Potential of the Aleurone Layer in Bread-Based Products: Comparison
Please note this is a comparison between Version 1 by Lucie Lebert and Version 2 by Jessie Wu.

The wheat aleurone layer is the main bran fraction. It is a source of nutritionally valuable compounds, such as dietary fibres, proteins, minerals and vitamins, that may exhibit health benefits. 

  • wheat aleurone
  • aleurone layer
  • endosperm

1. Histology and Functions of Aleurone Layer 

The aleurone layer is a tissue of wheat grain made of unicellular block-shaped cells (37–65 µm vs. 25–75 µm) [1][19]. Among the seven layers comprising the mature bran, the aleurone is the only one with the remaining living cells [2][20]. It is located between the endosperm and the nucellar epidermis (or hyaline layer) [3][4][10,21]. Although botanically part of the endosperm, it is considered by millers as a bran layer since it remains attached to the hyaline layer during milling [3][5][10,22]. It represents around 50% of wheat bran (or 75% w/w of its dry weight), making it the major bran layer [3][6][10,12]. Indeed, the aleurone layer is thick and can reach up to 65 µm [7][23]. It also corresponds to 5–8% (w/w) of the whole kernel [4][21].
Although the aleurone layer is singular in wheat, it can be found multi-layered in barley, rice and oats [3][10].
Multiple functions in the wheat grain are allotted to the aleurone layer, namely: accumulation and transport of nutrients for seed germination, decomposition of storage materials of the endosperm for embryo growth, and protection and maintenance of caryopsis activity [8][24]. More specifically, its major role is during germination, where it is involved in the synthesis and release of hydrolases in the endosperm, as induced by gibberellin [9][10][11,25]. These enzymes then break down starch polymers and proteins in starchy endosperm cells, which undergo programmed cell death [5][11][22,26]. To facilitate the transfer, the aleurone’s outer cell wall is degraded by endogenous hemicellulases, and only the inner resistant layer remains [10][12][25,27].
The aleurone layer is also involved in seed dormancy, induced by abscisic acid. At the same time, this hormone induces programmed cell death in endosperm cells [9][11]. Moreover, the aleurone layer serves as grain storage for metabolites, minerals and amino acids [11][13][14][9,26,28]. It is equally involved in the regulation of water diffusion and distribution through its cell-walls [9][15][11,29].
Finally, in the crease region of the grain, some modified aleurone cells, called transfer cells, also participate in grain filling via solute uptake [16][30]. Due to their arabinoxylan’s higher degree of arabinose substitution and lower degree of feruloylation, the transfer cells show specific cell wall hydration and porosity properties that are compatible with water diffusivity and uptake for grain filling [17][31].

