Biochemical markers: Efficient tools for the assessment of wheat grain tissue proportions in milling fractions

To produce safe and healthy whole wheat food products, various grain or bran dry fractionation processes have been developed recently. In order to control the quality of the products and to adapt these processes, it is important to be able to monitor the grain tissue proportions in the different milling fractions produced. Accordingly, a quantitative method based on biochemical markers has been developed for the assessment of grain tissue proportions in grain fractions. Grain tissues that were quantified were the outer pericarp, an intermediate layer (including the outer pericarp, the testa and the hyaline layer), the aleurone cell walls, the aleurone cell contents, the endosperm and the germ, for two grain cultivars (Tiger and Crousty). Grain tissues were dissected by hand and analysed. Biochemical markers chosen were ferulic acid trimer, alkylresorcinols, para-coumaric acid, phytic acid, starch and wheat germ agglutinin, for outer pericarp, intermediate layer, aleurone cell walls, aleurone cell contents, endosperm and germ respectively. The results of tissue quantification by hand dissection and by calculation were compared and the sensitivity of the method was regarded as good (mean relative errors of 4% and 8% for Crousty and Tiger outer layers respectively). The impact of the analytical variability (maximum 13% relative error on coarse bran) was also regarded as acceptable. Wheat germ agglutinin seems to be a promising marker of wheat germ: even if the quantification method was not able to quantify the germ proportions in milling fractions, it was able to classify these fractions according to their germ content. The efficiency of this method was tested, by assessing the grain tissue proportions of fractions exhibiting very different compositions such as flour, bran and aleurone-rich fractions obtained from three different grain or bran dry fractionation processes (conventional milling, debranning process, production of aleurone-rich fractions from coarse bran). By calculation of the composition of the different products generated, it was possible to study the distribution of the different tissues among fractions resulting from the different fractionation processes. This quantitative method is thus a useful tool for the monitoring and improvement of processes, and allows the effects of these processes to be understood and their adaption to reach the objectives.     

Wheat grain tissue proportions in milling fractions using biochemical marker measurements: Application to different wheat cultivars

Measurement of biochemical markers allows the quantification of wheat (Triticum spp.) grain tissue proportions in milling fractions. In order to evaluate the ability of extending this methodology to an unknown wheat grain batch, the variability of the markers in the different tissues was assessed on various wheat cultivars. Ferulic acid trimer amounts in the outer pericarp ranged from 0.97 to 1.67 μg mg⁻¹ (dm) with an average value equal to 1.31 μg mg⁻¹ (dm). Alkylresorcinols amounts in a composite layer, including the testa, the inner pericarp and the nucellar epidermis, ranged from 10.5 to 16.7 mg g⁻¹ (dm), with an average value equal to 14.0 mg g⁻¹ (dm). In the aleurone layer, phytic acid amounts ranged from 94.9 to 187.2 mg g⁻¹ (dm) with an average value equal to 152 mg g⁻¹ (dm) whereas, para-coumaric acid ranged from 0.08 to 0.29 μg mg⁻¹ with an average level of 0.18 μg mg⁻¹. In the embryonic axis, wheat germ agglutinin ranged from 879 μg g⁻¹ to 2086 μg g⁻¹ with an average value of 1487 μg g⁻¹. The impact of this variability on tissue proportion determination was evaluated and a strategy to decrease the prediction error was suggested. Percentages of the outer pericarp, intermediate layer (including the testa), aleurone layer and embryonic axis within grains were calculated and their variability discussed.     

Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition

Hand dissection of mature grains from two common wheats (Triticum aestivum L., cv. Caphorn and cv. Crousty) were performed to quantitatively assess their tissue composition and to obtain homogeneous samples of embryonic axis, scutellum, starchy endosperm, aleurone layer, hyaline layer, outer pericarp and a composite layer made up of testa+hyaline layer+inner pericarp. Polymeric neutral sugar and phenolic acid contents of the samples were determined and used to identify specific composition patterns in each tissue irrespective of the cultivar. The scutellum and embryonic axis showed the lowest amount of carbohydrates with similar relative amounts of arabinose and xylose (Ara+Xyl), but the scutellum differed from the embryonic axis in its high phenolic acid, in particular ferulate dehydrodimer, content. The peripheral layers of the grains were mainly composed of cell wall polysaccharides, chiefly arabinoxylans but with differing structures. The hyaline layer was mostly composed of arabinoxylan with extremely low Ara/Xyl ratio (0.1), with high amounts of ferulic acid monomers and hence very weakly crosslinked. The aleurone layer differed from the outer pericarp by its much lower Ara/Xyl ratio and lower amounts of ferulic acid dimers and trimers. High proportions of ether-linked phenolic acids (released by alkali at 170 °C) were detected specifically in the seed coat and tissues in the crease region. The possible application of biochemical markers found in the various tissues to monitor wheat grain fractionation processes is discussed.     

