Identification of disulfide bonds in wheat gluten proteins by means of mass spectrometry/electron transfer dissociation

Disulfide bonds within gluten proteins play a key role in the breadmaking performance of wheat flour. In the present study, disulfide bonds of wheat gluten proteins were identified by using a new liquid chromatography-mass spectrometry (LC-MS) technique with alternating electron transfer dissociation (ETD)/collision-induced dissociation (CID). Wheat flour was partially hydrolyzed with thermolysin (pH 6.5, 37 °C, 16 h), and the digest was subjected to LC-MS with alternating ETD/CID fragmentation. Whereas CID provided peptide fragments with intact disulfide bonds, cleavage of disulfide bonds was preferred over peptide backbone fragmentations in ETD. The simultaneous observation of disulfide-linked and disulfide-cleaved peptide ions in the mass spectra not only provided distinct interpretation with high confidence but also simplified the conventional approach for determination of disulfide bonds, which often requires two separate experiments with and without chemical reduction. By application of the new method 14 cystine peptides were identified. Eight peptides confirmed previously established disulfide bonds within gluten proteins, and the other six cystine peptides were identified for the first time. One of the newly identified cystine peptides represented a "head-to-tail" cross-link between high molecular weight glutenin subunits. This type of cross-link, which has been postulated as an integral part of glutenin models published previously, has now been proven experimentally for the first time. From the six remaining cystine peptides interchain disulfide bonds between α-gliadins, γ-gliadins, and low molecular weight glutenin subunits were established.     

Thermoformed wheat gluten biopolymers

The quantity of available wheat gluten exceeds the current food use markets. Thermoforming is an alternative technical means for transforming wheat gluten. Thermoforming was applied here to wheat gluten under chemically reductive conditions to form pliable, translucent sheets. A wide variety of conditions, i.e., temperature, reducing agents, plasticizers and additives were tested to obtain a range of elastic properties in the thermoformed sheets. These properties were compared to those of commercially available polymers, such as polypropylene. Elasticity of the gluten formulations were indexed by Young's modulus and were in the range measured for commercial products when tested in the 30-70% relative humidity range. Removal of the gliadin subfraction of gluten yielded polymers with higher Young's modulus since this component acts as a polymer-chain terminator. At relative humidity less than 30% all whole gluten-based sheets were brittle, while above 70% they were highly elastic.     

Isolation and identification of indigestible pyroglutamyl peptides in an enzymatic hydrolysate of wheat gluten prepared on an industrial scale

An enzymatic hydrolysate of wheat gluten was further digested in vitro with porcine pepsin and pancreatin to obtain an indigestible peptide. Indigestible pyroglutamyl peptide was isolated from the digest by strong cation-exchange, size-exclusion, and reversed-phase chromatographies. The pyroglutamyl peptide was digested with pyroglutamate aminopeptidase, and the digest was reacted with phenyl isothiocyanate. The resultant phenylthiocarbamyl (PTC) peptides were purified by reversed-phase HPLC by using binary gradient elution with ammonium acetate buffer, pH 6.0, and acetonitrile. The PTC peptides were analyzed with an automatic peptide sequencer on the basis of the Edman degradation method with a modified program. Some pyroglutamyl peptides were also analyzed by fast-atom bombardment ionization mass spectrometry without the pyroglutamate amino peptidase digestion. Consequently, pyroGlu-Asn-Pro-Gln, pyroGlu-Gln-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gly-Gln-Gly-Gln, pyroGlu-Gln, pyroGlu-Gln-Pro, pyroGlu-Ile-Pro-Gln, pyroGlu-Ile-Pro, pyroGlu-Gln-Pro-Leu, pyroGlu-Gln-Phe-Pro-Gln, pyroGlu-Ser-Phe-Pro-Gln, pyroGlu-Phe-Pro-Gln, and pyroGlu-Gln-Pro-Pro-Phe-Ser were identified.     

Properties of chemically and physically treated wheat gluten films

Chemical (vapors of formaldehyde), physical (temperature, UV and gamma radiation), and aging treatments were applied to wheat gluten films. Changes in film mechanical properties, water vapor permeability, solubility, and color coordinates were investigated. An aging of 360 h led to a 75 and 314% increase in tensile strength and Young's modulus, respectively, and a 36% decrease in elongation. Severe thermal (above 110 degrees C, 15 min) and formaldehyde treatments highly improved the mechanical resistance of the films. Under these conditions, up to 376 and 654% increase in tensile strength and Young's modulus and up to 66% decrease in elongation have been observed. Water solubility was only slightly modified, whereas water vapor permeability was not affected. Color coordinates of films heated above 95 degrees C changed to a great extent. An almost total insolubilization of proteins in sodium dodecyl sulfate occurred for heat- and formaldehyde-treated films, due to the modification of protein network leading to changes in properties of the films.     

