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: The separation of starch and gluten. 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.The hydrolysis of gluten. 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. Chapter 1 gives a general introduction of the thesis. In chapter 2, 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 G’, and a decrease in the maximum creep compliance Jc,max of 1–2 orders of magnitude. Addition of xylanases has the same effect on the dough consistency, G’ and Jc,max as an increase in water content of 2–5% (on a water basis). Tan δ is hardly and Jel not influenced by xylanase addition showing that the influence of xylanases on the mechanism of hydration is negligible. In chapter 3, 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). Chapter 4 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 chapter 5. 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 chapter 6, 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. Chapter 7 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.</p

Starch facilitates enzymatic wheat gluten hydrolysis

Wheat gluten can be hydrolyzed by either using (vital) wheat gluten or directly from wheat flour. This study investigates the influence of the presence of starch, the main component of wheat, on enzymatic wheat gluten hydrolysis. Wheat gluten present in wheat flour (WFG) and vital wheat gluten (VWG) were hydrolyzed at constant protein concentrations, but subsequently 5.6 times higher amounts of wheat flour. Nevertheless, WFG hydrolysis at 40% total solids resulted in significantly higher degrees of hydrolysis (DH%) than VWG hydrolysis at 7.2% solids. This difference increased to up to 4.5% in 6 h and diminished again for longer reaction times. Possible differences in the gluten composition and the presence of albumins and globulins in wheat flour could not explain the difference in DH% because the addition of starch to VWG increased the rate of hydrolysis similarly. Instead, it was concluded that starch granules impede gluten aggregation, which facilitates the hydrolysis. At higher solid concentrations of up to 70% wheat flour, the positive effect of starch disappeared, because WFG hydrolysis was hindered by mass transfer limitations and lower water activities

Wheat dough rheology at low water contents and the influence of xylanases

The effect of low water contents and xylanases on wheat dough rheology is reported. Farinograph, dynamic oscillation, and creep-recovery measurements were performed using water concentrations from 34 to 44.8% (total basis). A water reduction from 43.5–44.8% to 34% increased resistance upon mixing as evidenced by higher Farinograph Brabender Unit values, increased G' and G¿-values, and decreased the maximum creep compliance by 1–2 orders of magnitude. Addition of an endoxylanase with a higher selectivity for water-unextractable arabinoxylans and an endoxylanase with a higher selectivity for water-extractable arabinoxylans both resulted in lower G' and Farinograph dough consistencies and an increase in maximum creep compliance. The major influence of both xylanases was the release of water with possible water reductions of 2–5% (on water basis), though no distinct differences between the two xylanases were observed

Influence of high solid concentrations on enzymatic wheat gluten hydrolysis and resulting functional properties

Enzymatic hydrolysis at increased solid concentrations is beneficial with regard to energy and water consumption. This study examines the influence of the solid concentration on the enzymatic hydrolysis of wheat gluten and the resulting functional properties of the hydrolysate. Wheat gluten was mildly hydrolyzed at a solid concentration varying from 10% to 60% to degrees of hydrolysis (DH%) ranging from 3.2% to 10.2%. The gluten was susceptible to hydrolysis at all solid concentrations but the hydrolysis rate was influenced by increasing solid concentrations. Size-exclusion high-performance liquid chromatography revealed an increase in the ratio of peptides with a molecular mass >25 kDa for solid concentrations of 40% and 60%. The water solubility increased on hydrolysis and was independent of the solid concentration during proteolysis. The foam stability was not influenced by the solid concentration at low DH%. At DH% higher than 8%, high solid concentrations increased the foam stability, which might be related to the presence of more peptides with a molecular mass >25 kDa. In addition, we found increased reactor productivity. The results show the potential of hydrolyzing wheat gluten at high solid concentrations, which could lead to large savings for water and energy when applied industrially

Wheat dough rheology at low water contents and the influence of xylanases

The effect of low water contents and xylanases on wheat dough rheology is reported. Farinograph, dynamic oscillation, and creep-recovery measurements were performed using water concentrations from 34 to 44.8% (total basis). A water reduction from 43.5–44.8% to 34% increased resistance upon mixing as evidenced by higher Farinograph Brabender Unit values, increased G' and G¿-values, and decreased the maximum creep compliance by 1–2 orders of magnitude. Addition of an endoxylanase with a higher selectivity for water-unextractable arabinoxylans and an endoxylanase with a higher selectivity for water-extractable arabinoxylans both resulted in lower G' and Farinograph dough consistencies and an increase in maximum creep compliance. The major influence of both xylanases was the release of water with possible water reductions of 2–5% (on water basis), though no distinct differences between the two xylanases were observed

