2 and ADA.