Laying the foundations for dough-based oat bread

The motivation to perform this study was to generate the fundamentals to use oats for bread-making applications. This will offer consumers a healthier alternative product to wheat bread in their daily diet, because oat foods, especially through their high amount of soluble fibre (notably beta-glucans) contribute to the reduction of blood cholesterol levels and of blood glucose rise after the meal. Oats also have a high content of (poly-) unsaturated fatty acids that contribute to maintaining normal blood cholesterol levels. One specific target group that would benefit from the development of good quality oat bread are people with celiac disease (CD). Oats is widely consumed by them, even though its safety has been subject of some debate for a long time. Two peptides from oat avenins can be recognized as T cell epitopes by few CD patients, and differential signals of gluten-specific monoclonal antibodies and in-vitro T cells to oat varieties have suggested the existence of differences in immunogenicity. These health and food safety issues have been addressed in the General Introduction. Bread is consumed all over the world. So far, production of large-volume bread is only possible with wheat. The quality of existing oat bread is below to what consumers are used to with wheat bread. This is partly due to the lack of knowledge regarding the functionality of oats for other purposes than porridge and breakfast cereals, which are the most common applications. These applications do not represent a big technological challenge as bread does, because bread-making requires a system able to hold gas during proving and baking. In wheat, this is conferred by gluten proteins that form a viscoelastic network with the capacity to expand and to maintain itself after expansion. Oats lack gluten proteins with network-forming capacity. Current oat bread applications rely on batter systems and on the use of additives to increase viscosity for stabilization of gas cells. This thesis consists of two parts. The first part concerns the safety of oats for people with celiac disease (<strong>Chapter 2</strong>). This was studied by cloning and sequencing avenin genes from 13 <em>Avena </em>species with combinations of the three genomes (A, C, D) that are also present in the hexaploid cultivated <em>A. sativa. </em>We identified up to 10 avenin genes in a single hexaploid oat plant. Avenin proteins clustered in four groups of which two contained the two avenin CD epitopes. All <em>Avena </em>species examined harbored avenins of these two groups, so it is unlikely to find oat cultivars that are devoid of the avenin CD epitopes. None of the internationally agreed gluten CD epitopes from wheat, rye and barley were found to be present in oat avenins. Some epitope variants with two and three amino acid substitutions occurred, but they were predicted to not resist proteolysis in the gastro-intestinal tract and will therefore not be of clinical relevance. Perfect recognition sites of antibodies R5 and G12 (which are used in commercial gluten detection kits) were also not present in avenins. Thus, monoclonal antibody signals to oat are probably due to cross-reactivity or promiscuous recognition of avenin peptides, and such signals should not be interpreted as differences in immunogenicity of oat varieties for CD patients. The second part of this thesis focussed on the study of the technological properties of oats. Oats have been used as an addition to wheat-based dough or in an oat-based batter system. However, while for wheat the dough-making parameters necessary to obtain good quality bread have been defined through a long history of research, this is not the case for oats. To fill this gap, this thesis studied the technological properties of oats using a systematic approach. First, we developed a dough testing system that allowed us to assess the dough-making properties of oat flour in a standardized way (<strong>Chapter 3</strong>). For this we used wheat as a model. We reproduced various quality profiles of wheat flour using combinations of oat flour and vital gluten. Then, we selected a dough system made of 87.2% oat flour and 12.8% gluten as our standard dough test system. This dough system was sensitive to differences among oat cultivars. Thus, having developed a tool that could detect differences regarding dough-making properties among oat cultivars, the next step was to try to explain those differences in terms of compositional factors. We decided to start our exploration with beta-glucans, because these fibres are one of the oat components that attract interest because of their health benefits. We studied the impact of beta-glucans on dough rheology (<strong>Chapter 4</strong>) following two strategies: (i) using the developed standard dough system containing gluten; and (ii) by removing the gluten from the system and replacing these proteins by alternative network-forming compounds. In both systems, beta-glucans affected dough rheology. Increasing their concentration resulted in an increase of dough stiffness and in a reduction of dough extensibility. Beta-glucans negatively influenced the elastic properties that additional wheat gluten conferred to oat dough. Low beta-glucan (<2%) oat flour had better extensibility properties than oat meal dough or oat flour dough enriched with beta-glucans. The effect was governed by its concentration and its molecular weight (which determines viscosity). Medium-viscosity beta-glucans had a less negative impact than high-viscosity (high molecular weight) beta-glucans. Overall, our findings indicate that beta-glucans are a key component determining rheology of oat-based dough systems. <strong>Chapter 5 </strong>addressed the effect of particle size distribution on dough-making properties. We found that oat meal is not the best material for bread-applications because it produces a very stiff and short dough. Re-milling did not change this pattern. In contrast, complete removal of the bran from the oat meal did improve dough-making properties, which indicated that dough rheology was negatively impacted by the bran. Large and medium size bran particles were more harmful than fine bran particles. Large and medium bran contained 8% beta-glucans, while fine bran contained 1.6% only. We concluded that oat meal is not appropriate for bread applications. Fractionation of the milled product is an interesting alternative to produce low-beta-glucan flour for bread-making purposes, and the bran can be used to enrich other food products with beta-glucans. This chapter also addressed whether kilning and milling methods applied to oat grains could affect bread-making purposes. Infrared (IR) and steam kilning both affected dough-making properties of oat grains in the standard dough system. The effect of steam kilning was on water absorption only. Non-kilned and steam-kilned grains showed similar extensibility behavior. In contrast, IR kilning affected water absorption and harmed completely the dough extensibility properties of oat grains. Flour from IR kilned grains made a very stiff and short dough. Thus, IR kilning is definitely not suitable for bread applications. Finally, in <strong>Chapter 6</strong>, we addressed the need for good quality gluten-free oat bread. As further research is required for better understanding of the oat dough system, we studied the rheological properties of oat flour relevant for leavening with gluten alternatives. Whey protein particles (WPP) had appeared to be successful in enhancing viscoelastic properties of wheat starch dough, allowing loaves with specific volumes of ca 3.7 mL/g. We studied whether WPP could have a similar positive effect on oat flour dough. WPP increased the resistance to extension and the gas retention capacity of oat flour dough. However, in our small scale baking experiments, WPP did not increase loaf specific volume and had a negative effect on gas production. On the other hand, WPP improved crumb texture. WPP are promising as a structuring agent in oat dough, but the process should be further optimized. In the General Discussion we pay attention to the food safety issue of oats for people with coeliac disease. Our analysis across the genus <em>Avena </em>of avenin genes and proteins produced an important new and supporting argument to the safety of oats, as they appeared to contain none of the generally agreed celiac disease-related gluten epitopes from wheat, barley and rye. With this analysis we also could explain the positive signals for the presence of gluten (as described in the literature for several oat varieties on the basis of the R5 and the G12 antibody assay and on T cell tests) as being the result of cross-reactivity or promiscuity, without having clinical relevance. The data in this thesis therefore support the advice to gradually introduce the consumption of oats into the daily diet of people with coeliac disease. Further, we discuss the results and the consequences of our technological research on oat flour dough. It appeared that beta-glucans have a serious negative effect on the rheology of the oat dough, which indicates the need for further research on improvement of the balance between optimum application of beta-glucans for health (high amounts and of high molecular weight is better) and for baking quality (low amounts and of low molecular weight is better). Also the pre-treatment of oat flour (notably kilning and milling) and the application of whey protein particles to replace gluten require further optimization. Here the developed standard oat flour dough model system will be a useful tool.