Analysis of taste-active compounds in an enzymatic hydrolysate of deamidated wheat gluten

Hydrolyzed plant proteins are widely used as ingredients in culinary products for their glutamate-like ("umami") taste. Three hydrolysates were prepared from wheat gluten using different enzymatic approaches. Comparison of their taste profiles revealed the enzymatic hydrolysate of an acid-deamidated wheat gluten (WGH-3) to be the least bitter of all and to elicit an intense glutamate-like taste. Its umami taste intensity was similar to that of an enzymatic hydrolysate in which glutaminase had been employed to convert free glutamine to glutamic acid and which had a 3-fold higher concentration of free glutamate. Reconstitution studies based on the results of the chemical analysis of WGH-3 and sensory comparison of the model solution and WGH-3 indicated that other components in addition to glutamate and organic acids contribute to its glutamate-like taste. WGH-3 was fractionated by gel permeation chromatography and reversed phase high-performance liquid chromatography, and two fractions with a pronounced glutamate-like taste were obtained. In one of them four pyroglutamyl peptides were tentatively identified: pGlu-Pro-Ser, pGlu-Pro, pGlu-Pro-Glu, and pGlu-Pro-Gln. Apparently, these peptides were formed by cyclization of the N-terminal glutamine residues during the preparation of the hydrolysates.     

Variation in gluten strength and yellow pigment in Ethiopian tetraploid wheat germplasm

Ten released varieties and one hundred and eleven tetraploid (2n = 4x = 28, AABB) wheat accessions collected from different major wheat producing regions, consisting of totally 2904 entries were characterized for content of yellow pigment, gluten strength, thousand kernel weights, grain yield, percent of yellow berry, glumes color, awn color, seed color, beak shape and spike density. The objectives were to assess variation with respect to regions of origin, species and altitudinal classes and to study the interrelationships of the qualitative traits used for the study. The accessions and/or released varieties showed significant regional variation for all the traits used in the study, but clinal variation among altitudinal classes and species were significant (P ≤ 0.01) in 6 (60%) and 9 (90%) of the studied traits, respectively. The accessions revealed consistent variation (P ≤ 0.001) within both regions and altitudinal classes for all the traits used in the study. The sedimentation volume was positively correlated with content of yellow pigment, percent of yellow berry, thousand kernel weights, glumes color, awn color, beak shape, seed color and grain yield. The contents of yellow pigment was positively correlated with awn color, beak shape and grain yield, but negatively correlated with seed color and spike density. Generally wide variation was found in the germplasm. Particularly variation was high for the content of the yellow pigment as well as the gluten strength, which provides opportunities to be utilized for genetic improvement.     

Mechanisms of heat-mediated aggregation of wheat gluten protein upon pasta processing

During pasta processing, structural changes of protein occur, due to changes in water content, mechanical energy input, and high temperature treatments. The present paper investigates the impact of successive and intense thermal treatments (high temperature drying, cooking, and overcooking) on aggregation of gluten protein in pasta. Protein aggregation was evaluated by the measurement of sensitivity of disulfide bonds toward reduction with dithioerythritol (DTE), at different reactions times. In addition to the loss in protein extractability in sodium dodecyl sulfate buffer, heat treatments induced a drastic change in disulfide bonds sensitivity toward DTE reduction and in size-exclusion high-performance liquid chromatography profiles of fully reduced protein. The protein solubility loss was assumed to derive from the increasing connectivity of protein upon heat treatments. The increasing degree of protein upon aggregation would be due to the formation of additional interchain disulfide bonds.     

Polymerization kinetics of wheat gluten upon thermosetting. A mechanistic model

Size exclusion high-performance liquid chromatography analysis was carried out on wheat gluten-glycerol blends subjected to different heat treatments. The elution profiles were analyzed in order to follow the solubility loss of protein fractions with specific molecular size. Owing to the known biochemical changes involved during the heat denaturation of gluten, a mechanistic mathematical model was developed, which divided the protein denaturation into two distinct reaction steps: (i) reversible change in protein conformation and (ii) protein precipitation through disulfide bonding between initially SDS-soluble and SDS-insoluble reaction partners. Activation energies of gluten unfolding, refolding, and precipitation were calculated with the Arrhenius law to 53.9 kJ x mol(-1), 29.5 kJ x mol(-1), and 172 kJ x mol(-1), respectively. The rate of protein solubility loss decreased as the cross-linking reaction proceeded, which may be attributed to the formation of a three-dimensional network progressively hindering the reaction. The enhanced susceptibility to aggregation of large molecules was assigned to a risen reaction probability due to their higher number of cysteine residues and to the increased percentage of unfolded and thereby activated proteins as complete protein refolding seemed to be an anticooperative process.     

