Simultaneous Isolation of Wheat High Molecular Weight and Low Molecular Weight Glutenin Subunits

A procedure was established for isolating simultaneously high molecular weight (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) from chloroform-defatted wheat flour. Temperature conditions were optimised to obtain maximum yield and purity. A pre-extraction of gliadin was performed with 50% (v/v) propan-1-ol at room temperature. Glutenin subunits were solubilised from the residue at 60 °C with 50% (v/v) propan-1-ol containing 1% (w/v) dithiothreitol. The HMW-GS in the glutenin mixture were then precipitated selectively by increasing the propan-1-ol concentration to 60%. Upon addition of propan-1-ol to a final concentration of 85%, LMW-GS were precipitated. It was shown by densitometric scanning of SDS–PAGE gels that the isolated glutenin subunits were very pure. Furthermore, a partial separation of LMW-GS into B and C type subunits was observed when the propan-1-ol concentration was increased gradually.     

The Role of Dextrins in the Stickiness of Bread Crumb made from Pre-Harvest Sprouted Wheat or Flour Containing ExogenousAlpha-Amylase

Dextrins were extracted in water from bread made from pre-harvest sprouted wheat or standard flour supplemented with exogenousalpha-amylases. The dextrins were separated by gel permeation chromatography and the dextrin content (% of crumb weight) determined for different degree of polymerisation (DP) size classes; DP 1–2, DP 3–10, DP 11–50, DP 51–200 and DP >200. There were significant correlations between the dextrin content in each size class and crumb stickiness (r=0·84–0·91, 22 df ). The most significant correlation (r=0·96) was between total dextrin content and crumb stickiness. Addition of dextrins of various DP ranges from various sources to standard flour produced bread with sticky crumb. Again, the degree of stickiness was generally related to the amount of total dextrin in the crumb and not to size distribution of dextrins. In this instance, extensive enzymic hydrolysis of starch was not necessary to produce sticky crumb; the dextrins caused crumb stickiness directly. Addition of dextrins to reconstituted gluten–starch flour produced bread with unexpectedly low dextrin levels and correspondingly low stickiness scores. It is concluded that, to produce sticky crumb, high levels of dextrin of any size are necessary in the crumb; a sticky mass is produced when dextrins dissolve in the excess «free» water that is normally «bound» to starch, gluten and other insoluble components of bread crumb.     

新疆小麦1BL/1RS易位和 Dx5基因的多重PCR检测及其面筋品质分析

为了给新疆小麦面筋品质改良提供参考依据,利用多重PCR体系对267份新疆冬、春小麦品种中1BL/1RS易位和 Dx5基因的分布进行了检测,并测定了其中181份小麦品种的面粉蛋白质含量、湿面筋含量、Zeleny沉淀值以及面团特性等品质性状。结果表明,新疆小麦品种中,1BL/1RS易位品种有55份,占20.6%,含有 Dx5基因的品种有76份,占28.5%。冬小麦品种中1BL/1RS易位系分布频率（26.6%）显著高于春小麦（9.6%）,而春小麦品种中 Dx5基因的分布频率（31.9%）高于冬小麦（26.6%）。在新疆小麦农家品种、引进品种和自育成品种中,1BL/1RS易位和 Dx5基因的分布频率也存在明显差异。分析表明,1BL/1RS和非1BL/1RS小麦品种的主要面筋品质性状（如Zeleny沉淀值、峰值高度、8 min宽度等）达到显著性差异（P＜0.05）,1BL/1RS小麦中含 Dx5和不含 Dx5基因品种的面筋指数、Zeleny沉淀值、峰值时间和8 min面积等5个参数差异达到显著水平（P＜0.05）。多重PCR体系检测结果可靠稳定,节省实验经费和时间,提高了效率,可用于小麦分子辅助育种。     

Distribution of Water between Wheat Flour Components: A Dynamic Water Vapour Adsorption Study

