Malt hydrolysates for gluten-free applications: Autolytic and proline endopeptidase assisted removal of prolamins from wheat,barley and rye

Cereal based products intended for gluten sensitive individuals, particularly to celiac disease patients, tend to have poor organoleptic qualities and they contain low levels of healthy whole grain compounds. Adding whole grain ingredients, such as malt hydrolysates, could compensate these defects provided that the ingredients are adequately free from toxic prolamin epitopes. Here we demonstrate that the level of toxic prolamin epitopes in the malt autolysates (wheat, barley, rye) were substantially lower than in the native malts but too high to allow “very low in gluten” labelling. To further eliminate the residual levels of toxic prolamin epitopes, a proline-specific endoprotease from Aspergillus niger was added to the malt autolysates. In the resulting malt hydrolysates (of wheat and rye but not barley), the prolamins were indeed greatly reduced and were below the very low gluten limit of 100 mg/kg. Malt hydrolysates with adequately low gluten levels may potentially be used as novel ingredients within gluten-free foods.     

Laccase-mediated enhancement of the properties of regenerated fibers from wheat gliadin

In this study, laccase-mediated crosslinking was used to develop regenerated protein fibers from wheat gliadin with good mechanical properties and water stability. The oxygen consumption during laccase catalyzed oxidation of gliadin, molecular weight of gliadin, mechanical properties, water stability, thermal properties and morphology of gliadin fibers were tested to prove the effect of laccase as the crosslinker of gliadin. The rapid decrease of dissolved oxygen in gliadin solution indicated laccase was active in oxidizing gliadin. The results of SDS-PAGE and SEC demonstrated that laccase-mediated crosslinking reaction effectively occurred. The mechanical properties and water stability of the gliadin fibers with laccase treatment significantly were improved. The fibers from gliadin with laccase treatment exhibited a much smoother and more uniform surface was observed by SEM for the laccase-mediated modified gliadin fibers.     

Production of composites by using gliadin as a bonding material

In previous papers, a new technology that produces biopolymer composites by particle-bonding was introduced. During the manufacturing process, micrometer-scale raw material was coated with a corn (Zea mays L.) protein, zein, which was then processed to form a rigid material. The coating of raw-material particles with zein makes use of the unique property of this protein in aqueous ethanol solution. In this paper, it is shown that the behavior of a wheat (Triticum aestivum L.) protein, gliadin, is very similar to zein in aqueous ethanol. Size variation of aggregates in 45-65% aqueous ethanol was investigated with a turbidimeter in conjunction with Size Exclusion Chromatography. Confirming the resemblance of gliadin to zein in terms of aggregation behavior in aqueous alcohol, composites were fabricated by particle-bonding and their mechanical property was measured with a Universal Testing Machine. Test results showed that gliadin is a good substitute for zein in the production of composites.     

Fabrication and characterization of wheat gliadin hydrogels with high strength and toughness

Fabricating a hydrogel with high strength and toughness is still a challenge in many fields. Here, we prepared gliadin-based hydrogels by chemical cross-linking gliadin in acetic acid solution (GS) with glutaraldehyde (GA). Subsequently, the overall properties of the fabricated hydrogels were systematically investigated in terms of their mechanical properties, swelling ratio, weight loss, thermal stability, and the chemical/physical interactions in hydrogels. Results showed that the gliadin-based chemically cross-linked hydrogels exhibited excellent mechanical properties. The optimized hydrogel exhibited the compressive stress of 1.8 MPa at a strain of 70%, and an excellent self-recovery property after 30 cycles of loading-unloading treatments. The strength and toughness of the hydrogels could be tailored by adjusting the ratio of GS/GA. The chemical cross-linking (aldehyde-ammonia reaction) was the main molecular interaction in the hydrogels, including single-/multi-site crosslinking, and the hydrogen bond was the only physical cross-linking in the hydrogels. Moreover, the swelling ratio of the fabricated hydrogels performed a concentration negative-dependency in GA or GS concentration. And a higher GS concentration (40%) with an appropriate GA content (3.0%) could resist the degradation of hydrogels. In addition, the thermodynamic properties of hydrogels also improved by the GA addition. Overall, these findings suggested that gliadin can be applied for fabricating hydrogels with tunable mechanical properties, which will unlock the high-utilization of gliadin as biopolymer and biocompatible materials.     