Wheat bran tissue fractionation using biochemical markers

Phenolic acid analysis of hand-isolated outer grain layers and endosperm led to the identification of markers of pericarp and aleurone layers, respectively. A new dehydrotrimer of ferulic acid (DHT) was found to be concentrated in the outer pericarp of wheat bran whereas p-coumaric (p-CA) acid was mainly in the aleurone layer. Phytates were also used as a marker of aleurone layer and starch as a marker of starchy endosperm. Biochemical markers constitute an original method for determining the histological composition of any technological bran fractions. A pin milling process was applied to coarse bran produced by a conventional milling process. Three different fractions (B1, B2 and B3) were obtained by sieving the bran products and then the smallest bran particle fraction (B3) was air-classified to obtain two particle size fractions (B3a and B3b with a D50 of 83 and 7 μm, respectively). The biochemical composition of these fractions was used to calculate the distribution of tissues according to the sieving process. The dissociation behavior of individual bran tissues upon mechanical fractionation was investigated in relation to particle size and discussed according to their mechanical properties.     

Assessment of biochemical markers identified in wheat for monitoring barley grain tissue

The possible use of specific biochemical compounds identified in wheat grains was evaluated for monitoring barley grain tissues during fractionation. First barley grain anatomy was studied through microscopic observation and quantification of the relative proportion of each anatomical part in four distinct barley samples from both hulled and hulless genotypes. As expected from cereal phylogeny and irrespective of the possible presence of hull, common features were observed between barley and wheat grains, but the aleurone layer predominated in the outer layers. The specific location of the compounds identified in wheat was established. Phytic acid was specifically localized in the aleurone layer and alkylresorcinols in the composite layer containing the testa, even if their concentration differed from that observed in wheat grain tissues. Thus, these two markers identified in wheat can be used to monitor the corresponding barley tissues, independent of the presence of hulls. Conversely, phenolic compounds, either ferulic acid trimer or p-coumaric acid, cannot be used to monitor respectively the outer pericarp or the aleurone cell walls in barley grains. p-coumaric acid was identified as an efficient marker of the hull and could be used to distinguish hulled or hulless barley grains and to help monitor the dehulling process.     

Potential of dry fractionation of wheat bran for the development of food ingredients,part II: Electrostatic separation of particles

Wheat bran is a composite material made of several layers, such as pericarp, testa and aleurone. It could be fractionated into purified fractions, which might either be used as food ingredients, or serve as a starting material for extraction of bioactive compounds. The aim of this work was to evaluate the potential of using electrostatic separation as a way to obtain purified fractions from wheat bran. Ultrafine-ground bran obtained either by cryogenic grinding or by grinding at ambient temperature was used as starting material. The ultrafine bran was then charged by tribo-electrification and introduced in a chamber containing two high voltage electrodes, where bran particles were separated depending on their acquired charge, allowing positively and negatively charged fractions to be collected separately. The particle size distribution, microstructure and biochemical composition of the obtained fractions were studied. The charge of the particles was influenced by their biochemical composition: particles rich in highly branched and cross-linked arabinoxylans (pericarp) were separated from particles rich in β-glucan, ferulic acid and para-coumaric acid (aleurone cell walls). The testa and the intracellular compounds from aleurone were not highly charged, neither positively nor negatively. The most positively charged fraction represented 34% of the initial bran, and contained 62% of the ferulic acid present in the initial bran. The yield of the separation process was good (5.4% loss), and could be further increased.     

Phytase activity, phytate, iron, and zinc contents in wheat pearling fractions and their variation across production locations

Pearling is an effective method for evaluating the distribution of chemical components in wheat grain. Twelve pearling fractions (P₁–P₁₂) of wheat grain were obtained using two rice polishers for 10 cultivars (six soft red wheats and four hard white wheats) grown at two locations with different environmental conditions in Jiangsu Province, China. The results show that the effects of cultivar, location, and pearling on wheat flour phytase activity, phytate, iron, and zinc contents were all significant, with pearling having the greatest effect. All the four components showed a diminishing trend as pearling progressed from the outer layers to the inner part of wheat grain. Generally, the P₂ fraction (the outer 4–8% layer of wheat grain) had the highest phytase activity and phytate and iron contents, whereas the P₁ fraction (the outer 0–4% layer) ranked the highest for zinc content. Growing location had a large influence on grain phytase, phytate, and iron, but the differences between locations decreased as pearling level increased.     

