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Sub-aleurone in wheat and miller’s bran: An overlooked and unexploited source of gluten

Publication date: 2024-08-21

Author:

Hermans, Wisse
Courtin, Christophe

Keywords:

1S06222N#55361044

Abstract:

Bread is a complex food product that is, in essence, made from wheat flour, water, yeast, and salt. Despite starch being quantitatively the major constituent of wheat flour, gluten proteins are the main quality determinants of bread-making. Gluten proteins can form a viscoelastic network capable of retaining gas produced during fermentation, resulting in the characteristic texture and structure of bread. Often, extra commercial vital gluten is added to bread formulations, especially when using wholemeal flour. Whole grain products such as wholemeal bread are crucial towards the planetary health diet (Willett et al., 2019). The same applies for gluten due to its role in the protein shift. The question is: do we use wheat gluten efficiently? In a wheat kernel, gluten proteins occur in the starchy endosperm, which millers aim to recover as white flour. However, millers never achieve complete recovery and part of the starchy endosperm, and thus gluten, is lost in the miller's bran fraction, the by-product of wheat milling. This lost starchy endosperm is believed to be high in protein, and possibly gluten proteins, because of the presence of sub-aleurone cells. The sub-aleurone cells differ from other starchy endosperm cells in that they originate from aleurone cells. Although there are signs that sub-aleurone cells are high in protein, little is known about their exact protein content, protein composition, and functionality. The aim of this dissertation is (i) to investigate the protein content and composition of sub-aleurone cells and how these depend on cultivar and the level of nitrogen(N)-fertilisation, and (ii) to gain insight into the functionality of sub-aleurone proteins in bread-making and explore the potential valorisation of sub-aleurone proteins from miller's bran. The sub-aleurone protein content and composition were analysed using a sample set that consisted of three wheat cultivars (Claire (Cl), Apache (Ap), Akteur (Akt)), each grown at three levels of N-fertilisation (0, 150, 300 kg N/ha). To investigate the sub-aleurone protein content, the gradient in protein content within the starchy endosperm was determined using image analysis. From the outer to inner endosperm, this gradient could be described by a declining biexponential curve with protein levels up to 32.0% immediately beneath the aleurone due to the presence of sub-aleurone cells. The starchy endosperm region located from 0 µm to 100 µm beneath the aleurone layer was denoted as the sub-aleurone region (further referred to as sub-aleurone) because it was shown to approximate the region in which the sub-aleurone cells are found. The sub-aleurone protein content varied from 10.7% to 26.2%, whereas the protein content of the inner endosperm (600 µm beneath the aleurone to the centre of the starchy endosperm) from 6.3% to 9.8%. This demonstrates that the sub-aleurone protein content is high and varies strongly with the cultivar and N-fertilisation level. For Akt300, which had the highest sub-aleurone protein content, the sub-aleurone constituted approximately 16.4% of the starchy endosperm in weight while accounting for 27.8% of its protein. Cryosectioning, laser microdissection, and nanoLC-MS/MS revealed that the relative gluten content (number-based proportion of proteins being gluten) in the sub-aleurone is significantly higher than in the inner endosperm. The opposite is often claimed in literature. The relative gluten content (mass-based) in the sub-aleurone was estimated to vary from at least 81.2% to 87.3%, whereas that was 76.6% to 84.5% in the inner endosperm. In contrast to the protein content, the relative gluten content in the sub-aleurone was less dependent on the cultivar and N-fertilisation level than that in the inner endosperm. The differences in gluten composition between the sub-aleurone and inner endosperm were rather small and seemed to depend on the cultivar and N-fertilisation level. The unique protein expression in the sub-aleurone was attributed to its origin and/or relative position within the starchy endosperm. In a wheat milling experiment, the protein gradient within the starchy endosperm was shown to be reflected in the break fractions of the Bühler MLU-202 laboratory mill. The last break fraction and the coarse miller's bran fraction were the most enriched in sub-aleurone material. The challenge of recovering the sub-aleurone in the white flour could be explained by the relatively low puroindoline abundance in the sub-aleurone compared to the inner endosperm. Lab-scale-produced coarse miller's bran of Akt300 is estimated to contain about 28% of the starchy endosperm protein, which is approximately equivalent to the sub-aleurone protein contribution to the starchy endosperm. To investigate the functionality and valorisation potential of sub-aleurone proteins, sub-aleurone (protein) was isolated from Akt300 coarse miller's bran using dry fractionation techniques. Mild milling of the coarse miller's bran followed by sieving resulted in a sub-aleurone-enriched fraction (27.4% mass yield (on a coarse miller's bran basis), 22.4% protein, 63.2% starch, 3.1% arabinoxylan). Milling this fraction and air classifying it led to a coarse fraction (21.3% mass yield (coarse miller's bran basis), 20.1% protein, 66.2% starch, 2.7% arabinoxylan) and a fine fraction (4.9% mass yield (coarse miller's bran basis), 31.4% protein, 50.2% starch, 3.9% arabinoxylan), which was strongly enriched in sub-aleurone protein. A bread-making experiment with gluten isolated from the coarse, fine, and corresponding white flour fraction showed that the inherent functionality of the gluten in the sub-aleurone and in the corresponding flour was similar. This was also expected based on their similar gluten composition. Despite the higher level of bran contamination and lower relative gluten content in the fine and coarse fraction compared to commercial gluten, the fine, coarse, and commercial gluten fraction increased the specific bread loaf volume to a similar extent when the protein contents of the samples were standardised. The absence of wet processing in the production process, which is common practice in commercial vital gluten production from flour, seemed beneficial for gluten functionality. Commercial vital gluten production is also water- and energy-intensive. Finally, the effect of coarse miller's bran with different sub-aleurone protein contents on the quality of bran-enriched bread was investigated. In a bread-making experiment, it was shown that using coarse miller's bran with a high versus coarse miller's bran with a low sub-aleurone protein content increased the loaf volume by 22.4% for lab-scale-produced miller's bran. This concept was also proven for two commercial coarse miller's bran samples. Moreover, the bread loaf volume was strongly correlated (R² = 0.99) with the sub-aleurone protein content. Therefore, one can conclude that the sub-aleurone protein content is the most important factor regarding the functionality of coarse miller's bran in bread-making, more important than differences in hydration properties and particle size. To improve the resource efficiency in the industrial mills, three strategies were outlined. Particularly gluten use efficiency is becoming increasingly crucial due to the pressure on N-fertilisation levels. First, the isolation of sub-aleurone (protein) from miller's bran using dry fractionation techniques could increase the white flour yield or lead to the production of an alternative for commercial gluten. Second, miller's bran selection based on its (sub-aleurone) protein content could be a low-cost and low-effort strategy to reduce the reliance on commercial gluten supplementation for bran-enriched bread (including wholemeal bread) production. Currently, only less than 10% of miller's bran is used in human foods, and any form of bran selection is absent. Third, flour mill streams could be selected for the production of specialised flours. Future work could focus on investigating the sub-aleurone protein properties of wheat grown for commercial purposes and the valorisation of sub-aleurone from commercial miller's bran streams. Furthermore, the question of which mechanism hinders a clean separation between the sub-aleurone and aleurone layers remains unanswered.