β-Elimination reactions and formation of covalent cross-links in gliadin during heating at alkaline pH

The aim of this study was to increase insight into gluten polymerisation. While previous research on this topic focused on disulfide (SS) bonds, the present paper focuses on cross-links based on dehydro-protein formation through β-elimination reactions. Gliadin, the monomeric fraction of gluten containing no free sulfhydryl (SH) groups, was heated for 120 min at pH 8.0 and 130 °C, and cross-link formation was evaluated by determining extractability in sodium dodecyl sulfate containing buffer, reaction products of β-elimination reactions, and cross-links involving the latter. Heating decreased gliadin extractability. Reduction of SS bonds increased extractability of heated gliadin, but did not restore it to that of non-heated gliadin, suggesting contribution of both SS and non-SS bonds to gliadin cross-linking. Decreased SS levels and the presence of dehydroalanine and SH groups in heated gliadin samples indicated cleavage of SS bonds by β-elimination reactions. Some of the formed free SH groups were then involved in oxidation and/or SH–SS interchange reactions leading to intermolecular SS bonds. In addition, amino acid analysis revealed formation of an irreversible non-SS cross-link between dehydroalanine and the free SH group of cysteine, namely lanthionine. In conclusion, non-SS bonds may well contribute to the gluten network under specified reaction conditions.     

Influence of low soil nitrogen and phosphorus on gluten polymeric and monomeric protein distribution in two high quality spring wheat cultivars

The effect of low levels of nitrogen, phosphorus and a combination of the two on the distribution of polymeric and monomeric proteins in two high quality spring bread wheat cultivars was investigated for two consecutive seasons. Size exclusion-high performance liquid chromatography (SE-HPLC) was used to determine the quantity and relationships of monomeric and polymeric proteins, and their relationship with flour protein content (FPC) and SDS sedimentation volume (SDSS). The low nitrogen and combined low nitrogen and low phosphorus treatments had a much larger effect on the protein fractions than the low phosphorus treatment alone. The SDS-soluble large monomeric protein fraction and the percentage SDS-insoluble monomeric proteins, were significantly increased under low nitrogen and a combination of low nitrogen and low phosphorus treatments. The percentage SDS-insoluble large and total polymeric proteins was significantly reduced under low nitrogen and a combination of low nitrogen and phosphorus treatments. The SDS-soluble and -insoluble small polymeric proteins were significantly increased under both low nitrogen and a combination of low nitrogen and low phosphorus treatments. The low nitrogen treatment consistently caused the lowest FPC and SDSS values. Under low nitrogen conditions, there was a significant positive correlation between the SDS-soluble gliadins and SDSS, and FPC.     

Valorization of rice by-products: Protein-phenolic based fractions with bioactive potential

Rice bran (RB) is an important by-product from the rice production with a high valorization potential. The aim of the present work was to explore this potential by considering combined protein and phenolic fractions and enzymatic hydrolysis. The influence of different protocols in the recovery/fractioning of protein were evaluated, including defatting procedures, in rice brans from two very different locations: Portugal and Colombia. The different protein fractions achieved were characterized. Proteins were further hydrolyzed using trypsin and their hydrolysates were characterized in terms of degree of hydrolysis and peptide size. Moreover, the total phenolic content (TPC) and phenolic compounds’ profile on extracted fractions and hydrolysates of RB from Portugal and Colombia were determined. The functional potential was assessed in terms of antioxidant and antihypertensive activities. Though the protein concentrates had already significant potential bioactivities, protein hydrolysates showed that the TPC, antioxidant and antihypertensive activities significantly increased with the digestion by trypsin. Hydrolysates of RB from Portugal presented a higher antioxidant and hypertensive activities in comparison with hydrolysates of RB from Colombia, moreover it presented a higher content of TPC. This study indicated that the exploitation potential of rice bran can be increased with simple fractioning procedures, that combined protein/phenolic fractions are important to deliver functionality and that hydrolysis can be a relevant tool to release that functionality. Furthermore, regional differences in rice bran composition are also important factors to consider. Overall, this knowledge can be used to increase the industrial potential of valorization for this by-product.     

Impact of microwave heating on hemagglutinins,trypsin inhibitors and protein quality of selected legume seeds

Selected legume seeds (dry and soaked) including faba beans, peas, chickpeas, soybeans, lentils and common beans containing 8 and 25% moisture, respectively, were subjected to microwave heating, and to a conventional cooking method to determine the heating effect on toxic compounds and protein quality. Trypsin inhibitors, hemagglutinins and available lysine were analyzed, and laboratory rats were used to determine digestibility and protein efficiency ratio (PER). Results indicated that microwaving destroyed trypsin inhibitors to a similar degree to that observed in beans cooked using the conventional method without affecting PER for raw seeds with low antinutrients content (faba beans, peas, chickpeas and lentils). Microwave-heated soaked soybeans had a higher amount of destroyed trypsin inhibitors, along with a higher PER, compared with microwave-heated dry soybeans. Microwave heating of common beans failed to destroy hemagglutinins and trypsin inhibitors, and consequently their digestibility and PER values were poor. Finally it was concluded that microwave heating constitutes an adequate method for destroying hemagglutinins and trypsin inhibitors without affecting protein quality of most legume seeds, except for common beans that despite of this process retained the antinutritional substances.     