The water adsorption properties of hard and soft wheat flours and flour components (starch, damaged starch, gluten, soluble pentosans, and insoluble pentosans) were determined at 25 °C using a controlled atmosphere microbalance. At different levels of relative humidity (from 10% to 95%), changes in sample mass (i.e., water gain) were continuously measured versus time and described using exponential models (R²≥0·994). Water adsorption isotherms were constructed for wheat flours and flour components and described using Guggenheim-Anderson-de Boer models (R²≥0·997). It was not possible to distinguish between the selected hard and soft wheat flours by their isotherms. The water-soluble pentosans had the highest water adsorption capacity. The theoretical distribution of water between the flour components (calculated using the Guggenheim-Anderson-de Boer parameters) was starch, 88%; gluten, 10%; and pentosans, 2%.     

Production of Proteases by Fusarium Species Grown on Barley Grains and in Media Containing Cereal Proteins

The production of proteases by the cereal plant pathogens Fusarium culmorum, F. graminearum and F. poae was followed through seven days of cultivation. The fungi were grown in mineral and in gluten culture media, and on autoclaved barley grains. The proteolytic activities of each sample were analysed at pH 2·2, 5·0 and 8·0 and the pH optima of the most active proteases were determined. All of the fungi grown in the gluten medium produced proteases that were active at pH levels between 6 and 10 and were most active at about pH 9·0. Fusarium poae also produced acid protease(s) with pH optima between 3.0 and 3.5 when grown in the gluten medium. No protease activity was detected in the cultures that were grown in the mineral medium, except that a small amount was formed after the glucose substrate was depleted. When grown on the barley grain medium the Fusarium species produced protease activities that were similar to the neutral and alkaline ones present in the gluten cultures, but no pH 2·2 protease activity was detected. The alkaline proteases had some characteristics that were similar to those of chymotrypsin.     

Functional Properties of Low Mr Wheat Proteins. I. Isolation, Characterization and Comparison with Other Reported Low Mr wheat Proteins

A group of low M_r wheat proteins with characteristic extractability behavior was isolated using two different isolation procedures. The proteins were extractable with water, salt solution and 70% (v/v) ethanol. After water extraction of flour and separation of gluten, a substantial proportion of these proteins was still extractable from gluten using 70% (v/v) ethanol. Based in their amino acid compositions, M_rs and IEF patterns, the isolated proteins resemble closely most of the alpha -amylase/protease inhibitors described in the literature. This was confirmed by enzyme inhibition studies in which it was shown that they inhibited mammalian, but not wheat, bacterial and fungal alpha-amylases. All proteases tested were inhibited by the low M_r proteins. Their M_rs and their high cysteine contents (6·5-8·1 mol%) indicated that the proteins contain four to five disulphide bonds. Free thiol groups were not detected in the proteins. Upon reduction, the M_r increased from 7-8000 to 14-19000. Furthermore, the disulphide bonds were highly reactive as determined by their reaction with the thiol-specific label monobromobimane. This suggests that the low M_r wheat proteins may play a role in thiol group/disulphide bond exchange in wheat proteins.     

25份澳大利亚小麦种质的性状表现及利用

为给黄淮南片冬麦区小麦育种提供信息,对25份澳大利亚小麦种质于2016-2018年度在新乡进行了部分农艺性状调查,对其品质性状及优质亚基等进行了分析。结果表明,25份澳大利亚小麦种质多表现为春性,综合农艺性状较差;千粒重低于黄淮麦区高产对照品种周麦18,变化范围为31.4 g～43.1 g;湿面筋含量、面筋指数、稳定时间、弱化度的变化范围分别为28.0%～39.1%、55.2%～96.5%、4.0 min～25.0 min、5%～100%。从澳大利亚小麦种质中,筛选出了具有湿面筋含量高、面筋指数高、稳定时间长、面粉粉色白等优良品质特性的材料,鉴定出了含5+10亚基、7+8~*亚基(7超量表达亚基)、17+18亚基、13+16亚基、低PPO活性基因及 Wx-B1基因缺失等优异品质材料,筛选出了含有 Rht-D1b矮秆基因以及 Lr34和 Lr46抗叶锈基因的材料。利用澳大利亚小麦种质为改良亲本,与黄淮麦区表现突出的小麦品种配制三交或四交组合,按常规系谱方法对优良性状进行定向选择,培育出了综合农艺性状表现好、品质表现突出的小麦新品系。     

