Starch isolation method impacts soft wheat (Triticum aestivum L. cv. Claire) starch puroindoline and lipid levels as well as its functional properties

Wheat (Triticum aestivum L.) kernel hardness is a major quality characteristic, which has been ascribed to the presence of puroindolines a and b. These proteins occur in higher levels at the surface of water-washed starch granules from soft wheat cultivars than at that of starch from hard wheat cultivars. In the present study, prime starch was isolated from flour from soft wheat (cultivar Claire) using a dough ball or batter based separation method. Starch isolated with the dough ball method contained lower levels of puroindolines, as well as of other starch granule associated proteins and lipids than that isolated with the batter method. Similar patterns of puroindoline and lipid levels after starch isolation can presumably be related to (polar) lipid binding by puroindolines. Both isolated starch fractions showed comparable differential scanning calorimetry thermograms, whereas higher levels of starch surface associated components restricted starch swelling. Necessary controls demonstrated that the observed differences did not arise from artefacts associated with hydration, fractionation or freeze-drying in the experimental protocols. Apparently, proteins and lipids at the starch granule surface impact water absorption and, as such, starch swelling, but they do not affect starch granule internal phenomena such as melting of the crystalline amylopectin chains.     

Variation in polar lipid composition among near-isogenic wheat lines possessing different puroindoline haplotypes

The exact mechanism underlying wheat (Triticum aestivum L.) kernel hardness is unknown. Similar to puroindoline proteins, polar lipids are present on the surface of starch granules. The objective of this research was to determine the specific polar lipid species present on the surface of wheat starch from near-isogenic wheat lines that have different puroindoline haplotypes and endosperm hardness. Four near-isogenic wheat lines were used in this study, all derived from the soft cultivar Alpowa. Direct infusion tandem mass spectrometry was used to identify the lipid species in whole-meal, flour and starch samples. Endosperm hardness had no significant effect on the polar lipid contents in wheat whole-meal, a slight influence on the polar lipid contents of the flour fractions and a significant influence on the polar lipid composition of the polar lipids located on the surface of wheat starch. The greatest quantities of polar lipids on the starch-surface occurred when both puroindoline proteins were present in their wild-type form. Starch-surface polar lipid content dramatically decreased when one of the puroindoline proteins was null or if pin-B was in the mutated form. The least amount of polar lipids was present when pin-B was in its mutated form and pin-A was in its wild-type form.     

Puroindolines co-localize to the starch granule surface and increase seed bound polar lipid content

Endosperm texture in wheat is controlled by the Pina and Pinb genes that comprise the Hardness (Ha) locus. Studies have shown that soft and hard varieties differ in the amount of starch bound polar lipids but have not addressed whether PINs are directly involved and whether the presence of one particular PIN affects seed polar lipid levels and cellular localization. Here, we determined the effect of overexpressing PINA or PINB on seed bound polar lipids and PIN localization. F₃ recombinants homozygous for either a Pina or Pinb null Ha locus with or lacking a transgenically added Pina or Pinb were analyzed for grain hardness, PIN abundance, and seed bound polar lipid levels. Overexpressed PINs resulted in reduced hardness, increased starch bound PINs, and increased seed bound polar lipids. Addition of PINA to the PINA nulls or PINB to the PINB nulls resulted in higher bound polar lipid levels than the addition of the alternative PIN. Both PINs localized to the starch surface in the presence or absence of the other protein. Our results indicate that PIN overexpression results in reduced endosperm texture and increased seed bound polar lipids and that PINs independently localize to the surface of starch granules.     