Mechanical properties of outer layers from near-isogenic lines of common wheat differing in hardness

The mechanical properties of the combined outer layers from near-isogenic wheats differing by hardness were determined. Results from traction tests showed significant differences between the isogenic lines, outer layers from grains of the soft type showing higher extensibility. Determination of the mechanical properties of the corresponding component tissues revealed significant differences between the isolated tissues from soft or hard wheat grains. It also allowed analysis of their respective contribution to the properties of the combined peripheral tissues using a simulation of their rupture as unseparated tissues. According to the results, if the component layers displayed similar maximum lineic force to rupture, the rupture of combined outer layers occurs when the least extensible individual tissue breaks. The major cell wall biochemical components of the combined outer layers and of their component tissues were analysed. The phenolic acid composition of soft wheat pericarp contained more ferulic acid in either monomeric or polymeric forms than the pericarp from hard wheat. Arabinoxylans in walls of the soft wheat pericarp appeared 1.6 times more cross-linked by ferulic acid dehydrodimers than walls of hard wheat. These differences in arabinoxylan cross-linking may explain the observed differences in pericarp mechanical properties.     

Physical aspects of the biopolymer matrix in wheat bran and its dissected layers

The objectives of this work were to 1) determine the physical structure of untreated wheat bran and the differences in physical structure between its dissected layers; 2) evaluate how bran hydration affected bran crystallinity and polymer order; and 3) determine how enzymatic treatment of wheat bran affected its physical structure. For the first time, X-ray diffraction (XRD), small angle X-ray scattering (SAXS), solid-state ¹³C cross-polarization magic-angle spinning nuclear magnetic resonance (¹³C CP/MAS NMR), and polarized light microscopy with a waveplate were used to study the physical structure of wheat bran and its dissected layers. The XRD and solid-state ¹³C CP/MAS NMR both confirmed the presence of crystalline cellulose in untreated bran, enzymatically treated bran, and dissected bran layers. The outer pericarp had the highest crystallinity of the dissected bran layers and showed negative birefringence. The aleurone layer was low in cellulose content and completely amorphous, yet the cell walls in the aleurone layer showed strong positive birefringence. The treatment of destarched and deproteinated bran with the Updegraff reagent removed amorphous material, leaving its crystalline cellulose structure intact. Hydration of the outer pericarp increased its crystallinity index and CP/MAS NMR resonance intensity, which indicated a possible increase in polymer order. The SAXS also confirmed that cell wall polymers, possibly aggregated cellulose microfibrils, increased in order as a result of hydration.     

花生果种皮特异表达基因AhPSG13的克隆和表达研究

为了克隆在花生果种皮中特异表达的基因,本实验从花生果皮种仁抑制消减文库中筛选出一个277bp的EST序列并进行研究,通过构建花生果皮全长cDNA文库,获得此目的基因G13的全长为1369bp,开放阅读框从第28个碱基始至1122个碱基止,预测分子量为39865.59,等电点5.73。生物信息学分析表明该基因编码的蛋白具有4个跨膜结构域和多个活性位点,可能与细胞内DNA转录有关;该蛋白与多种豆科作物的半胱氨酸蛋白酶具有较高同源性,推测为花生半胱氨酸蛋白酶相关基因;RT-PCR研究该基因表达,结果显示该基因在果种皮中特异表达,于30d果皮中表达量最大。本研究克隆的AhPSG13基因序列已经登陆到GenBank,登录号为FJ475061。     

Quantities of phenolic compounds and their impacts on the perceived flavour attributes of rye grain

The use of whole grain rye products, beneficial to one's health, could be substantially extended if the typical intensively bitter flavour of rye could be modified without losing the characteristic rye-like flavour. The aim of the study was to evaluate the contribution of non-volatile phenolic compounds on the perceived flavour. Rye grain was milled into five milling fractions. The levels of phenolic compounds, i.e. the phenolic acids, alkylresorcinols and lignans, of the fractions were analysed and related statistically to sensory flavour profiles by partial least-squares (PLS) regression. The non-bound (free) phenolic acids are suggested to be most flavour-active. Cereal and intense flavour and aftertaste were related to vanillic and veratric acids, alkylresorcinol C_23:0, and other lignans except for pinoresinol. The perceived bitterness of the bran fractions was suggested to result from pinoresinol and syringic acid. Sinapic and ferulic acids, alkylresorcinols, except for alkylresorcinol C_23:0, and syringaresinol seemed to cause the germ-like flavour. Phenolic acids, alkylresorcinols and lignans distributed with a similar pattern in the rye grain as the flavour attributes: the phenolic compounds were clearly located in the outer bran fractions being intense in flavour, but not in the mild-tasting inner layers of the grain.     