Protein Inhibitors ofAlpha-amylase in Mature and Germinating Grain of Rye (Secale cereale)

The activities of endogenous (R-type) and exogenous acting (D-type) protein inhibitors ofalpha-amylase and the activities ofalpha- and total amylase were determined in milling fractions of rye. High D-type amylase inhibitor activities were detected in the embryo (255 IU/g) and in the endosperm fraction (64·9 IU/g), low inhibitor activities were found in the aleurone layer fraction (25·9 IU/g). The highest R-typealpha-amylase inhibitor activity was found in the aleurone layer fraction (32·6 IU/g), and the lowest value in the epidermis containing fraction (5·0 IU/g). The D- and R-typealpha-amylase inhibitor activities varied with growing conditions. D-type amylase inhibitor activities were found to be high in those samples which grew under drought conditions and low in samples cultivated under wet and cool weather. Higher R-typealpha-amylase inhibitor activities were found in rye genotypes cultivated under wet conditions and lower values under dry weather. There were small variations inalpha-amylase inhibitor activities between sprout-stable and sprout-sensitive rye genotypes. The D- and R-typealpha-amylase inhibitor activities of all varieties were stable during 72 h of germination. Similar soil conditions will therefore lead to differentialalpha-amylase inhibitor activities depending on weather conditions during growth.     

高分子量麦谷蛋白亚基评分系统的改进及应用

对 191个冬、春小麦品种的品质分析结果表明 :拉伸曲线的最大抗延阻力能比较客观地反映面筋的强度 ,但该项试验所需的样品量较大 (至少需要 30 0g面粉 )。高分子量麦谷蛋白亚基 (HMW GS)组成受遗传基因的控制 ,麦谷蛋白电泳分析所需的样品量少 (只需半粒小麦即可 ) ,适宜育种的早世代分析。选用各高分子量麦谷蛋白亚基的最大抗延阻力均值的差异建立的HMW GS组成评分系统 ,可以对小麦品种的面筋强度进行早期预测     

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.     

Wx-1基因变异和优质HMW-GS组合对中筋小麦品质的影响

高分子量谷蛋白亚基(HMW-GS)和Wx蛋白是两个影响小麦理化品质和制成品品质的重要因素。为研究HMW-GS和Wx基因变异对小麦理化品质、面条感官评分和质构特性的影响,以镇麦9号和扬糯麦1号为亲本的2个高代系为试验材料,研究了优质HMW-GS 5+10及Wx基因缺失对小麦理化品质及面条加工品质的效应。结果表明,品系1与品系2的湿面筋含量和SDS沉淀值与镇麦9号相近,但显著好于扬糯麦1号。相较于镇麦9号,品系1与品系2的直链淀粉含量分别降低了1.5%和2.5%,其峰值黏度和稀懈值均显著高于镇麦9号,膨胀势也高于镇麦9号。5+10亚基显著提高了面条质构参数中的硬度和咀嚼性,而Wx基因缺失显著提高了面条的软硬度评分和光滑性评分。综合来看,具有5+10亚基和 Wx-D1缺失型的品系2具有较高的SDS沉淀值、面团形成时间和稳定时间、较低的直链淀粉含量、较高的峰值黏度、稀懈值和膨胀势,蛋白质和淀粉综合品质表现较好。品系2面条总评分最高,显著高于镇麦9号和扬糯麦1号,其主要体现在适中的面条软硬度和较好的光滑性。推测Wx基因缺失和优质HMW-GS聚合可显著提高小麦淀粉品质和蛋白质品质,有效改善面条的蒸煮品质和感官评分。     

Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale

Flour samples of 14 wheat cultivars previously characterised by rheological measurements and by baking tests on a micro-scale (Kieffer et al.: Journal of Cereal Science27 (1998) 53–60) were analysed for the relative amounts of gluten protein types using a combined extraction/HPLC procedure. Regression analysis was used to find relations between wheat properties and protein quantities. The results indicated that the maximum resistance of dough and gluten and the gluten index were strongly dependent on the quantity of glutenin subunits (GS) in flour; additionally they were influenced by the ratio of gliadin to glutenin subunits. Within the family of glutenin proteins, the correlation coefficients for high-molecular-weight (HMW) and low-molecular-weight (LMW) GS were in a similar range, but twice the amount of LMW GS was necessary to get the same resistance as with HMW GS. Among HMW GS, the contribution of x-type GS was more important than those of y-type GS. The extensibility of dough and gluten was mainly dependent on the ratio of gliadin to total glutenin subunits, to HMW GS and LMW GS. Dough development time showed the highest correlation with total HMW GS and x-type HMW GS. Bread volume was influenced by the total amount of gluten protein more than by the amount of protein in different groups or of different types, probably because of the rather low range of flour protein content (8·7–12·0 %) within the set studied. Significant differences between gliadins and glutenins with respect to their effects on bread volume could not be detected. The correlation between bread volumes and the quantity of gluten proteins was higher, when dough was mixed to optimum.     

谷朊粉对不同筋力小麦粉及面包品质的改良效果

为了探讨谷朊粉对我国不同筋力小麦粉面包加工品质的影响,选用师栾02-1(强筋)、石优17(中强筋)、矮抗58(中筋)和济麦22(弱筋)等四种不同筋力的小麦粉进行谷朊粉添加试验,谷朊粉添加水平分别占小麦粉质量的0%、1%、3%、5%、7%、9%。对不同处理的小麦粉样品进行面包烘焙试验和理化性质测试,结果表明,谷朊粉的添加使面包体积迅速增大,对感官特性在一定范围内具有改良作用,谷朊粉添加过量会出现面包芯孔洞变大等不良影响;不同筋力小麦粉的最适添加量不同,师栾02-1、石优17、矮抗58和济麦22的最适添加量分别为面粉质量的1%～2%、3%～5%、7%、9%。     

Salt-Induced Disaggregation/Solubilization of Gliadin and Glutenin Proteins in Water

The effect of salt concentration used in preparing gluten, on the subsequent dissolution of gluten in water, was examined. Flour from a Canadian hard red spring wheat cultivar, Katepwa, was used to prepare glutens using three different solvents, i.e. distilled deionized water (DDW), 0·2% NaCl solution and 2% NaCl solution. The isolated wet glutens were extracted sequentially with DDW, providing four water soluble fractions and an insoluble residue. The amount of protein in each fraction was determined and respective compositions were assessed electrophoretically under reducing and non-reducing conditions. Surprisingly, DDW extracts of gluten prepared with 2% NaCl contained almost all the gliadins, except some ω-gliadin components, and most of the polymeric glutenin. For the gluten prepared with 0·2% NaCl, most of the gliadin, but only a small portion of glutenin, was extracted. For gluten prepared with DDW, only part of the gliadins and almost no glutenin was extractable with water. The DDW solubilities of gluten proteins prepared in DDW, 0·2% NaCl and 2% NaCl were 27, 52, and 85%, respectively, after four sequential extracts with DDW. The large increases in the solubility of gliadin and glutenin proteins in DDW when the gluten is prepared in salt solution (after removal of most of the salt) can be explained on the basis of a salt-induced conformational change of the proteins, which renders water a more effective solvent.     