2. Extraction of the Aleurone Layer and Its Challenges

The aleurone layer’s potential can only be revealed if it is first extracted. However, no universal process yet exists. In addition, as the grain’s milling properties (friability) are dependent on its constituents, themselves related to the culture conditions and genetic background, the process must constantly adapt to the raw material. Therefore, this represents a major hurdle in the utilisation of the aleurone layer and in the exploration of its properties.
Another challenge arises from its composition. The aleurone layer, although botanically part of the endosperm, is considered by millers as belonging to the bran fraction. Since it is tightly adhered to the pericarp, it is usually removed from the endosperm during conventional milling. This tight adherence to seed coats also makes it difficult to separate the aleurone from the rest of the bran [3][6][10,12]. Consequently, most existing processes for the retrieval of the aleurone layer start with bran material. Multiple procedures have been patented [18][19][20][21][22][14,111,112,113,114] and two companies have mainly been known to produce and commercialise aleurone-rich flour: Bühler A.G. and Cargill Limited (through Horizon Milling with the GrainWise brand). However, it seems that most existing processes result in the obtention of aleurone-rich flour that is either not highly concentrated [23][13] or low yielding [18][14]. A summary of the composition of aleurone-rich fractions issued from the existing processes mentioned below, available in the literature, is shown in Table 1. As shown in the table, the purity of the obtained aleurone-enriched fraction depends greatly on the extraction process performed, which explains the discrepancies observed in their composition when compared to that of the pure hand-isolated aleurone layer. These differences in composition may also arise from the use of different analytical methods among publications.
Most of these processes extract the aleurone layer from bran components by dry-fractionation, a succession of mechanical or physical unitary steps [3][24][25][10,115,116]. Many researchers first aim to dissociate the different bran tissues, which can be conducted by grinding. They include a separation step that then enables sorting out the particles according to their size, mass, density or electrostatic properties [25][116]. The obtained milling fraction can thus be added to basic wheat flour to enrich it with the aleurone layer [24][115].
The aleurone layer is extensible, similar to the intermediate strips of wheat bran, and it has an elastoplastic rheological behaviour. Its mechanical characteristics are impacted by the degree of feruloylation of its arabinoxylans (AX), particularly by the presence of ferulic acid (FA) dehydrodimers [26][44]. Hence, the mechanical stress generated by dry fractionation processes first affects the aleurone cell walls, which crack, allowing the cell contents to be released [27][117]. According to Rosa et al. [28][118], the velocity of phytic acid release could thus be used as a marker to estimate aleurone cell opening.
Electrostatic separation of aleurone from other bran tissues is an interesting process since the aleurone layer presents unique electrostatic properties compared to the other strips. However, this process can be influenced by multiple parameters, such as particle size, composition, microstructure and moisture content [29][68].
The main advantage of physical or mechanical extraction methods is that they do not require the use of chemical products that can interact with the matrix and decrease the product’s purity and phytochemicals’ functionality [25][116]. Compared with wet processes (chemical and enzymatic treatments), they also enable higher energy efficiency [3][25][10,116]. However, the succession of unit operations may impact the antioxidant and secondary metabolites of the aleurone layer [30][3]. Moreover, grinding generates various particles from bran tissues of different sizes and densities, which are hard to differentiate, hence the reported end-product’s low purity [25][116].
This type of process was used in the patented method by Stone and Minifie [18][14], who first used hammer-milling in wheat bran containing 34% of aleurone cells, followed by sieving, electrostatic fractionation, and a final separation through an electric field. A 95% purity of aleurone cells was obtained with a 10% yield [18][29][14,68]. Nonetheless, alternative methods exist: humidification then micro-grinding of wheat bran with a friction roller mill [22][114]; sequential pearling cycles in a vertical abrasive polishing machine [31][74]; centrifugal impact milling [29][68]; ultrafine grinding and electrostatic separation [32][119]. Different outcomes have been reported with these processes, with varying aleurone purity and yield.
However, there are limited studies on the extraction of the aleurone layer by wet processes (chemical and enzymatic treatments). For instance, the maceration of wheat bran in chemical reagents, such as organic solvents, has been tested but not in an industrial scale [3][10].
Nonetheless, dry and wet processes can be coupled. A patented method isolated aleurone by successive steps of cleaning, steaming, stabilising, roller-milling, sieving, fine grinding and air-classifying. However, the end-product still contained 36.5% starch, demonstrating low purity [33][34][87,120]. In addition, the patent deposited by Kvist et al. [35][121] subjected wheat bran to several enzymatic treatments, wet milling steps, sequential centrifugation, and ultrafiltration. Other researchers coupled successive steps of milling, sieving, air classification and centrifugation with benzene-carbon tetrachloride mixtures at laboratory scale [3][10].
Although many experiments have been conducted and sometimes patented to extract and isolate the aleurone layer, the challenge of measuring end-product purity has arisen. Thus, researchers have defined biochemical markers to differentiate grain parts. These biochemical markers can be used to determine the extent to which the aleurone layer is extracted from other grain components. Starch, phytate, p-coumaric acid, alkylresorcinols and FA trimer are used to estimate the proportion of the endosperm, aleurone cell content and cell-walls, intermediate layer and outer pericarp, respectively [36][37][38][46,65,122]. However, the relative amount of grain tissue can only be calculated when compared to the reference values. The latter were values of the same parameters from pure isolated tissues of identical wheat cultivars. Thus, it limits their utilisation for characterisation since pure fractions are obtained from hand-isolated tissues, a long and laborious process. Moreover, these markers are susceptible to natural variability among wheat cultivars due to the culture conditions and genetic background [39][40][50,123]. As an alternative to these biochemical markers, microscopy analyses can be performed to estimate the purity of the extracted fractions [24][115].

