Deliverables An efficient method to determine molecular weight of cereal beta-glucan and information about the effect of bread-making on molecular weight distribution and structure of the beta-glucan A system for detection of beta-glucan molecular weight in extracts from cereal products was developed. The system includes high-performance size-exclusion chromatography with detection based on specific binding of Calcofluor to beta-glucan. Fractions of beta-glucan with a narrow molecular weight distribution were used to calibrate the system. Inactivation of endogenous beta-glucanases was a crucial step in the analysis. Weight average molecular weight for molecules with a weight greater than 20,000 dalton and molecular weight distribution were determined. Molecular weight and structure units of beta-glucan in dough and bread made from hull-less barley/wheat flour were studied. Only small differences in molecular weight distribution and structure units of milling fractions from barley were detected. During bread making the molecular weight of the beta-glucan was reduced and the molecular weight distribution was polymodal, indicating an enzymatic degradation of the molecular weight. To retain high molecular weight of the beta-glucan in the bread, which is important for the cholesterol-lowering effect of the beta-glucan, it is important to keep the mixing and fermentation times as short as possible when baking the bread. Technology to produce consumer acceptable breads with increased levels of health promoting dietary fibre components arabinoxylan and beta-glucan Today the market for soft barley based breads is very small, mainly due to the poor baking performance of the barley flour. The main goal with the baking trials was to develop healthy, tasty barley based soft breads. The breads were optimised in terms of volume, staling and crumb structure as well as for organoleptic properties. It was found that it is possible to make breads with up to 40% whole-grain hull-less barley flour, using appropriate mixtures of wheat and hull-less barley flour and different baking aids such as an uninhibited endoxylanase. Results showed that it was possible to produce breads with increased levels of total and soluble arabinoxylan and beta-glucans by the combined use of hull-less barley flour and a bread-making active endoxylanase. During development of the industrial bread-making experiments, one important parameter to consider was problems with sticky doughs at different steps in the process. These problems were reduced by different adjustments in recipes and the baking process itself. It was found that most breads baked according to the Solfibread concept had relatively good baking performance. Out of five different breads developed, the barley based tea cake had the highest consumer acceptance. Among consumers, barley taste was generally regarded as positive and seemed to work out best in breads with a slightly sweet taste. Finally it can be concluded that the Solfibread concept could be a possible way to add tasty and healthy bread products to the market. Purified and biochemically characterised barley endoxylanase inhibitors By using classical biochemical and affinity chromatographical techniques, two types of endoxylanase inhibitors were purified and characterised from barley, i.e. the Hordeum vulgare endoxylanase inhibitor (HVXI) and the endoxylanase inhibiting protein (XIP)-type inhibitor. HVXI occurs as two molecular forms, both with a molecular weight (MW) of ca. 40,000. It strongly inhibits endoxylanases from glycoside hydrolase family 11, while no inhibition is found against those of family 10. In contrast to HVXI, the barley XIP-type inhibitor is monomeric with a MW of ca. 30,000 and shows a strong inhibition against fungal endoxylanases, while bacterial endoxylanases were not inhibited at all by the barley XIP-type inhibitor. Both types of inhibitors play probably an important role in plant protection mechanisms against microbial endoxylanases. Therefore, selection of barley cultivars with high endoxylanase inhibitor levels may in the future lead to decreased levels of fungicides. Due the presence of endoxylanase inhibitors in barley, the activity and functionality of endoxylanases in barley-based processes (e.g. bread-making, beer production, feed industry) will be influenced. Inhibition sensitive endoxylanases will progressively lose their activity during processing, resulting in the need of higher endoxylanase dosages compared to inhibition insensitive endoxylanases. While endoxylanase inhibitors are undesirable in bread-making, they are beneficial in the refrigerated dough industry because they can reduce the action of endogenous endoxylanases, which form an undesirable brown liquid during dough storage. Endoxylanase-based affinity chromatography for efficient one-step purification of endoxylanase inhibitors Initially, the isolation of barley endoxylanase inhibitors was based on an elaborate classical biochemical purification method. This, combined with the fact that the level of endoxylanase inhibitors in barley is approximately ten times lower than in wheat, resulted in difficulties when trying to obtain larger quantities of inhibitor for in-depth studies. Therefore, a new technique was developed for the purification of endoxylanase inhibitors based on affinity chromatography with immobilised endoxylanases from Bacillus subtilis and Aspergillus niger. The main advantages of this procedure over the classical biochemical method are speed on the one hand and the possibility to isolate two types of barley endoxylanase inhibitors, i.e. Triticum aestivum endoxylanase inhibitor (TAXI)-type and endoxylanase inhibiting protein (XIP)-type inhibitors, on the other hand. The choice of the endoxylanases that are immobilised on the affinity columns is such that TAXI-type inhibitors are retained at the first column (with B. subtilis endoxylanase) and XIP-type inhibitors are only retained on the second column (with A. niger