Total plant sterols,steryl ferulates and steryl glycosides in milling fractions of wheat and rye

The total plant sterol, steryl ferulate and steryl glycoside contents in wheat and rye milling fractions show that there are significant differences in both sterol content and composition between various milling fractions collected from a commercial mill. Total sterols were analysed by gas chromatography after acid and alkaline hydrolyses. The steryl conjugates were first extracted with acetone, fractionated on solid phase extraction (SPE) cartridges and then analysed individually (steryl glycosides by gas chromatography and steryl ferulates by reverse phase-high performance liquid chromatography with UV detection. Differences in sterol contents of the wheat samples were greater than in the rye samples. The highest total sterol content was found in wheat germ, but surprisingly high sterol contents, that were comparable to bran, were found in some flour fractions. Contents of steryl ferulates were high in the bran fractions contributing up to 17% of total sterols. The variation in the content of steryl glycosides in the samples was lower and contributed less than 10% of total sterols. These results show that much of the bioactive components may be lost when certain flour fractions produced in common flour milling procedures are discarded. However, some of these fractions with significantly high sterol contents could possibly be introduced into milling products used in breadmaking and the food industry without greatly compromising consumer acceptability.     

Fractionation of cereal flour by sedimentation in non-aqueous systems. I. Development of the method and chemical characterisation of the fractions

A method for the fractionation of wheat, rye, and barley flours without using aqueous solvents was developed. The separation of protein and starch was based on differences in their densities. Therefore, ball-milled flour was suspended in a mixture of inert solvents (toluene/tetrachoroethene) with a density of 1.47 g/cm³ and centrifuged. Owing to its higher density, the starch fraction was obtained as sediment whereas the protein fraction (PF) formed a layer on the surface of the solvent because of its lower density. The PF was enriched in a solvent mixture with a density of 1.355 g/cm³ yielding a middle fraction (sediment) and the enriched PF (upper layer). The latter was then defatted with toluene (0.87 g/cm) providing a lipid fraction in addition. The influence of ball milling under air or in the sedimentation solvent on the yield and the purity of the fractions was studied. Three varieties of wheat, and one rye and barley variety were fractionated by the optimised method and the obtained fractions were characterised by chemical methods e.g. gel permeation chromatography, SDS electrophoresis, and a combined extraction/HPLC method.     

Fractionation of cereal flour by sedimentation in non-aqueous systems. II. Rheological characterisation and baking performance of the protein fraction

A previously described method for the non-aqueous fractionation of cereal flours by sedimentation in non-aqueous solvents was carried out using flours of three wheat cultivars differing in baking performance, as well as one rye and one barley flour. The method was based on differences in the densities of starch (higher) and protein (lower). Thus, suspending finely milled flour in an inert solvent mixture with a density in between the densities of starch and protein yielded a sedimented starch fraction and a protein-rich fraction at the surface of the solvent. Further purification of this upper fraction provided a protein fraction, a middle fraction, and a lipid fraction. The protein fractions were examined by means of rheological methods such as micro-extension tests and creep-recovery tests. They also were reconstituted to standard flour with a protein content of 13.5%, which was used for micro-scale baking tests. Compared to aqueous isolated gluten, the hydrated protein fractions from wheat were much more extensible and had a lower resistance to extension. The baking performance of the wheat protein fractions was superior to gluten and comparable to the native wheat flours. The protein fraction from rye gave a wheat-like bread crumb, whereas the barley protein was not suited for bread making.     

四个不同粒重水稻品种颖果发育的比较

 以粒重差异较大的4个水稻品种为供试材料,采用树脂切片、酶解胚乳细胞和显微观察等方法,比较研究了品种间在颖果生长、胚乳细胞增殖、果皮和胚乳结构等方面的差异,探讨了影响颖果生长的因素。 大粒品种颖果发育时间较小粒品种长,其胚乳细胞数、胚乳干质量及单个胚乳细胞平均干质量均高于小粒品种。在粒重相近的情况下,籼稻颖果发育和淀粉积累快于粳稻。与小粒品种相比,大粒品种子房壁细胞中淀粉粒多,子房壁细胞生长的持续时间长,果皮及背部维管束衰亡迟。 小粒品种胚乳外层细胞在花后7 d已转化成糊粉层细胞,大粒品种胚乳外层细胞要在花后10 d才转化成糊粉层细胞。 大粒品种的库容大和生理活性期长是其颖果能显著增大的生理原因。     