Amylose and Lipid Contents,Amylopectin Structure,and Gelatinisation Properties of Waxy Wheat (Triticum aestivum) Starch

Starch was isolated from the endosperm of three recently developed waxy wheat lines and their parents. Their amylose and lipid contents, amylopectin structures and gelatinisation properties were evaluated. The endosperm starch from waxy wheat lines is composed essentially of amylopectin. The apparent amylose (1·2–2·0 g/100 g) and lipid contents (0·12–0·29 g/100 g) are much lower than the apparent amylose (26·0–28·4 g/100 g) and lipid contents (1·05–1·17 g/100 g) of their non-waxy parents. The amylopectin of waxy wheat lines is structurally identical to that of the parents. The peak gelatinisation temperature and gelatinisation enthalpy for waxy starch are significantly higher than for non-waxy starch, but the gelatinisation enthalpy for the amylopectin fraction of waxy starch is nearly identical to that of non-waxy starch.     

Surface properties and locations of gluten proteins and lipids revealed using confocal scanning laser microscopy in bread dough

Surface properties of gluten proteins were measured in a dilation test and in compression and expansion tests. The results showed that monomeric gliadin was highly surface active, but polymer glutenin had almost no surface activity. The locations of those proteins in bread dough were investigated using confocal scanning laser microscopy and compared with polar and nonpolar lipids. Added gluten proteins participated in the formation of the film or the matrix, surrounding and separating individual gas cells in bread dough. Gliadin was found in the bulk of dough and gas ‘cell walls’. Glutenin was found only in the bulk dough. Polar lipids were present in the protein matrix and in gas ‘cell walls’, as well as at the surface of some particles, which appeared to be starch granules. However, nonpolar lipid mainly occurred on the surface of particles, which may be starch granules and small lipid droplets. It is suggested that the locations of gluten proteins in bread dough depends on their surface properties. Polar lipid participates the formation of gluten protein matrix and gas ‘cell walls’. Nonpolar lipids may have an effect on the rheological properties by associating with starch granule surfaces and may form lipid droplets.     

小麦籽粒发育时期Puroindolines蛋白与硬度的关系

为探讨Puroindolines蛋白的表达特点与籽粒硬度的关系,采用改进的SDS-PAGE凝胶分析了不同硬度小麦品种的籽粒在各个发育时期Puroindolines蛋白的表达.结果表明,不同硬度的小麦籽粒中总Puroindolines(PinA和PinB)蛋白的表达量差异不大,但与胚乳淀粉颗粒结合的Puroindolines蛋白量差异非常明显:在籽粒发育的不同时期,软质小麦籽粒淀粉粒表面结合的Puroindolines蛋白量显著高于硬质小麦;基因型同为野生型但硬度有差异的品种,籽粒较软的材料其淀粉粒表面结合的Puroindolines蛋白量也明显高于较硬的材料,说明该蛋白的结合特性是决定籽粒硬度的直接原因.结果还表明,胚乳中水溶性戊聚糖与籽粒硬度关系密切.     

The composition of floury and vitreous endosperm affects starch digestibility kinetics of the whole maize kernel

Maize grain starch is the major energy source in animal nutrition, and its high digestion and utilization largely depend on endosperm traits and the structure of the starch-lipoprotein matrix. The aim of this work was to determine floury and vitreous endosperm traits and its relation to starch digestibility rate. In total, kernels of 30 hybrids were manually dissected, and amylose, total zein and starch and non-starch lipids were determined in both vitreous and floury endosperm. Starch digestibility of the whole kernel was determined based on glucose released during a two-step in vitro pig model of enzymatic digestion, and starch digestibility rate was calculated according to the first-order kinetics. The vitreous endosperm of tested hybrids had higher contents of amylose (204.6 vs 190.4 g/kg), zein (63.2 vs 40.4 k/kg) and starch lipids (5.6 vs 4.9 g/kg), and lower content of non-starch lipids (7.3 vs 9.6 g/kg) than floury endosperm. Digestibility coefficients varied among hybrids, and starch digestibility rate varied from 0.73 to 1.63 1/h. Lipids in both vitreous and floury endosperm negatively correlated with the most of digestion coefficients, whereas zein correlated in vitreous and amylose in the floury endosperm (P < 0.05). Starch digestibility rate negatively correlated with all traits, except amylose content in vitreous endosperm. As a result, a linear regression model with four variables including contents of zein and starch lipids in vitreous and zein and amylose in floury endosperm can predict more than 65% variability of starch digestibility rate of tested hybrids.     