Rheological properties of wheat flour processed at low levels of hydration: Influence of starch and gluten

The rheological properties of wheat flour under processing such as extrusion (with 28% moisture content, wet basis) are influenced by the molecular changes its components undergo during processing. But, there was no simple relationship between the wheat-flour characteristics and their rheological properties. In order to investigate the quantitative and qualitative effects of the individual flour components on rheological properties, model blends of wheat starch and wheat gluten with different starch/gluten ratios were studied. The effects of gluten and starch quality were also investigated by using different gluten types and by modifying the amylose content of starch, respectively. The shear viscosity of the blends, determined by capillary rheometry under controlled conditions (35% moisture content, 140 °C), was observed to be modified by both gluten and amylose content. The changes undergone by wheat gluten under these conditions were analysed by HPLC, to determine the levels of unextractable polymeric proteins, and by Lab-on-a-Chip analysis of protein composition, to follow the polymerisation of protein under processing. This study indicated that in low hydrated products in the molten state, shear viscosity is affected by the structure of the blends as determined by fluorescence microscopy and by the molecular changes occurring during processing.     

The Role of Gluten Proteins in the Baking of Arabic Bread

Fractionation and reconstitution/fortification techniques were utilised to study the role of gluten in Arabic bread. Glutens from two wheat cultivars of contrasting breadmaking quality were fractionated by dilute HCl into gliadin and glutenin. Gluten, gliadin and glutenin doughs from the good quality flour had higher G ′ and lower tan δ values than those from the poor quality flour at all the frequencies examined. Interchanging the gliadin and glutenin fractions between the reconstituted flours showed that the glutenin fraction is largely responsible for differences in the breadmaking performance. Fortification of an average quality flour with the gliadin and glutenin fractions from the poor and good quality flours, at the levels of 1% and 2% (protein to flour mass), induced marked differences in the mechanical properties of bread. The resilience of the loaves was not adversely affected by the addition of gliadins and increased, with a concomitant significant (p<0·05) improvement in quality, at the 2% level of fortification with gliadins from the good quality flour. Addition of glutenin resulted in loaves with leather-like properties that became particularly apparent at the higher level of fortification; the observed deterioration in quality paralleled the increase in the elastic character of the doughs. It is suggested that highly-elastic doughs are not compatible with the rapid expansion of gases at the high-temperature short-time conditions employed in the baking of Arabic bread and that there exists a threshold in dough elasticity beyond which a rapid decline in quality takes place.     

Fatty Acid Composition of Three Rice Varieties Following Storage

The petroleum ether extractable lipids (PEE-L) and aqueous propan-1-ol extractable lipids (PWE-L) of three varieties of rice were determined gravimetrically and characterised by fatty acid profiles. The content of PEE-L (22·5–28·2 mg g⁻¹) was higher than that of PWE-L (7·4–11·5 mg g⁻¹) in brown rice with the situation reversed in milled rice (3·0–4·5 mg g⁻¹vs. 7·2–8·7 mg g⁻¹). The ratio of unsaturated fatty acid to saturated fatty acid was about two times higher in PEE-L than that in PWE-L for both brown and milled rice reflecting the selective complexation of saturated fatty acids. Rice storage at 37 °C resulted in some minor but statistically significant changes in the fatty acid profile. In the case of brown rice, the only notable changes were a reduction in the amounts of oleic and linoleic acids in the aqueous propan-1-ol extractable fatty acid fraction (PWE-FA) following storage at the higher temperature. Milled rice of all three varieties showed a decrease in linoleic acid content of PEE-L following storage at 37 °C for 4 and 7 months compared to storage at 4 °C. There was no change in fatty acid contents of PWE-L of milled rice when stored at 4 and 37 °C for 4 and 7 months. This implies that the PWE-L (or bound lipids) were more stable than PEE-L (or free lipids) during storage.     

Biochemical Characterisation of a Novel Polymeric Protein Subunit from Bread Wheat (Triticum aestivum L.)