Antioxidant properties of wheat and rye bran extracts obtained by pressurized liquid extraction with different solvents

Rye and wheat brans are valuable sources of bioactive compounds, which could be used for the development and commercialization of high added value functional ingredients such as dietary antioxidants. The aim of this study was to evaluate antioxidant potential of rye and wheat bran using different polarity solvents. Cereal brans were ground to four different particle size fractions and extracted at 10.3 MPa pressure and 80 °C temperature by consecutive application of hexane, acetone and methanol:water (80:20%). The highest extract yield was obtained from rye bran using methanol-water; particle size in most cases had a significant effect. Antioxidant potential of extracts was assessed by ABTS^+•, DPPH^• scavenging, ORAC and total phenols content (TPC) assays. Extraction solvent had a major influence on TPC and antioxidant activity of the extracts. The most active extracts were obtained using methanol:water; rye bran extracts, in general, were stronger antioxidants than wheat bran extracts. For the majority of assays, reduction of particle size resulted in higher antioxidant activity values. However, ABTS^+• scavenging was found to decrease by decreasing particle size of rye bran used for extraction.     

Sulphur nutrition differentially affects the distribution of asparagine in wheat grain

Asparagine is known to accumulate in wheat grain under conditions of sulphur deficiency, leading to increased levels of acrylamide formation during processing. Analyses of milling fractions and of the outer layers of the grain prepared by hand dissection showed that the highest levels of asparagine were present in the bran fractions and in particular the aleurone layer, when grain were grown with sufficient sulphur supply. However, even mild S deficiency resulted in disproportional increases in the asparagine contents of white flour fractions, implying that optimisation of yield in a conventional milling system is not an appropriate strategy for processing grain from sulphur-deficient crops.     

Pseudocereals as alternative sources for high folate content in staple foods

Total folate content was determined in the pseudocereals amaranth (four varieties), quinoa and buckwheat in comparison to four cereal species (eight wheat varieties, four barley varieties, one oat variety, one rye variety). Amaranth and quinoa were found to possess very high total folate contents: in amaranth, total folate ranged from 52.8 to 73.0 and in quinoa it was 132.7 μg/100 g dm, about ten times as much as in wheat. The bran fractions contained on average 124% of total folate, while only 57% on average was present in the flour fractions.     

Evaluation of Tissue Dissociation of Durum Wheat Grain (Triticum durum Desf.) Generated by the Milling Process

The three major botanical components (starchy endosperm, aleurone layer and pericarp) of eight durum wheat samples exhibited significantly different compositions and concentrations in phenolic acids. The starchy endosperm, the aleurone layer and the pericarp were respectively characterised by a low content in ferulic acid (FA), a high content intrans -sinapic acid (t -SA), and a high content in ferulic acid dehydrodimers (DHD). These three chemical markers can be exploited to differentiate the three grain botanical parts within milling fractions and to evaluate the milling efficiency, particularly the separation between bran and endosperm. The histological dissociation of the wheat grain generated by the milling process can be investigated further into details using the three phenolic acids markers. A separability index (S i) was proposed in order to quantify the ease of dissociation of endosperm from bran. Differences in S i values between wheat varieties grown under various agricultural conditions demonstrated the relevant variability of this character. The structural and molecular factors implied in the control of tissue dissociation are discussed in details.     

黑麦MIKC型MADS-box家族基因的鉴定和表达分析

MIKC是MADS-box蛋白家族中一类保守的转录因子家族,参与调控植物的开花时间和花器官发育。通过对黑麦MIKC基因家族的分析,为研究黑麦各时期组织器官的功能奠定基础。利用PFam和BLASTP两种方法确定黑麦MIKC家族共47个蛋白序列,将以上蛋白序列利用TBtools软件进行理化性质、进化关系、保守基序和基因结构、共线性及聚类分析等生物信息学分析。将黑麦47个MIKC基因分为12个亚家族,共线性分析发现黑麦与小麦的共线性基因多于黑麦与水稻间的共线性基因,说明黑麦与小麦的亲缘关系更近。聚类结果结合黑麦物种内共线性分析发现,ScMIKC31基因仅在穗子表达,于其他植物组织器官及发育时期均无表达,选用课题组材料进行RNA-seq验证,结果与生物信息学结果一致,推测ScMIKC31基因为黑麦中与开花有关的基因。以上结果说明MIKC家族基因在黑麦中存在功能分化,为进一步研究该家族基因的功能提供信息参考。