Potential use of exogenous lipases for DATEM replacement to modify the rheological and thermal properties of wheat flour dough

The effect of two lipases and DATEM at different concentrations on the rheological and thermal properties of white and whole wheat flour doughs was determined by farinograph, extensograph, texture analyzer and differential scanning calorimetry. Lipases provided similar or better results than that of DATEM on decreasing softening degree and stickiness, and increasing stability, maximum resistance to extension and hardness. Extensibility and energy did not change with lipases, but decreased with DATEM. Lipases and DATEM slightly modified the transition temperatures and enthalpies of ice-melting and starch gelatinization, but significantly increased the moisture and unfrozen water contents, and decreased the solid concentration in unfrozen water. The greatest effect of lipases was observed on decreasing the peak temperature and increasing the enthalpy of amylose–lipid complex dissociation. The changes in the rheological and thermal properties were almost independent of the concentration levels tested, and were mostly reduced when whole wheat flour was used. The improvement in the dough properties by lipases is possibly related to the in situ formation of surface active compounds from endogenous flour lipids. Presumably, these compounds are already located in the right positions in dough, where they might have higher interaction potentials than DATEM with the dough components.     

Impact of lipases with different substrate specificity in wheat flour separation on the properties of the resultant gluten

Wheat gluten was isolated in a laboratory dough-batter flour separation process in the presence or absence of lipases differing in hydrolysis specificity. The obtained gluten was blended with wheat starch to obtain gluten-starch (GS) blends of which the water and oil binding capacities were investigated. Furthermore, GS blends were mixed into dough and processed into model breads, of which dough extensibility and loaf volume were measured, respectively. In comparison to GS blends prepared with control gluten, oil binding capacity was higher when GS blends contained gluten isolated with Lecitase Ultra (at 5.0 mg enzyme protein/kg flour), a lipase hydrolyzing both non-polar and polar lipids. Additionally, dough extensibility and total work needed for fracture were lower for dough prepared from GS blends containing gluten isolated with Lipolase (at 5.0 mg enzyme protein/kg flour), a lipase selectively degrading non-polar lipids. In GS blend bread making, this resulted in inferior loaf volumes. Comparable GS blend properties were measured when using control gluten and gluten isolated with YieldMAX, a lipase mainly degrading N-acyl phosphatidylethanolamine. In conclusion, properties of GS blend model systems are altered when gluten prepared in the presence of lipases is used to a degree which depends on lipase specificity and concentration.     

Mechanism of gas cell stabilization in breadmaking. II. The secondary liquid lamellae

The study for the first time demonstrates that flour lipids at their natural levels do not affect dough rheology as measured by bubble inflation, thus indicating the presence of liquid lamellae as an independent secondary gas cell stabilizing mechanism in bread dough. The liquid lamellae, stabilized by adsorbed surface active compounds, plays its role during the later proving and early baking stage, when discontinuities occur in the gluten–starch matrix surrounding gas bubbles. To study this secondary stabilizing mechanism, different lipid fractions were added incrementally to the defatted flours. No effects were observed on the rheological properties of the dough. However, large effects on the loaf volume were measured. The additives used were the total flour lipid and its polar and non-polar fractions and the fatty acids palmitic, linoleic and myristic. Polar lipids and palmitic acid had positive or little effect on loaf volume, respectively. Non-polar lipid, linoleic and myristic acids had negative effects on loaf volume. The different effects of the lipid fractions are thought to be related to the type of monolayer that is formed. Polar lipid and palmitic acid form condensed monolayers at the air/water interface whereas non-polar lipid, linoleic and myristic acids form expanded monolayers.     

