A lipase-based approach to the functionality of wheat (Triticum aestivum L.) endogenous lipids and puroindolines in wheat bread (dough) making.

Bread is a staple food in many parts of the world. It provides consumers with energy, dietary fiber, vitamins and minerals. Evidently, consumers expect bread to be of high sensory quality. While lipids in wheat flour are only present in low levels (2.0 – 3.0%), they tremendously affect bread loaf volume and crumb structure. Lipases are lipid degrading enzymes catalyzing the hydrolysis of (phosphodi)ester bonds of glycero(phospho)lipids. They are also excellent research tools since they allow for selective modification of wheat endogenous lipids without affecting other flour constituents. Some researchers have successfully applied lipases to study the role of wheat endogenous lipids in bread making. While the impacts of different wheat flour lipids on loaf volume and crumb structure are quite well established, information on the functional effects of enzymatically released hydrolysis products in bread making is still very scarce. Furthermore, in depth understanding of possible synergistic effects and appropriate balances between wheat endogenous lipids and their enzymatically released hydrolysis products is missing.

The impact of wheat flour lipids (and their enzymatically released hydrolysis products) on bread loaf volume and crumb structure is largely if not entirely related to the stability of gas cells in bread dough. It has been proposed that they have an indirect impact on gas cell stability by affecting the strength of the gluten-starch matrix, i.e. the primary support of expanding gas cells in wheat flour dough. In addition, lipids have been considered to have a direct impact on gas cell stability by being present as surface-active agents in the liquid films which surround expanding gas cells in dough and provide a secondary stabilization mechanism. Observations that flour lipids and lipases have no significant impact on dough bulk rheology have contributed to the current belief that the impact of wheat endogenous lipids and their enzymatically released hydrolysis products is exclusively related to their direct action as surface-active components. This is supported by observations that the effects of lipids on loaf volume and crumb structure broadly match their theoretical ability to (de)stabilize gas/liquid interfaces. While such observations indicate a direct impact of lipids on gas cell stability, the presence and action of such compounds at gas cell interfaces in dough has never been experimentally proven.

Against this background, this doctoral dissertation aimed to unravel the role of wheat endogenous lipids and their enzymatically released hydrolysis products in bread making. Hereto, several lipases were used as research tools in bread making with several flour samples.

In this dissertation, lipase-induced changes in the dough lipid population were related to those in bread loaf volume. This provided insights in the impact that wheat endogenous lipids and their enzymatically released hydrolysis products have on bread quality. It was found that the conversion of endogenously present lipids into their corresponding lysolipids has a positive effect on loaf volume and crumb structure. Such conversion however unavoidably releases free fatty acids which have a deleterious effect on bread quality. The positive effect of lipases in bread making is thus restricted by the unavoidable release of detrimental free fatty acids. When appropriate lipase levels are applied in bread making, the detrimental effects of free fatty acids are overruled by the more outspoken beneficial effects of conversion of endogenous lipids into their corresponding lysolipids. At such levels, improved loaf volumes and crumb structures are obtained. An appropriate balance between different types of lipids is thus crucial in bread making. The impact on bread loaf volume as a function of lipase level from a qualitative viewpoint is the same for all lipases applied in this dissertation.

The quantitative impact of enzymatically altering lipids on loaf volume did not only depend on which lipase but also on which flour was used in bread making. It was hypothesized that puroindolines modify lipase impact on loaf volume in bread making. Although it was clear that puroindolines neither restrict the availability of lipids as a substrate for lipases (due to their lipid-binding properties) nor act as lipase inhibitors in bread making, this hypothesis could not be further explored. It thus remains unclear whether differences in flour puroindoline level and/or type lie at the basis of flour dependent lipase impact on loaf volume. It would seem that differences in flour lipid level and/or composition would also be important in this context. However, natural variations in flour triacylglycerol levels are likely not at the basis of optimal lipase levels (strongly) depending on the flour used because inclusion of different oils up to a level of 1.0% did not notably affect the optimal lipase level in bread making.

In this dissertation, a mechanism whereby wheat endogenous lipids and their enzymatically released hydrolysis products affect bread quality is proposed. It entirely relies on the ability of lipids to directly stabilize gas cells in dough by aligning at gas/liquid interfaces. In our view, optimal gas cell stability is achieved when the level of lipids promoting lamellar mesophases, yielding stable condensed monolayers, is maximal while maintaining an appropriate balance between, on the one hand, lipids promoting hexagonal I mesophases and, on the other hand, nonpolar lipids and lipids promoting hexagonal II or cubic mesophases. Such lipid populations can be obtained in bread making by applying lipases which hydrolyze lipids promoting hexagonal II or cubic mesophases into lipids promoting lamellar mesophases.

Although this mechanism is in line with recent views of other researchers and fully explains loaf volume increases obtained with several lipases in bread making with several flours, its validity could not be proven by relating lipase-induced changes in dough liquor composition and properties to such changes in loaf volume. At this point, it is unclear whether this is due to lipases having no positive impact on the stability of gas cell interfaces during bread making or to dough liquor not being an appropriate model system for gas cell interfaces in dough.

An indirect impact on gas cell stability of wheat endogenous lipids and their enzymatically released hydrolysis products in bread making was demonstrated by evaluating the effect of lipase on dough rheology with a recently developed uniaxial dough extension test. Including lipase in the bread recipe significantly increased dough extensional viscosity upon extension. Altogether, we suspect that the impact of wheat endogenous lipids and their enzymatically released hydrolysis products on bread quality results from both their indirect and direct effect on gas cell stability.

Finally, lipases were demonstrated for the first time to influence gluten agglomeration and yield as well as the properties of the resultant gluten when applied in dough-batter wheat flour separation. The findings indicated that wheat flour lipids (and their enzymatically released hydrolysis products) affect dough (and/or batter) rheology. It was shown that lipases selectively degrading nonpolar lipids can be used as processing aids in wheat flour separation to improve gluten agglomeration and yield.

In conclusion, the results presented in this doctoral dissertation show that wheat endogenous lipids greatly contribute to bread loaf volume. In spite of being minor constituents in flour, they are excellent targets for improving bread quality. We were able to propose in detail which reactions are preferably catalyzed by a bread making lipase. Consider it very likely that the impact of wheat endogenous lipids and their enzymatically released hydrolysis products results from both indirect and direct effects on gas cell stability during bread making. Finally, enzymatic modification of endogenous lipids not only affects bread making but also dough-batter wheat flour separation processes.