endoxylanase). Evaluation of novel endoxylanases and identification of an uninhibited endoxylanase showing superior performance in Solfibread-making Project foreground work had shown that the TAXI type inhibitors from cereals significantly affect the functionality of microbial xylanases in in-vitro assays. Furthermore, the inhibitors seemed to effect the xylanase functionality in bread-making applications too. To elucidate this effect further, Danisco included five Bacillus xylanase variants in the SOLFIBREAD project. These xylanases had been developed using site-directed mutagenesis and reflected a broad range of xylanase inhibitor sensitivity from highly inhibited to completely uninhibited. Bacillus xylanases were chosen, since they are only inhibited by TAXI type inhibitors - not XIP type inhibitors. Evaluating the xylanase variants in a dough model system, showed some highly interesting results. We can conclude that the TAXI type inhibitors from cereals in vitro - but also in dough systems - significantly affect the Bacillus wildtype xylanase and three of the five new variants brought into the project by Danisco. Due to the difference in inhibition sensitivity between the xylanases, the xylanases affected the dough rheology differently. The novel un-inhibited xylanase, brought into the project by Danisco, stabilised the dough system significantly more than the other xylanases. Furthermore, the novel uninhibited xylanase was able to solubilise significantly more water un-extractable arabinoxylan, creating more soluble fibres in the final product, than any of the other xylanases. Due to the mechanism described above, the novel un-inhibited Bacillus xylanase was able to optimise the Wheat-Barley flour - making it possible to produce consumer acceptable bread products. Isolated and characterised hull-less barley proteins Gluten proteins from wheat flour have the unique property to form a visco-elastic network after hydration and mixing. They are storage proteins and can be divided in glutenins and gliadins. In general, glutenin polymers have elastic properties while gliadins have viscous properties. The poor baking performance of barley based breads can partly be ascribed to barley proteins. Therefore, proteins from hull-less barley flour were fractionated, characterised and compared to those of wheat flour. Higher protein yields were obtained with the Osborne fractionation method than with the salt precipitation method. Furthermore, data showed that the glutenin fraction of wheat flour had a higher molecular weight distribution than that of hull-less barley flour, whereas the gliadin fraction of hull-less barley flour had a higher molecular weight distribution than the wheat flour gliadin fraction. Because of their higher molecular weight, wheat flour glutenin polymers were more elastic than those of hull-less barley, while hull-less barley flour gliadins had very viscous properties. A rapid screening method to determine endoxylanase substrate selectivity The functionality of endoxylanases is affected by different parameters such as the endoxylanase substrate selectivity or the relative activity of endoxylanases towards water-unextractable (WU-AX) and water-extractable arabinoxylans (WE-AX). Previous research already showed the importance of substrate selectivity in biotechnological processes such as bread-making and gluten-starch separation. A laborious and time-consuming method with native substrates was used to determine the substrate selectivity of endoxylanases. In this project, a rapid screening method using microtitre plates with chromogenic substrates was developed to determine this parameter. The results were consistent with those obtained with the elaborate method. A high substrate selectivity factor indicates that the endoxylanase preferentially hydrolyses WU-AX over WE-AX, while endoxylanases with lower factors more readily hydrolyse WE-AX. The present rapid screening method is designed for use in routine applications by adaptation of the traditional enzyme assays in tubes to microtitre plate format, involving semi-automated multichannel pipettes and a microplate reader. The developed method is fast, accurate, reliable and reproducible and only small amounts of reagents and samples are needed. The screening method can be used for both standard evaluations and scientific research. It can, for example, be used for establishment of the structure-function relationship of natural and engineered enzymes. Hull-less barley milling technology with analytical characterisation of resultant milling fractions During the last years, a number of new barley varieties have been introduced to the market. One such example is hull-less barley which can be used in food without pearling. One objective with this project was to optimise milling of hull-less barley, both in laboratory and in industrial-scale, in terms of production of health promoting barley flours with appropriate functional properties. Milling of hull-less barley was successful and resulted in higher milling yields in the industrial-scale millings than in the laboratory-scale millings. The final industrially milled products were sifted flour and whole-grain flour. The contents of health-promoting fibre components (beta-glucan, arabinoxylan) were generally lower in the white flours than in the shorts and bran fractions. The alpha-amylase, beta-glucanase and arabinoxylanase activities, as well as endoxylanase inhibitor activity, were low in the white flours and higher in the shorts and bran. Low activities of the cell-wall degrading enzymes may be beneficial from a baking point of view if the objective is to produce bread with a high content of high molecular weight soluble fibre. The white flour fractions had lower contents of antioxidants. In order to increase the content of health-promoting components in foods, inclusion of whole grain or bran and shorts fractions of hull-less barley would provide the greatest benefits. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