Endosperm Texture in Wheat

One of the fundamental means of classifying wheat is through its endosperm texture. It impacts significantly on the milling process affecting among other things flour particle size and milling yield. Hardness in wheat is largely controlled by genetic factors but it can be affected by the environment and factors such as moisture, lipid, and pentosan content. The principal genetic locus controlling endosperm texture in wheat, Ha, is located on the chromosome 5D. At this locus several genes, notably the puroindolines, have been identified. Puroindolines are the major components of the 15 kDa protein band associated with starch granules that is more abundant in soft wheats than in hard. Recently the puroindolines have been shown to enhance grain hardness in rice. In this review we discuss the structure of hard and soft wheat endosperm with particular emphasis on when differences in endosperm texture can be detected in the developing seed. The role of the environment and other factors that may affect the endosperm texture is also examined together with the role of the puroindoline genes at theHa locus. Finally, we compare endosperm hardness in wheat and in barley.     

面粉高白度小麦种质资源筛选及其品质分析

为了挖掘面粉白度高、品质优的小麦种质,对1 248份小麦种质资源进行了面粉白度测定,并对面粉白度值超过80的小麦种质的籽粒硬度、淀粉RVA糊化特性以及面团揉混特性等有关品质性状进行了分析。筛选出面粉白度值≥80的小麦种质195个,其籽粒硬度指数分布范围为15～77;淀粉RVA糊化特性参数峰值黏度范围为1 030～3 407cP,稀懈值范围为629～1 522cP,回生值范围为5～1 546cP,糊化温度范围为65.3～88.2℃,最终黏度范围为755～3 870cP;面团形成时间范围为1.3～4.2min,衰落角范围为3～35度,沉降值范围为21～58.7mL。研究结果还表明,面粉中类胡萝卜素含量低,则面粉白度就高,仅靠延长面粉储存时间不会大幅度提高面粉白度。筛选出的14个高白度优异小麦种质可用于培育中强筋面粉高白度小麦新品种。     

Composition and surface properties of dough liquor

The composition and surface properties of dough liquor isolated by ultracentrifugation have been characterised. Addition of ascorbate had no effect and salts only a limited effect, on the yield, protein content and composition of the dough liquor. Fourier transform infrared spectroscopy (FT-IR) revealed the presence of proteins, lipids, starch oligosaccharides together with the non-starch polysaccharide, arabinoxylan. At high dilution the dough liquor air:water interface was dominated by protein, with surface tensions of around 55 mN/m and high surface elasticity. As the concentration was increased, surface tensions dropped to around 40 mN/m for undiluted dough liquor. This was accompanied by the interface becoming less elastic, and indicated that dough liquor lipids were interacting and disrupting the protein films in concentrated dough liquor. Dough liquors from de-fatted flours remained elastic and gave surface tension values of around 50–55 mN/m even at low dilution, indicating that removal of the lipids gave rise to a purely protein stabilised interface. Addition of salt to the dough had the greatest effect on the surface properties, both reducing surface tension and reducing surface elasticity, probably because the charge screening effect of the salt improved the dispersion of lipids in the dough liquor, thus enabling it to disrupt the protein films more effectively. These results indicate that the aqueous phase of bread doughs lining the gas cells would give rise to a mixed protein:lipid interface. Such interfaces are unstable, and would contribute to the instability of the foam structure of risen dough. In addition they show that dough ingredients may modify gas cell stability (and hence may affect crumb structure), by altering the composition and properties of the aqueous phase of doughs.     

Flour constituent interactions and their influence on dough rheology and quality of semi-sweet biscuits: A mixture design approach with reconstituted blends of gluten, water-solubles and starch fractions

