Variation in polar lipids located on the surface of wheat starch

It is unknown whether starch isolated before dough development has the same surface lipid composition as starch isolated after dough development. The abundance of starch surface polar lipids is related to the physical hardness of the endosperm, but the variation in specific lipid classes and molecular species is unknown. The objective of this study was to determine the variation in polar lipids present on the surface of wheat starch granules. The experimental wheat lines used are, within each set, near-isogenic to each other but vary in endosperm hardness. Starch was isolated using two different processes: a dough and a batter method. Direct infusion electrospray ionization tandem mass spectrometry was used to identify and quantitatively determine the polar lipid species in wheat flour and on starch. Wide ranges in starch surface polar lipid concentrations were observed between the starch isolation methods. Starch isolation method provided a greater source of variation than did wheat kernel hardness. When dough is optimally mixed, lipids originally on the surface of wheat starch are dissociated, whereas in a batter system, starch surface lipids stay associated with the starch surface. The predominant starch surface polar lipids were digalactosyldigylcerol (DGDG), monogalactosyldigylcerol (MGDG) and phosphatidylcholine (PC) polar lipid classes.     

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.     

Fluorimetric studies of the interactions of wheat puroindolines with polar lipids on the surface starch granules

The interactions of puroindolines with polar lipids were investigated using polarization of fluorescence probes preincorporated into a liposomal bilayer containing PC, PI, PS, MGDG, DGDG, and sulfolipids. The intrinsic fluorescence of Trp residue method was also used. Regardless of the kind of lipid used for liposome preparation, proteins interacted with the liposomes. Conformational changes of the proteins were observed simultaneously with the change in the molecule packing in the lipid bilayer of the liposomes. Puroindoline interactions with the surface of the liposomes have explicit importance for the net charge of this surface. The strong interaction between the proteins and lipids takes place in the presence of a ligand with a negative charge. The obtained results confirm that lipids take part in puroindoline–starch granule surface interactions.     

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.     

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.     

Dough/crumb transition during French bread baking

The modifications occurring during dough to crumb (D/C) transition of French bread (350 g) were studied in an instrumented pilot-scale oven for doughs with different contents of minor components, soluble, lipids and puroindolines. Internal temperature measurements showed that, for most compositions, complete D/C transition occurred between 55 and 70 °C, after 5 min of baking, and coincided with maximum loaf expansion. Differential scanning calorimetry (DSC) in excess of water performed on samples taken during baking (3 and 5 min) showed that starch gelatinization and melting developed continuously during D/C transition for various contents of the soluble fraction in dough. Dynamic thermomechanical analysis (DMA) on dough showed that dough stiffened between 60 and 70 °C, as seen by the increase of elastic modulus E′ by more than one decade, for all dough compositions. Relating these changes to the results of baking experiments, D/C transition was assigned first to gluten reticulation and, to a lesser extent, to continuous starch granule swelling.     

糯小麦籽粒淀粉粒度分布特征

为进一步加深对糯小麦淀粉特性的认识,选用2个糯小麦和2个普通非糯小麦品种为材料,利用激光粒度分析仪分析了糯小麦完熟籽粒中淀粉的粒度分布特征,及其与普通非糯小麦品种的差异。结果表明,糯小麦淀粉粒的粒径分布范围为1.0~43.7 μm,不同粒径淀粉粒的数目比例分布呈单峰曲线变化,体积和表面积分布均呈双峰曲线变化;糯小麦籽粒中存在2种类型淀粉粒,即A型大淀粉粒（直径≥10 μm）和B型小淀粉粒（直径<10 μm）,B型淀粉粒的数目、体积和表面积比例均高于A型淀粉粒。与普通非糯小麦相比,糯小麦籽粒中B型小淀粉粒的数目、体积和表面积比例较高,淀粉粒的平均粒径和中位粒径较大。糯小麦与普通非糯小麦不同粒径淀粉粒的数目、体积和表面积比例分布曲线存在差异。糯小麦B型淀粉粒处的峰高显著高于普通非糯小麦,A型淀粉粒处的峰值低于后者。糯小麦体积和表面积分布中B型淀粉的峰值粒径显著小于普通非糯小麦。     

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.     