Drying temperature and relative humidity effects on wheat gluten film properties

The mechanical and physical properties of glycerol-plasticized wheat gluten films dried at different temperatures (20, 50, and 80 degrees C) and relative humidities (35 and 70% RH) were investigated. Dispersion of wheat gluten was prepared at pH 11 in aqueous solution. Films were obtained by casting the wheat gluten suspension, followed by solvent evaporation in a temperature and relative humidity controlled chamber. Decreasing relative humidity altered most of the mechanical properties. At 35% RH, tensile strength increased when drying temperature increased. However, at 70% RH, tensile strength decreased when temperature increased. Thickness of the films decreased by increasing temperature. Hypothetical coating strength increased with increasing drying temperature at 35% RH. However, at 70% RH, a maximum value was observed at 50 degrees C. Films produced at 80 degrees C exhibited low solubility in aqueous solution. Addition of 1.5% (w/v) sodium dodecyl sulfate increased solubility of all of the films except the film dried at 50 degrees C and 70% RH. Overall, drying temperature and relative humidity affected mechanical and physical properties of the wheat gluten films. However, the effect of drying temperature was more pronounced than the effect of relative humidity.     

Development and characterization of biodegradable films made from wheat gluten protein fractions

Gliadins and glutenins were extracted from commercial wheat gluten on the basis of their extractability in ethanol and used to produce film-forming solutions. Films cast using these gliadin- and glutenin-rich solutions were characterized. Glycerol was used as a plasticizer, and its effect on the films was also studied. Films obtained from the glutenin fraction presented higher tensile strength values and lower elongation at break and water vapor permeability values than gliadin films. Gliadin films disintegrated when immersed in water. The GAB isotherm model was used to describe the equilibrium moisture sorption of the films. The glycerol concentration largely modified mechanical and water vapor barrier properties of both film types.     

脱酰胺与双酶协同作用提高小麦面筋蛋白酶解效率

为了探讨了不同脱酰胺处理和双酶协同作用方式对小麦面筋蛋白酶解效率及其产物抗氧化活性的影响,该文研究了小麦面筋蛋白在各种预处理方式和酶解条件下的蛋白回收率、水解度、抗氧化性能及肽分子量分布情况。结果显示,单独热处理(90℃,30 min)小麦面筋蛋白对其酶解效率无显著影响,而采用添加0.5 mol/L柠檬酸溶液进行热处理(质量分数为5%,90℃,30 min)可显著(P0.05)提高其蛋白回收率。此外,酶制剂添加顺序及双酶共同水解作用时间对酶解效率均具有较大影响:加入谷氨酰胺酶预先水解对小麦面筋蛋白的深度水解有促进作用;一定时间内的双酶协同作用有利于酶解的进行,但较长时间的双酶作用反而会抑制酶解效率。采用谷氨酰胺酶(质量分数为0.2%)对经柠檬酸加热处理的小麦面筋蛋白作用12 h后再加入胰酶(质量分数为0.6%)共同作用7 h可使蛋白回收率达70.74%,水解度达到9.88%;另外,酶解产物的自由基清除能力ABTS+(2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)+)值与氧化自由基吸收能力(ORAC,oxygen radical absorbance capacity)值分别达到478.95 mmol/g和213.85μmol/g,提示该酶解产物是一种潜在优秀食品抗氧化剂。研究结果可为拓宽小麦面筋蛋白的应用领域,以及高效制备抗氧化活性肽提供方法和理论指导。     

钾对强筋小麦产量和品质的调控效应

采用盆栽试验方法研究了钾对强筋小麦产量和品质的影响。结果表明，施用钾肥能增加小麦株高、穗长、千粒重及穗粒数，因而显著提高小麦产量，小麦增产10．2％～24．8％。施用钾肥能提高小麦籽粒蛋白质含量和湿面筋含量，但对籽粒沉降值的影响不大。     