The aim of this study was to identify the biochemical parameters that alter the soft wheat flour functionality for biscuit-making quality. A 9-point simplex centroid was used to investigate the effect of varying the ratios of gluten, water-solubles and starch-fractions isolated from three different flour grades (patent, middle-cut and clear flours) which exhibited a wide range of compositional and functionality characteristics on the dough rheological behaviour and the semi-sweet biscuit quality parameters. The amounts of soluble and insoluble proteins and pentosans as well as the endogenous lipids in each flour fraction were quantified. Dough consistency, elongational viscosity, hardness, half-relaxation time, relaxation rate constant, cohesiveness and springiness as well as biscuit density, firmness, tearing force and spatial frequency for the different flour fraction combinations were also assessed. Regression models have been developed to predict the responses of the rheological attributes of the dough as well as the biscuit quality characteristics to the compositional changes of the flour blends; in addition to the main linear terms (concentration of starch, gluten and water-solubles isolated from the different flour grades), significant interaction terms were identified which cannot be neglected in any prediction scheme for the dough and biscuit properties. Contour plots were drawn in an effort to better understand the overall property responses of the dough and biscuits. Significant relationships among certain dough rheological parameters and biscuit characteristics were found, implying a functional role for the total, soluble and insoluble proteins, pentosans and lipids in biscuit making.     

Buckwheat Seed Milling Fractions: Description, Macronutrient Composition and Dietary Fibre

The structure of the mature buckwheat achene and groat is discussed in relation to milling fractions and nutritional composition. Whole groats contain 55% starch, 12% protein, 4% lipid, 2% soluble carbohydrates, 7% total dietary fiber (TDF), 2% ash, and 18% other components (organic acids, phenolic compounds, tannins, phosphorylated sugars, nucleotides and nucleic acids, unknown compounds). The composition of the milling fractions reflects the relative abundance of seed tissues. Starch is concentrated in the central endosperm. Protein, oil, soluble carbohydrates and minerals are concentrated in the embryo. Commercial «Fancy» flour, a light-coloured flour, is mostly central endosperm and contains 75% starch, 6% protein, 1% lipid, 1% soluble carbohydrates, 3% TDF, 1% ash, and 13% other components. Although the embryo traverses the central endosperm, during milling parts of the embryo separate with the aleurone and seed coat in the bran fraction. Bran, with little central endosperm, contains 18% starch, 36% protein, 11% lipid, 6% soluble carbohydrates, 15% TDF, 7% ash, and 7% other components. Buckwheat bran also is a rich source of TDF and soluble dietary fibre (SDF), particularly bran with hull fragments (40% TDF of which 25% is SDF), while bran without hull fragments has 16% TDF of which 75% is SDF.     

The effects of puroindoline b on the ultrastructure of endosperm cells and physicochemical properties of transgenic rice plant

Endosperm texture is an important factor governing the end-product quality of cereals. The texture of wheat (Triticum aestivum L.) endosperm is controlled by puroindoline a and b genes which are both absent in rice (Oryza sativa L.). It has been reported that the endosperm texture of rice can be modified by puroindoline genes. The mechanism, however, by which puroindolines affect the ultrastructure of rice endosperm cells remains to be investigated. In this study, we observed the ultrastructure of endosperm cells and the morphology of isolated starch granules of the transgenic rice expressing the puroindoline b gene. SEM and TEM observations indicated that compound starch granules were embedded within the matrix material in non-transgenic rice, Nipponbare, whereas they were surrounded by spaces in the transgenic rice. The morphology and size of each starch granule were not different between non-transgenic and the transgenic rice. However, the transgenic rice flour showed smaller particle size, higher starch damage, and lower viscosity during gelatinization than that of non-transgenic rice. These results confirm that puroindoline b reduces the grain hardness in rice. Moreover, the results also suggest that puroindoline b functions at the surface of compound starch granules, and not on polygonal starch granules in rice endosperm.     

Variation in Granule Bound Starch Synthase I (GBSSI) loci amongst Australian wild cereal relatives (Poaceae)