3. Application to Breadmaking

3.1. Aleurone Bread Nutritional Profile

According to past reviews and experiments, there are many benefits to incorporating an aleurone-rich flour into bread and bakery products, starting with an improved nutritional profile of the end-products. This amelioration is related to increased dietary fibre (DF) and protein (mainly albumin and globulin) content at the expense of readily digestible carbohydrates [23][32][50][55][56][13,15,119,131,136]. The enhancement of minerals, including phosphate, magnesium, manganese and iron, and bioactive compounds such as phenolic acids, antioxidants, phytoestrogens and sterols, also increase the value of the obtained end-products [56][57][16,136]. This improved composition confers the end product a nutritional profile similar to that of whole wheat products [47][128], while equally making it a good source of fibre [6][12]. However, the nutritional benefits of aleurone-rich products are accompanied by increased phytate content, which is known for its antinutritional effect [58][57].

3.2. Aleurone Bread Dough Characteristics

Despite these beneficial nutritional properties, the incorporation of the aleurone layer for breadmaking leads to changes during dough formation, which affects the sensory attributes of the end-product. With its high DF content (Table 1), the aleurone layer impairs dough hydration properties. The AXs and β-glucans contained by the aleurone layer compete for water with the proteins forming the gluten network, thus increasing the water absorption of the dough and retarding the dough development time [56][59][136,137]. The water retention capacity is also affected by fibres that take up a large amount of water (3.5 to 6.3 times their weight for water-extractible AX and 6.7 to 9.9 for water-unextractable AX) by binding through hydroxyl groups, resulting in a longer mixing stability due to the alteration of the gluten structure [50][56][60][6,131,136].
In addition, the presence of these fibres has a diluting effect on the starch granules. Damaged starch content is then decreased as well as the falling number. The latter effect is further reduced by the increase in α-amylase activity in the presence of calcium. Indeed, this metalloenzyme requires calcium for its performance, which is provided by the aleurone layer (Table 1). In addition, these observed properties seem to increase in relation to the aleurone-rich flour dosage [56][136].
The effect of aleurone incorporation on starch also influences the pasting properties of dough. Multiple studies have shown a decrease in peak viscosity, as well as in the retrogradation of dough [50][51][131,132]. This might not only stem from the presence of fibres that interfere with starch granule swelling and increased amylase activity but also from the combined effect of other aleurone constituents. For instance, the presence of fat and FA can also impact pasting properties, in addition to an already low starch content [51][61][132,138].
Nevertheless, the aleurone dough exhibits higher Rapid Visco Analyzer (RVA) parameters, revealing a strong gel ability greater than that of whole wheat flour [50][131]. According to Bucsella et al. [56][136], this could be due to the swelling of fibres, which form a strong gel despite the lower starch content. This gel is described as being more resistant to heat and mechanical stress.
The gluten network can also be strengthened following the addition of aleurone-rich flour to bread dough (up to 40%). However, according to Mixolab (Chopin) measurements, the dough development time is increased due to the presence of fibres that compete for water and hinder gluten network formation by intercalating between the proteins, resulting in a more heat-stable and stress-resistant dough [50][131].
This increase in dough stability is also depicted by firm elastic-like behaviour due to the stronger gluten complex [50][56][131,136]. The increased protein content (albumin) and the strengthening effect of AX binding to gluten via the oxidative dimerization of FA also contribute to these observed effects [62][139]. Instrumentally, this translates into an increase in dough stability and break time, as well as delayed weakening [56][136].
Despite the aforementioned beneficial traits observed due to the aleurone components, most of them are dose dependent. Excessive addition of aleurone-rich flour to the dough can lead to deleterious effects on dough rheology.