A new wheat endosperm protein subunit that was found in accessions belonging to different collections was identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Insoluble in 0·5 M NaCl, 70% ethanol, dimethyl sulphoxide (DMSO) and 50% propan-1-ol, it appeared in the pellet corresponding to the polymeric proteins along with high (HMW) and low molecular weight (LMW) glutenin subunits (GS). In the reduced form, it had an electrophoretic mobility between those two types of glutenin subunits. The apparent M_r of this novel protein was estimated by SDS-PAGE to be 71 000. N-terminal sequence and amino acid analyses indicated a composition similar to the ω-gliadins encoded by genes located on chromosome 1B. This protein can be ascribed to the D-subunits of LMW-GS with at least one cysteine residue that allows it to form part of the polymeric structure of glutenin, as shown by reaction with a fluorogenic reagent specific for sulphydryl groups. Fractions collected after size exclusion high-performance liquid chromatography (SE-HPLC) fractionation and further characterised by SDS-PAGE, confirm that the protein participates in the glutenin polymeric structure. An increase in its concentration was observed in fractions collected within the polymeric peak as elution time increased, implying that a larger amount of this protein is present in small size polymers. The role of this protein in the complex relationship between endosperm proteins and quality parameters is discussed in relation to its likely role as a chain terminator.     

RP–HPLC and Capillary Electrophoresis of Subunits from Glutenin Isolated by SDS and Osborne Fractionation

The polypeptide subunits present in SDS-unextractable glutenin, the glutenin macropolymer (GMP) and the 70% (v/v) ethanol unextractable protein, the Osborne glutenin fraction, of various cultivars were separated by RP–HPLC and capillary electrophoresis (CE) under denaturing (urea and SDS, respectively) and reducing conditions. In addition, the SDS-extractable protein was separated by CE. HighM_rglutenin subunits were well separated by CE, while the separation of lowM_rglutenin subunits was better by RP–HPLC. HighM_rglutenin subunits separated by RP–HPLC were collected and separated by CE. The subunits were identified unequivocally using the combined information from these two techniques and from SDS–PAGE patterns using the cvs. Spring and Troy Spring. By both RP–HPLC and CE it could be demonstrated for flour from three wheat cvs. (Camp Remy, Obelisk and Rektor) and a blend of flour from two of those cvs. (Camp Remy/Obelisk) that the highM_rglutenin subunit content of the GMP was 29–31%. In contrast, the SDS-extractable protein consisted of 4–6% highM_rglutenin subunits, which accounted for 14–23% of the highM_rglutenin subunits in flour. Interestingly, the SDS-extractable highM_rglutenin subunits consisted mainly (90–96%) of x-type subunits whereas, in the GMP, only 70–75% of the highM_rsubunits were x-type subunits. Although the SDS extractable protein was not separated by RP–HPLC, results similar to those obtained by CE could be inferred from the subtraction of the contents of glutenin subunits of the GMP from the contents in the Osborne glutenin fraction. The results suggest that x- and y-type highM_rglutenin subunits may have a different role in the structure (size and composition) of glutenin polymers.     

宁夏引黄灌区冬小麦谷蛋白亚基组成和1BL/1RS易位分析及品质性状研究

为全面了解宁夏引黄灌区冬小麦品质概况,为冬小麦品质改良和粮食生产提供理论依据。选用冬小麦主栽品种、亲本材料和高代品系30份。用SDS-PAGE分析了其中18份材料的HMW—GS、LMW—GS组成和1BL／1RS易位系分布状况。并对23个冬小麦品种的营养与加工品质进行了研究。结果表明,2#、7＋9、2＋12、Glu-A3a、Glu-A3c、Glu-B3h和Glu—B3j在宁夏引黄灌区冬小麦中分布较广,1BL／1RS易位系分布相当普遍。分布频率为27．8％。参试品种的籽粒硬度、面粉PPO活性、SDS沉淀值、形成时间、稳定时间和评价值的变异范围较大,变异系数分别为25．37％、33．68％、21．42％、26．58％、43．95％和29．31％。多数冬小麦品种（系）的千粒重、出粉率和湿面筋含量低于对照宁春4号。而蛋白质含量、SDS沉淀值、吸水率和稳定时间优于宁春4号。供试品种（系）中。HMW-GS品质评分、籽粒硬度、蛋白质含量、SDS沉淀值均较高,并且不含1BL／1RS易位系的有烟优361、济麦20、鲁875067和923—9等,可用于宁夏引黄灌区冬麦品质改良。