A complex cascade of enzymes is responsible for the development of starch granules in grain endosperm. Granule Bound Starch Synthase I (GBSSI), encoded by the Waxy gene, is a key enzyme of starch synthesis and determines the accumulation of amylose in the starch granules. The complete genomic GBSSI sequence was ascertained for eight Australian cereal wild relatives (CWR) to determine diversity within the gene. A phylogeny derived from the coding sequence of the entire Waxy gene was compared to established phylogenetic relationships. Starch granule morphology observed in conjunction with this phylogeny suggests that small polygonal starch granules arranged as compound granules are the ancestral state, evolving subsequently to bimodal starch granules and to larger simple granules. Genomic sequence length varied within the species from 2800 to 3572 bp. Most variation occurred within the intron sequences, the largest insertion showing strong homology to a retrotransposon. One wild species was determined to have a deletion in the 3′-end of exon 1 resulting in a putatively non-functional allele. Alignment of the amino acid sequence showed strong homology throughout the central fragments of the gene but broad variation in the transit peptides. All putative functional alleles maintained the reported active sites for glycogen synthesis, though with variations in other highly conserved areas of the gene. These variations within the wild relatives of cultivated cereals may provide novel sources of genetic diversity for future cereal improvement programs.     

Scanning Electron Microscopic Study of Dough and Chapati from Gluten-reconstituted Good and Poor Quality Flour

Hard and soft wheat flours, which were used in the study, resulted in good and poor quality chapatis respectively. Gluten was isolated and interchanged among the two whole wheat flours and studied by scanning electron microscopy for its influence on structural characteristics of dough and its relation to chapati-making quality. Microscopic observations clearly indicated that larger gluten strands covered starch granules in hard wheat flour dough, while gluten was short and starch granules exposed in dough prepared from soft wheat flour. Greater film forming ability of gluten in hard wheat flour dough manifested in long and bulky starch strands interwoven with protein matrix in its chapati crumb. Higher moisture retention and starch gelatinization as a consequence of greater film forming ability of gluten in hard wheat flour resulted in pliable and soft textured chapati.     

Effect of Barley and Barley Components on Rheological Properties of Wheat Dough

The effects of addition of whole barley and barley components (starch, β-glucans and arabinoxylans) on rheological properties of dough prepared from wheat flours with variable gluten quality (cv. Glenlea, extra-strong; cv. Katepwa, very strong; cv. AC Karma, strong; and cv. AC Reed, weak) were investigated in these studies using Mixograph and dynamic rheological measurements. Whole barley meal, starch and non-starch polysaccharides from hulless barley with variable starch characteristics (normal, high amylose, waxy, and zero amylose waxy) were tested. Upon addition of either β-glucans or arabinoxylans, significant increases in peak dough resistance, mixing stability, and work input were recorded in all flours. The addition of starch to various wheat flours reduced the strength of the respective flour-water doughs. The improvement of dough strength upon addition of waxy or zero amylose waxy barley meal was associated with the high content of total and soluble β-glucans present in barley samples. The addition of arabinoxylans or β-glucans increased the G′ of wheat doughs; arabinoxylans had a greater effect than β-glucans. Starch substantially decreased the elastic modulus of dough prepared from cv. Glenlea but waxy and high amylose starches increased the G′ of dough prepared from cv. AC Karma. A combination of the high amounts of non-starch polysaccharides and unusual starch characteristics in barley seems to balance the negative effects associated with gluten dilution brought about by addition of barley into wheat flour.     

灌浆期遮阴对小麦胚乳淀粉粒度分布及糊化特性的影响

为了解灌浆期遮阴对小麦胚乳淀粉粒度分布及淀粉糊化特性的影响,研究了灌浆期对扬麦13、宁麦13和烟农19进行30%遮阴后籽粒的淀粉粒度分布与糊化特性的变化。结果表明,小麦灌浆期遮阴显著降低了<10μm的B型淀粉粒体积和表面积占比,增加了>10μm的A型淀粉粒的体积和表面积占比,其中A型淀粉粒中主要增加了10~20μm淀粉粒的体积和表面积占比;遮阴处理对淀粉粒数目分布无显著影响。遮阴处理后,峰值粘度、低谷粘度、稀懈值、最终粘度、回升值等糊化参数指标均显著降低。小麦籽粒硬度、容重、出粉率等指标也因遮阴处理而显著降低。综上所述,遮阴处理改变了小麦籽粒淀粉粒粒度分布,降低了淀粉糊化参数和加工品质。