基于管式模型的小麦面筋系统流变行为的解析

利用动态流变仪对8种小麦面筋系统的进行小形变试验,研究在振动模式下的频率扫描时,面筋系统流变学特性,从分子动力学的角度进行解析,并引入Pierre Gilles de Gennes等人的管式模型理论基础性地对面筋流变行为进行阐述。试验表明：面筋蛋白转变温度在52.6～58.5℃间;0.1～40℃间,频率扫描下,时间-温度叠加法算出20℃时表观活化能在4602.57～6511.16 J/mol间,不同样品间有较大的差异;Arrhenius模型和WLF模型在拟合面筋系统叠合曲线的效果上一致。通过SPSS分析相关性表明：在面筋蛋白转变温度下,固定频率的温度扫描的弹性模量和在不同温度下的频率扫描的弹性模量成显著的正相关;面筋蛋白的转变温度与表观活化能呈较显著的正相关。该试验结果为小麦面筋相关制品的开发提供了参考。     

砂姜黑土强筋小麦施肥技术研究

通过田间试验研究了施用不同肥料和不同施肥方法等对强筋小麦养分吸收和产量品质的影响。结果表明:在氮肥用量相同时,氮肥后移小麦产量和品质均好于全部基施(习惯施肥)处理;高氮和硫酸铵处理能提高强筋小麦的品质。磷酸二铵提高小麦产量效果好于过磷酸钙,而对子粒品质影响则过磷酸钙好于磷酸二铵;高量磷肥虽然不能进一步提高小麦产量,但能改善小麦的品质。增施有机肥和钾肥可促进小麦对N、P、K养分的吸收,显著提高小麦产量和品质,是砂姜黑土区优质高产强筋小麦重要施肥技术。锌肥能提高小麦产量和品质,含硫肥料有改善小麦品质的作用。试验表明,在砂姜黑土上,施用有机肥,稳定磷肥用量,加大氮、钾肥用量,配施锌肥和硫肥,分期施用氮肥(追肥量占总氮量比例在40%以上)有利于强筋小麦的优质高产。     

Occurrence of the free and Peptide forms of pyroglutamic acid in plasma from the portal blood of rats that had ingested a wheat gluten hydrolysate containing pyroglutamyl peptides

In order to determine pyroglutamic acid levels in plasma, we developed a method based on precolumn derivatization of the carboxyl group of pyroglutamic acid with 2-nitrophenylhydrazine. Eight-week-old male SD strain rats were administered 200 mg of an acidic peptide fraction obtained from a commercial wheat gluten hydrolysate containing 0.63 mmol/g pyroglutamyl peptide. After administration, significant amounts of free pyroglutamic acid were observed in the ethanol-soluble fraction of the plasma from the portal vein. In addition, pyroglutamate aminopeptidase digestion of the ethanol-soluble fraction liberated significant amounts of pyroglutamic acid, which indicated the presence of the pyroglutamyl peptide. The presence of the pyroglutamyl peptide in the plasma was further confirmed by size exclusion chromatography. The levels of free and peptide forms of pyroglutamic acid increased significantly and reached a maximum (approximately 40 nmol/mL) at 15 and 30 min after administration, respectively.     

Glass transition of wheat gluten plasticized with water, glycerol, or sorbitol

The glass transition temperature of wheat gluten, plasticized with water, glycerol, or sorbitol, has been studied using dynamical mechanical thermal analysis. For the three plasticizers studied, the general behavior of the glass transition temperature broadly followed the Couchman-Karasz relation using a wheat gluten DeltaC(p)() of 0.4 J g(-)(1) K(-)(1). Compared on such a fractional weight basis, it could be concluded that the plasticizing effect of glycerol and sorbitol on wheat gluten proteins is less important than the plasticizing effect of water. A continuous curve was obtained with the three plasticizers when the evolution of the glass transition temperature was presented on a fractional molecular basis. This was related to the similar chemical structure of these three components containing hydroxyl groups.     

肥密互作对强筋春小麦品质的影响

针对某些地区小麦生产中存在的加大种植密度和增加施肥量问题,以优质春小麦品种“龙麦26”为材料,设置施肥量与密度互作试验,分析其对高寒地区强筋春小麦品质形成的影响。结果表明,种植密度和施肥量对小麦子粒品质和面粉品质的影响不具交互效应,但施肥量和种植密度对小麦千粒重和子粒容重的影响显著。稀植可提高千粒重,但降低了子粒容重,较高的施肥量降低了子粒容重和千粒重;适宜的施肥量可提高子粒中蛋白质的百分含量和湿面筋含量,同时获得较高的沉淀值和降落值。研究认为,在600～750万株/hm2的种植密度基础上400 kg/hm2左右的施肥量是高产优质的合理组合。