Variation in Friabilin Composition as Determined by A-PAGE Fractionation and PCR Amplification, and its Relationship to Grain Hardness in Bread Wheat

A-PAGE fractionation of starch granule proteins from 63 bread wheat cultivars with contrasting grain texture characteristics revealed two prominent polypeptides and three minor ones, approximately 15 kDa in size. These proteins were found to be encoded by genes on the short arm of chromosome 5D. The two major friabilin components were assumed to correspond to puroindolines a and b (pinA and pinB), as suggested by PCR amplification of genes coding for pinA, glycine-type or serine-type pinB. Two electrophoretic patterns for pinA (presence vs absence) and three patterns for pinB were obtained by A-PAGE. In cultivars with pinA (allele Pina-D1a), pinB was found to be encoded by wild-type Pinb-D1a, serine-type Pinb-D1b or by the novel glycine-type b1 allele. Cultivars lacking pinA (allele Pina-D1b) were shown to contain eitherPinb-D1a or the novel b2 allele, both alleles coding for glycine-type pinB. The intensity of pinB in A-PAGE gels was found to be associated with grain hardness as determined by the SKCS method. Cultivars lacking pinA had the highest SKCS values, suggesting that both pinA and pinB may affect grain texture. In the presence of pinA, cultivars with wild-type allelePinb-D1a had soft grain texture, whereas those with alleles Pinb-D1b or b1 showed increased grain hardness. It is suggested that allele b1 affects the interaction of pinB with starch granules because of a sequence mutation different from the glycine-to-serine change.     

Wheat (Triticum aestivum L.) puroindoline functionality in bread making and its impact on bread quality

Wheat puroindolines (PINs) spontaneously adsorb at air/water interfaces and show excellent foaming properties. They can positively impact bread quality, in which the formation of stable foam is important for product quality. The impact of endogenous PINs on bread quality was studied by preparing gluten–starch blends from isolated gluten and starch fractions with different PIN levels, which allowed largely retaining the interaction between PINs and flour components. Our results indicate that blends with high PIN levels yielded more homogeneous crumb structures with fine gas cells than bread made with blends containing medium or low PIN levels. However, the mechanism by which PINs exert this crumb improving effect is not clear. Varying PIN levels impacted neither dough extensibility nor did it result in different PIN levels in dough liquor. Lipid removal yielded bread with a less homogeneous crumb gas cell distribution, indicating that lipids also are required to obtain good crumb structure.     

A novel hardness-related and starch granule-associated protein marker in wheat: LMW-GS-‘S’

Starch granule (SG)-associated proteins are involved in starch synthesis and the interaction between SGs and the endosperm protein matrix. In this study, SG proteins were sequentially extracted with the chaotropic reagent, urea from 1 M to 4 M, and then profiled using an integrated proteomic approach including one- and two-dimensional electrophoresis, mass spectrometry and antibody-based enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the SG-associated proteins were dominated by granule-bound starch synthase (GBSS), gliadin, low molecular weight glutenin subunits (LMW-GS), serine protease inhibitors, α-amylase inhibitors and puroindolines. A protein with an apparent molecular mass of 50 kDa, expressed in cultivar hard wheat Kukri but not in soft wheat Triller was identified as a novel member of the ‘S’ group of LMW-GS, designated as LMW-GS-‘S’. Further characterization using a broad wheat population revealed that LMW-GS-‘S’ was selectively expressed in hard wheat cultivars while deleted in all soft wheats tested. Its relationship with hardness was confirmed by its expression in tetraploid durum wheats, which are among the hardest wheats around the world. Monoclonal antibody (MAb) F8-14E6 against LMW-GS-‘S’ was developed and used in an ELISA to screen 90 Glu allele-defined doubled haploid Janz/Kukri wheat lines. The allele that encodes LMW-GS-‘S’ was mapped to GluB3h (p < 0.001).     