3.3. End-Product: Aleurone Bread Characteristics

The addition of aleurone-rich flour to bread-making has an impact on end-product quality, although the results of the researcher’s findings are contradictory. This may be related to the aleurone-enrichment level, the purity of this material as well as the bread formulation process, compiled in Table 2.
Some report a decrease in loaf volume, accompanied by reduced height and increased weight [23][55][57][63][13,15,16,17]. For instance, Bagdi et al. [55][15] observed a diminution of 27% of the specific volume for a bread prepared with 100% of aleurone-rich flour compared to a control white bread, as well as a reduction in loaf height of 13%. Using the same breadmaking process (ICC Standard Method 131), Bartalné-Berceli et al. [57][16] obtained a height decrease of 15 and a 7.2% weight increase with a bread containing 25% of aleurone-rich flour compared with a control white bread. These tendencies are further incremented with a higher aleurone flour input (50%), where almost half of the height was decreased and 3.6% of the weight was increased compared with the control.
Other studies have observed a higher loaf volume than white bread upon aleurone incorporation or have found insignificant changes. The texture in these experiments was also reported to be softer than white bread, which means that the crumb was less dense [6][56][64][12,136,140]. Indeed, Tian et al. [64][140] described an increase of 40.91% in bread specific volume using aleurone-rich flour (modified GB/T 35869-2018 procedure with 54.11% of aleurone layer content). However, this beneficial effect could be attributed to the presence of hemicellulases (at 40 mg/kg) that enable the formation of water-extractible AX (WEAX) from water-unextractable AX (WUAX). Similar results were obtained from breads made with a sourdough preparation (MSZ-6369-8:1988) incorporating an aleurone fraction, even though the observed volume increase was not significantly different from that of the control bread [56][136]. Breads made from straight dough and sponge dough processes with additives containing aleurone-rich flour (20%) also show this beneficial trait [6][12].
Overall, it seems that these beneficial effects could be related to the presence of hemicellulose-degrading enzymes—either endogenous (sourdough) or exogenous (as an additive)—or additives or a special breadmaking process, each enabling the revelation of the aleurone layer’s full technological potential.
Unlike for the volume and texture of the breads, the appearance of the end-product is equivocal: the crumb colour (measured by colorimetry) is darker than white bread, even brownish, which can be a limiting factor for some consumers. However, it is still lighter than whole wheat products [23][55][58][13,15,57].
As for the taste of the bread, diverging results also occur. Whereas some report a flavour similar to that of white bread, especially when a long fermentation process takes place [6][47][12,128]; others describe a bread that is more bitter and sour, even rye-like [23][55][13,15]. In addition to the last finding, Amrein et al. [58][57] outlined a gritty mouth-feel, which is a limiting factor for the consumers of the study. However, the smell of the products is reported to be more intense and sour [23][55][13,15].
Overall, bread made of aleurone-rich flour in different proportions showed similar properties to that of white breads but with the nutritional profile of whole wheat breads. More thorough experiments on the relevance of aleurone addition in cereal products compared to other wheat kernel layers should be conducted, as the diversity of breadmaking methods and starting materials is great in the existing studies. Nevertheless, the results are still contradictory and lead to either a decrease or increase in end-product consumer acceptability. The use of special breadmaking technologies or additives, such as cell wall degrading enzymes, could thus reveal the aleurone layer’s full technological potential.