Properties of protein concentrates and hydrolysates from Amaranthus and Buckwheat

Protein concentrates and pepsin hydrolysates were made after isoelectric precipitation of the proteinaceous liquor from wet-milling of grain of five Amaranthus and one buckwheat genotype. The Amaranthus protein concentrates exhibited better solubility, foaming, and emulsification than two commercial soy protein controls. Many protein properties depend on solubility, and Amaranthus protein concentrates were more soluble than soy protein isolate. The buckwheat protein concentrate was highly soluble with excellent emulsification, but poor foaming ability. Partial pepsin hydrolysis further improved solubility of the protein concentrates and also altered their foaming property. Fractionation and subsequent characterization of protein concentrates revealed that glutelins, albumins, and globulins predominated, with prolamins present in minor quantity. SDS-PAGE showed that globulins and glutelins were comprised of several subunits with varying molecular weights from relatively high to low while albumins were mostly of low molecular weight. The prolamin fraction of the buckwheat concentrate was comprised of intermediate to low molecular weight subunits while those of Amaranthus concentrates were only of low molecular weight. This study demonstrated the feasibility of producing potentially useful functional protein concentrates as by-product of Amaranthus and buckwheat starch extraction.     

Effect of hydrostatic pressure and temperature on the chemical and functional properties of wheat gluten: Studies on gluten, gliadin and glutenin

The effect of hydrostatic pressure (0.1–800 MPa) in combination with various temperatures (30–80 °C) on the chemical and physical properties of wheat gluten, gliadin and glutenin was studied. Chemical changes of proteins were determined by extraction, reversed-phase high-performance liquid chromatography (HPLC), sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy, thiol measurement and studies on disulphide bonds. Rheological changes were measured by extension tests and dynamic stress rheometry. Treatment of gluten with low pressure (200 MPa) and temperature (30 °C) increased the proportion of the ethanol-soluble fraction (ESF) and decreased gluten strength. The enhancement of both pressure and temperature provoked a strong reduction of the ESF and the thiol content of gluten. Within gliadin types, cysteine containing α- and γ-gliadins, but not cysteine-free ω-gliadins were sensitive to pressure and were transferred to the ethanol-insoluble fraction. Disulphide peptides isolated from treated gluten confirmed that cleavage and rearrangement of disulphide bonds were involved in pressure-induced reactions. Increased pressure and temperature induced a significant strengthening of gluten, and under extreme conditions (e.g. 800 MPa, 60 °C), gluten cohesivity was lost. Isolated gliadin and glutenin reacted differently: solubility, HPLC and SDS-PAGE patterns of gliadin having a very low thiol content were not influenced by pressure and heat treatment; only conformational changes were detected by CD spectroscopy. In contrast, the properties of isolated glutenin having a relatively high thiol content were strongly affected by high pressure and temperature, similar to the effects on total gluten.     

Fractionation of oats into products enriched with protein,beta-glucan,starch, or other carbohydrates

A modified wet method was developed to fractionate ground oat groats into 4 fractions enriched with beta-glucan (BG), protein, starch, and other carbohydrates (CHO), respectively. Effects of defatting oats and centrifuge force for separation were also investigated. Results show that, depending on the two factors, nutrient concentrations in the corresponding fractions ranged 28.53–44.84% (dry matter) for BG, 72.41–92.62% for protein, 79.13–81.69% for starch, and 37.47–42.16% for other CHO. Nutrient recoveries from each fraction were 39.82–51.20% for BG, 60.36–72.08% for protein, 77.04–87.25% for starch, and 24.87–29.68% for other CHO. For the protein fraction, defatting improved the protein content but did not on its recovery. For the starch fraction, the effect of defatting was just opposite, improving the starch recovery but not the starch content. Centrifuge force increased the BG content in the BG fraction when the defatted sample was used. The cumulative recovery of each component in the combined fractions was not affected by oil removal or centrifuge force. Overall, the wet method described was relatively effective in recovering the major nutrients from oats into their respective fractions, while it alleviated the problems of viscous slurry upon mixing ground oat groats with an alkaline solvent and the difficulty of slurry separation.     