Spectroscopic Characterisation of the Lipid-binding Properties of Wheat Puroindolines

The lipid-binding properties of wheat puroindolines (PINs) make these proteins likely candidates to play an important role in the stability of lipid films in the gas cells of bread dough, an important aspect of bread making. Therefore, the interaction between PINs and water-soluble model lipids was investigated by fluorescence emission and circular dichroism (CD) spectroscopy. The secondary structure of PIN-a and PIN-b in solution, as determined by far-UV CD measurements, resembled that of plant non-specific lipid transfer protein (LTP). However, PINs contain a tryptophan-rich loop located at the exterior of the protein. It was shown by fluorescence emission spectroscopy and near-UV CD spectroscopy that this domain is involved in the lipid binding. Fluorescence titration experiments of PIN and its synthetic tryptophan-rich domains with the zwitterionic lipidn-hexadecylphosphocholine (C16PN) revealed that the binding was cooperative. For the binding of the charged lipids,n-hexadecacylphosphoglycol (C16PG) and hexadecyl-trimethylammoniumbromide (CTAB), a clear effect of ionic strength of the solution was obtained. Organisation of lipids into micelles was not a prerequisite for binding to PINs. Most likely, wheat PIN binds to monomeric lipid molecules via its tryptophan-rich domain, rendering the protein more hydrophobic. The overall secondary structure of wheat PINs did not change significantly upon binding to lipids.     

Study of the mechanism of improvement due to waxy wheat flour addition on the quality of frozen dough bread

Waxy wheat flour (WWF) was substituted for 10% regular wheat flour (RWF) in frozen doughs and the physicochemical properties of starch and protein isolated from the frozen doughs stored for different time intervals (0, 1, 2, 4 and 8 weeks) were determined to establish the underlying reasons leading to the effects observed in WWF addition on frozen dough quality. Using Nuclear Magnetic Resonance (NMR), Differential Scanning Calorimeter (DSC) and X-ray Diffraction (XRD) among others, the gluten content, water molecular state, glutenin macropolymer content, damaged starch content, starch swelling power, gelatinization properties, starch crystallinity and bread specific volume were measured. Compared to RWF dough at the same frozen storage condition, 10% WWF addition decreased dry gluten and glutenin macropolymer contents and T₂₃ proton density of frozen dough, but increased the wet gluten content, T₂₁ and T₂₂ proton density. 10% WWF addition also decreased damaged starch content, but increased starch swelling power, gelatinization temperature and enthalpy, crystallinity of starch and bread specific volume of frozen dough. Results in the present study showed that the improvement observed due to WWF addition in frozen dough bread quality might be attributed to its inhibition of redistribution of water molecules bound to proteins, increase in damaged starch content and decrease in starch swelling power.     

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.     

小麦淀粉的粒度分布、组分及糊化特性对氮硫肥的响应

为明确氮硫肥对小麦淀粉粒度分布及主要理化特性的影响,以强筋小麦品种西农9718和中筋品种陕农138为材料,采用不同氮硫肥水平进行处理,系统分析了小麦A、B淀粉的粒度分布、淀粉组成、膨胀势及糊化特性对氮硫肥的响应。结果表明,强筋和中筋小麦品种的淀粉粒度分布基本一致。A淀粉粒体积分布占总淀粉的74.95%～75.43%,但淀粉粒数量远少于B淀粉粒。A淀粉的直链淀粉、峰值黏度、低谷黏度、崩解值、最终黏度以及回生值高于B淀粉,而B淀粉有更高的支链淀粉、膨胀势和糊化温度。不同氮硫肥处理改变了小麦淀粉的粒度分布,进而影响淀粉的组成和糊化特性。在施氮(230kg.hm-2)条件下,强筋品种西农9718和中筋品种陕农138的A、B淀粉分别在硫肥施用量为46和56kg.hm-2时有较低的直链淀粉含量、较高的膨胀势及较好的糊化特性。陕农138淀粉的粒度分布、组成及糊化特性对氮硫肥的响应比西农9718更敏感。适当的氮、硫肥配施有利于改善小麦的淀粉品质。     

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.     