Starch facilitates enzymatic wheat gluten hydrolysis

Wheat gluten can be hydrolyzed by either using (vital) wheat gluten or directly from wheat flour. This study investigates the influence of the presence of starch, the main component of wheat, on enzymatic wheat gluten hydrolysis. Wheat gluten present in wheat flour (WFG) and vital wheat gluten (VWG) were hydrolyzed at constant protein concentrations, but subsequently 5.6 times higher amounts of wheat flour. Nevertheless, WFG hydrolysis at 40% total solids resulted in significantly higher degrees of hydrolysis (DH%) than VWG hydrolysis at 7.2% solids. This difference increased to up to 4.5% in 6 h and diminished again for longer reaction times. Possible differences in the gluten composition and the presence of albumins and globulins in wheat flour could not explain the difference in DH% because the addition of starch to VWG increased the rate of hydrolysis similarly. Instead, it was concluded that starch granules impede gluten aggregation, which facilitates the hydrolysis. At higher solid concentrations of up to 70% wheat flour, the positive effect of starch disappeared, because WFG hydrolysis was hindered by mass transfer limitations and lower water activities

Shear-induced starch-gluten separation at very low water content aided by xylanases

This study examines the influence of extremely low water content on shear-induced starch–gluten separation and how endoxylanases influence the separation by releasing water associated with arabinoxylan. Shearing was performed at a water content ranging from 34% to 43.5% (w/w). It was possible to concentrate gluten to 60% protein content in local gluten clusters and most of the concentration occurred within 5 min. Contrary to higher water concentrations, a water conten

Formulierung in festen Matrices: Dispergieren von Öltropfen in plastifizierten Stärkenmatrices

Emulgieren ist ein Fertigungsprozess, der aus der Lebensmittel-, Pharma- und Kosmetikindustrie nicht wegzudenken ist. Mit Hilfe moderner Emulgiertechniken lassen sich eine Vielzahl von Produkten herstellen. Die Neuauflage des Werkes „Emulgiertechnik" liefert hierfür das notwendige Wissen. Emulgierverfahren, aber auch die technologischen Grundlagen, werden anschaulich und praxisnah erläutert

Factors Impeding Enzymatic Wheat Gluten Hydrolysis at High Solid Concentrations

Enzymatic wheat gluten hydrolysis at high solid concentrations is advantageous from an environmental and economic point of view. However, increased wheat gluten concentrations result in a concentration effect with a decreased hydrolysis rate at constant enzyme-to-substrate ratios and a decreased maximum attainable degree of hydrolysis (DH%). We here identified the underlying factors causing the concentration effect. Wheat gluten was hydrolyzed at solid concentrations from 4.4% to 70%. The decreased hydrolysis rate was present at all solid concentrations and at any time of the reaction. Mass transfer limitations, enzyme inhibition and water activity were shown to not cause this hydrolysis rate limitation up to 50% solids. However, the hydrolysis rate limitation can be, at least partly, explained by a second-order enzyme inactivation process. Furthermore, mass transfer impeded the hydrolysis above 60% solids. Addition of enzyme after 24 h at high solid concentrations scarcely increased the DH%, suggesting that the maximum attainable DH% decreases at high solid concentrations. Reduced enzyme activities caused by low water activities can explain this DH% limitation. Finally, a possible influence of the plastein reaction on the DH% limitation is discussed

Formation of oil droplets in plasticized starch matrix in simple shear flow

This paper describes the effect of simple shear flow on the formation of triglyceride oil droplets in a plasticized starch matrix. An in-house developed shearing device was used that enabled the application of controlled shear flow and rheological characterization of the native maize starch–triglyceride blends at shear stresses of up to 37 kPa. Due to the high viscosity of starch matrix, the viscosity ratio of the continuous starch phase and the dispersed triglyceride phase varied between 10-7 and 10-5. It was possible to create small droplets with a droplet diameter of 2.1 µm using simple shear flow only. An increase in shear rate had no influence on droplet diameter. However, an increase in oil content led to a vast increase in droplet diameter indicating the occurrence of coalescence. The results further show that the maximum stable droplet size in plasticized starch is significantly smaller (up to 100 times) than the predicted values for a Newtonian matrix. The differences of plasticized starch to Newtonian matrices are discussed in detail