3.4. Underlying Mechanisms

4.2.4. Underlying Mechanisms

Many of the adverse or positive technological effects due to the addition of aleurone-rich flour to bakery products stem from its unique composition and, more specifically, its high protein and DF content. Indeed, studies investigating the effect of DF, AX and bran incorporation into bakery products showed similar properties to those described in aleurone-enriched products.
Most experiments on this subject describe that the addition of fibres to bread dough increases dough development time, water absorption and strength. However, it also seems to weaken the dough’s tolerance to mixing and fermentation [65][141]. This results for most studies in a reduction in loaf volume [65][66][67][68][69][5,141,142,143,144], an increase in crumb firmness [67][68][69][142,143,144], and a darkened crumb appearance [65][67][68][69][141,142,143,144].
Hypotheses exist to explain the mechanisms underlying these results, which corroborate those of aleurone-rich products. First, fibres with their high water binding capacity might compete for water with starch and gluten, thus keeping wheat proteins from sufficient hydration for the formation of the gluten network [65][66][67][70][71][72][73][74][5,141,142,145,146,147,148,149]. Another explanation is that fibres dilute gluten, thus affecting its gas-holding capacity [65][66][67][70][71][72][73][74][5,141,142,145,146,147,148,149]. Nonetheless, this impairment in gas retention that causes a loss of loaf volume could also be due to the shortened and lowered resistance to dough extension upon DF addition, which increases the concentration of soluble cell wall materials and disrupts the gluten network [67][75][142,150].
In addition to the previous mechanisms of action that hint at a physical mode of action, a chemical hypothesis also exists that states that FA linked to DFs could mediate AX–AX and AX–protein cross-linking (through FA–tyrosine linkages), thus impacting gluten properties [71][72][73][74][146,147,148,149]. This would be possible in the presence of oxidants or enzymes (such as laccase and peroxidases) that provoke the dimerization of FA, thus creating covalent linkages between AX chains. Moreover, this dimerization increases the water retention capacity of AXs, which directly affects the gluten network [60][6].
More specifically, studies conducted on the addition of AXs to bakery products could be helpful in understanding the mechanisms underlying the aleurone-enriched bread properties, since they represent a large part of this layer. Similar to the addition of general DFs, an increase in the water absorption of the dough is reported due to the high water retention capacity of AXs, which increases dough consistency [76][151]. According to Berger and Ducroo [60][6], to reach the same dough consistency as the control dough on the Brabender farinograph, 0.5 to 2% of additional water should be incremented per percent of AX supplemented.
As for the negative effects on gluten network formation due to AX addition, they could stem from the steric hindrance of the increased batter viscosity that limits components mobility, thus decreasing the formation of gluten aggregates and starch entrapment in its matrix [76][151]. Nonetheless, the observed effects are not as important as the extent of their addition and their molecular size, but most importantly, depend on the breadmaking quality of the initial flour used for the experiments [77][152].
Furthermore, the water extractability of AXs is also a determining factor in the adverse effects it causes on bread and bakery products. For instance, WUAXs seem to generate more deleterious side effects upon their addition than WEAXs. This might explain the contradictory results with aleurone applications since it mainly contains WUAXs, which can be transformed into WEAXs during breadmaking.
The use of WUAXs in bakery products is often reported with breads of lower volume, coarser crumb and higher firmness [78][39]. To explain this phenomenon, there are three hypotheses: (i) WUAXs form physical barriers for wheat proteins during dough development [78][39]; (ii) these AXs form intrusions in the gas cells during fermentation [78][79][39,153]; (iii) the WUAXs compete for water with the gluten network, thus impairing its formation and leading to a fracture effect that increases dough resistance to extension [43][60][78][6,7,39]. The latter hypothesis is believed to be more accurate since a correction of dough hydration (2% per percentage of AXs added) improves its extensibility [60][6].
In contrast, the use of WEAXs in bread dough yields contradictory results, even though they are mostly beneficial. Globally, a finer and more homogenous breadcrumb is depicted, with a bread that is softer [78][39]. The loaf volume is also impacted, but contradictory results are obtained. The observed higher volume is usually obtained with the use of high molecular weight WEAXs [78][79][80][39,153,154]. The underlying mechanisms explaining these effects include an increase in liquid film stability and thus in dough aqueous phase viscosity [43][78][7,39]. Moreover, WEAXs of higher molecular weight can form a secondary network, weaker than gluten, which enforces the latter and stabilises it through the dimerization of FA and by physical entanglement, either of gluten or between WEAXs [78][39]. By increasing the dough’s gas retention capacity, the resulting breads become higher [60][6]. According to [60][6], the higher the WEAXs molecular weight, the highest beneficial effect is observed.

4. Application to Other Food Products

4.3. Application to Other Food Products

Although the incorporation of aleurone-rich flour into bakery products have mostly been studied, its application to other product categories exists. For instance, Cargill Limited [47][128] developed cereal flakes and extruded snacks with 35% of aleurone-rich flour, as well as high-protein bars containing 20%. Ready-to-eat cereals enriched in aleurone were also studied by Byrne [81][155].
Other applications entail aleurone-enriched pasta and noodles. Whereas the former was described as healthier than wholemeal spaghetti due to higher protein, fat, and DF content, its consumer acceptance was decreased. Although it showed improved quality characteristics (lower water uptake, higher cooked pasta firmness, higher tensile strength and lower stickiness), the darker, more intense, bitter and sour taste of the pasta influenced consumer appreciation of the product [61][138]. The second used a combination of aleurone-rich flour and transglutaminase, which resulted in noodles with less cooking loss and the best sensory evaluation when compared to traditional noodles [49][130].
Finally, Yang et al. [53][134] used aleurone-enriched flour and cell wall degrading enzymes for the production of Chinese buns. They found that the action of enzymes promoted the WEAX content while also increasing the water availability to the gluten-forming proteins, resulting in softer dough, especially when enzyme activities were combined.
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