Mechanism of gas cell stabilization in bread making. I. The primary gluten–starch matrix

Expansion of dough and hence bread making performance is postulated to depend on a dual mechanism for stabilization of inflating gas bubbles. Two flours were used in this study, one from the wheat variety Jagger (Jagger) and the other from a composite of soft wheat varieties (Soft). Thin liquid lamellae (films), stabilized by adsorbed surface active compounds, act as an auxiliary to the primary gluten–starch matrix in stabilizing expanding gas cells and this mechanism operates when discontinuities begin to appear in the gluten–starch matrix during later proving and early baking stages. Contributions of the liquid lamellae stability to dough expansion were assessed using flours varying in their lipid content. Incremental addition of natural lipids back into defatted flour caused bread volume to decrease, and, after reaching a minimum, to increase. Strain hardening is a key rheological property responsible for stabilizing the primary gluten–starch matrix. Jagger gave higher test-bake loaf volume than Soft and higher strain hardening index for dough. The different lipid treatments were found to have negligible effects on strain hardening index. Image analysis of crumb grain revealed that differences in number of gas cells and average cell elongation with different lipid treatments were insignificant. The evidence agrees with a dual mechanism to stabilize the gas cells in bread dough. To understand dough rheology at a molecular level, rheological properties of doughs were varied by addition of flour protein fractions prepared by pH fractionation. Fractions were characterized by SE-HPLC and MALLS. The molecular weight distribution (MWD) of fractions progressively shifted to higher values as the pH of fractionation decreased. Mixograph dough development time paralleled the MWD. However, the strain hardening index and the test-bake loaf volume increased with increasing MWD up to a point (optimum), after which they declined. At a given strain rate, the behavior at the optimum is thought to result from slippage of the maximum number of statistical segments between entanglements, without disrupting the entangled network of polymeric proteins. Shift of MWD to molecular weight higher than the optimum results in a stronger network with reduced slippage through entanglement nodes, whereas a shift to lower molecular weights will decrease the strength of the network due to a lesser number of entanglements per chain.     

The tryptophan-rich domain of puroindoline is directly associated with the starch granule surface as judged by tryptic shaving and mass spectrometry

The starch granule surface is a frontline of microbial attack and defence, operating in the background of normal starch granule metabolism. Puroindoline, a wheat protein which binds starch granule surfaces, contains a unique tryptophan-rich domain likely responsible for this property, though direct evidence is lacking. To test puroindoline’s tight association, prime starch granule extracts were water-washed 8 or 20 times and residual puroindoline removed using a solution of 50% isopropanol/50 mM NaCl. We found that this solvent was consistent in the amount of protein extracted from wheat flour and washed starch, regardless of initial protein content. Relative quantification of puroindoline following water-washing was performed using dot blot. Washing more than 8 times did not further reduce puroindoline content of starch granules suggesting a strong association with the starch granule surface. To identify the tryptophan-rich domain tightly associated with the starch granule surface, a combination of in situ tryptic digestion and mass spectrometry was used. Following digestion and water-washing, 50% isopropanol/50 mM NaCl was used to remove tightly-associated peptides for identification by mass spectrometry. Using this method, we identified the tryptophan-rich domain of puroindoline directly bound to the starch granule surface of wheat.     

Genetic and environmental factors affecting grain texture in common wheat

Thirteen wheat cultivars grown in six locations were compared for kernel weight, protein content and grain texture, as determined by the Single Kernel Characterization System (SKCS). Moreover, puroindolines a (Pin-A) and b (Pin-B) bound to starch were quantified by densitometric scanning of A-PAGE fractionations. All cultivars shared allele Pina-D1a coding for wild-type Pin-A, and differed from each other in allele composition at Pinb-D1 coding for Pin-B. Cultivars with Pinb-D1a exhibited soft grain and high amounts of Pin-A and Pin-B compared to cultivars with Pinb-D1b or Pinb-D1d. Significant genetic variation for grain hardness and Pin-A level was detected in soft cultivars. The ratio between Pin-A and Pin-B levels in soft cultivars was approximately 6:5, whereas it varied between 9:5 and 10:1 in hard cultivars. Protein content was significantly correlated with Pin-B content (r=0.34) and SKCS value (r=0.36) in soft wheats. Significant correlations (0.68 and 0.73 for soft and hard wheats, respectively) were observed between Pin-A and Pin-B levels. Grain hardness was not correlated with puroindoline levels and Pin-A/Pin-B ratio in both textural classes. By contrast, kernel weight was found to act as a major environmental factor affecting grain texture in both soft and hard wheats.     

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.