Enzyme-assisted separation and hydrolysis of gluten : options for intensification

The food industry is one of the largest water consumers in industry. Using large amounts of water, however, is undesirable from an environmental point of view because freshwater is a scarce good in many regions of the world and undesirable from an economic point of view because high water loadings require high amounts of energy for dehydration and signify high amounts of wastewater. This thesis uses wheat, one of the major crops in human nutrition, to study the influence of low water concentrations on two relevant processes in wheat processing: <strong>The separation of starch and gluten</strong>. Separation is often performed using 10–15 L water per kg dry matter. Instead, starch and gluten can be separated by inducing shear using 0.5 L water per kg dry matter. In this thesis we make use of xylanases to hydrolyze arabinoxylan present in wheat, thereby releasing the water associated with arabinoxylan. In doing so, shear-induced starch–gluten separation is performed at even more concentrated conditions. The influence of arabinoxylan hydrolysis in wheat dough at low water contents is studied in chapters 2 and 3.<strong>The hydrolysis of gluten</strong>. Hydrolysis is currently performed using approximately 4 L water per kg dry mater. In this thesis we perform gluten hydrolysis at solid concentrations of up to 70%, thereby investigating the changes in the hydrolysis reaction and the functionality of the resulting hydrolysates. Wheat gluten hydrolysis at low water contents is studied in chapters 4, 5 and 6. This thesis consists of seven chapters. <strong>Chapter 1 </strong>gives a general introduction of the thesis. In <strong>chapter 2</strong>, wheat dough rheology at low water contents below 40% and the influence of xylanases is studied. A reduction in water content from 43.5–44.8% (representing optimal Farinograph water absorption) to 34% (the lowest water content where a dough forms) results in a non-linear increase in the dough consistency, elastic modulus <em>G</em>’, and a decrease in the maximum creep compliance <em>Jc,max </em>of 1–2 orders of magnitude. Addition of xylanases has the same effect on the dough consistency, <em>G</em>’ and <em>Jc,max </em>as an increase in water content of 2–5% (on a water basis). <em>Tan </em><em>δ</em> is hardly and <em>Jel </em>not influenced by xylanase addition showing that the influence of xylanases on the mechanism of hydration is negligible. In <strong>chapter 3</strong>, shear-induced starch–gluten separation with the help of xylanases is studied at water contents from 43.5% to 34%. Addition of xylanases at the standard water content of 43.5% results in a slurry without any separation. As a result, lower water contents are used. At water contents below 40%, the local formation of gluten clusters is observed with and without xylanases addition. However, opposed to shear-induced separation at 43.5% water without xylanase, the gluten patches do not migrate to the center of the cone because of the densely packed dough and an inhomogeneity in the shear field. Nevertheless, gluten clusters can be concentrated up to 60% (N×5.7) protein. Similar to chapter 2, xylanase addition allows water savings of 3–5% (on a water basis). <strong>Chapter 4 </strong>introduces enzymatic wheat gluten hydrolysis at high solid concentrations and describes the influence of high-solid hydrolysis on the resulting functional properties of the gluten hydrolysates. Wheat gluten can be hydrolyzed at solid concentrations of up to 60% (w/w). The water solubility of the dried hydrolysates is independent of the solid concentration during hydrolysis, just like the foam stabilizing properties at degrees of hydrolysis (DH%) below 8% At DH% above 8%, high solid concentrations even increase the foam stabilizing properties of the resulting hydrolysates, which is related to the presence of more peptides with a molecular mass >25 kDa. Furthermore, an increase in solid concentration results in an increase of the volumetric productivity. Despite the advantages of high-solid gluten hydrolysis, we also observe lower hydrolysis rates in high-solid gluten hydrolysis compared to low-solid gluten hydrolysis at constant enzyme-to-substrate ratios. The factors causing this hydrolysis rate limitation are investigated in <strong>chapter 5</strong>. It is shown that enzyme inhibition, the water activity, and mass transfer limitations do not impede the hydrolysis up to 50% solids. However, the hydrolysis rate limitation can be explained by a second-order enzyme auto-inactivation rate along with the higher enzyme concentrations used. At solid concentrations above 50%, the hydrolysis rate further decreases due to mass transfer limitations. Furthermore, the addition of enzyme after 24 h at high solid concentrations hardly increases the DH%, suggesting that the maximum attainable DH% decreases at high solid concentrations. This DH% limitation is explained by a reduced enzyme activity due to a decline in water activity. Based on the findings in chapters 4 and 5, a direct hydrolysis of gluten present in wheat flour at high solid concentrations is investigated in <strong>chapter 6</strong>, thereby omitting the starch–gluten separation. At a constant protein concentration, the protease activity is higher for wheat flour hydrolysis (at 40% total solids) than for vital wheat gluten hydrolysis (at 7.2% total solids) in the initial 6 h of hydrolysis, despite the high starch content in wheat flour and consequently lower water content. This is related to the starch granules in wheat flour, preventing the aggregation of (native) gluten. At wheat flour concentrations above 50% and for longer reaction times the positive effect of starch disappears. This is explained by mass transfer limitations and reduced water activities in the wheat flour slurry or dough, respectively. <strong>Chapter 7 </strong>summarizes and generalizes the main findings of this thesis and compares the current status in starch–gluten separation and gluten hydrolysis with the concentrated separation and hydrolysis processes developed in this study. Water and energy savings of at least 50% are possible when separating and hydrolyzing at concentrated conditions. In the end, future prospects in high-solid wheat gluten hydrolysis are briefly discussed.