Effects of protein oxidation on gelatinization characteristics during rice storage

Gliadin and glutelin are major rice storage proteins. In this study, we evaluated changes in these proteins to determine the influence of pasting properties on rice storage. Notably, the physical and chemical properties of these proteins changed steadily. We analyzed protein oxidation indexes and determined correlations between the protein oxidation index and gelatinization property index. The results showed that significant oxidation of gliadin and glutelin occurred during the process of rice storage (P < 0.05). Protein oxidation had a significant impact on pasting properties. For glutelin, changes in the structure and function of the protein during rice storage had a significant impact on the five rice pasting property indexes, including peak viscosity (PV), holding strength (HS), breakdown, final viscosity (FV), and setback (SB; P < 0.05). In particular, the levels of carbonyl compounds and active sulfur and the surface hydrophobicity of glutelin were significantly correlated with rice pasting property indexes (P < 0.05). However, gliadin only significantly affected three indexes, i.e., PV, HS, and FV (P < 0.05). Thus, these findings suggested that the carbonyl compounds, active sulfur, and surface hydrophobicity of glutelin could be used as sensitive indexes for changes in rice quality evaluation.     

小麦新品种川农16与99E18的SSR及贮藏蛋白差异分析

为了更好地识别川农16的遗传基因特性,为充分利用优异材料99E18改良川农16提供理论依据,应用简单重复序列(SSR)标记、酸性聚丙烯酰胺凝胶电泳(A-PAGE)和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对小麦新品种川农16与高抗优质材料99E18间的遗传差异进行了检测与分析。SSR标记检测结果表明川农16与99E18在DNA水平上存在明显差异。A-PAGE分析显示川农16与99E18间至少有12条醇溶蛋白差异带。SDS-PAGE分析表明川农16和99E18的高分子量谷蛋白亚基组成分别为(1,20．5 10)和(1,7 8,5 10)。     

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

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

河西灌区小麦地方品种醇溶蛋白的遗传多样性分析

为了挖掘小麦地方品种的潜力,采用APAGE方法分析了河西灌区70份小麦地方品种的醇溶蛋白,研究了醇溶蛋白的遗传多样性。结果表明,供试材料中共分离出迁移率不同的醇溶蛋白谱带91条,品种间变异幅度为11～26条,平均为18.33条,变异系数为16.08,具有16、17、19、20条谱带的品种最多。在91条醇溶蛋白谱带中,B01号谱带出现频率最高,为97.14%;B77和B03号谱带出现的频率也较高,分别为92.86%和88.57%。B72和B91号谱带出现频率最低,分别只出现1次,频率均为1.43%。其余谱带多态性很高。在不同分区中醇溶蛋白谱带的分布存在较大差异,ω区出现的谱带最多,β区次之,γ区第三,α区出现的谱带最少。供试材料之间遗传距离的变化范围为0.24～1.00,平均值为0.66。说明河西灌区小麦地方品种间存在丰富的醇溶蛋白遗传多样性,在小麦品质育种中具有一定利用价值。     

安徽小麦品种醇溶蛋白遗传多样性及其与品质性状的相关性研究

为了解安徽省种植小麦品种醇溶蛋白对其品质性状的影响,利用酸性聚丙烯酰胺凝胶电泳(A-PAGE)技术对137份安徽小麦品种进行醇溶蛋白谱带分析,同时测定了面粉的理化特性、面粉粉质特性以及面筋相关指标。结果表明,137份安徽省种植小麦品种共分离出2690条醇溶蛋白谱带,统计后得出不同迁移率的谱带共96条。其中,出现次数最高的是ω-14.3,出现概率为77.70%;出现次数最低的是α-81.7和α-89.3,出现概率均为0.72%。供试材料的遗传距离(GD)为0.13~0.81,所有材料的遗传距离平均值为0.70,说明供试材料的遗传差异较大。在GD=0.73的水平上将所有材料分为7个类群。57条不同迁移率的谱带与136项次品质性状的相关系数达到极显著或显著水平。其中,谱带β-68.0与面团粉质质量指数和面筋指数呈显著正相关,谱带α-73.4与面粉色泽L*值和白度值呈显著正相关,与b*值呈显著负相关。本试验筛选出这些对若干面粉及面团品质性状有显著影响的谱带,可为小麦品质育种工作提供参考。     

离子束介导大豆DNA转入普通小麦后代的麦谷蛋白和醇溶蛋白分析

为了研究离子束介导大豆DNA转入普通小麦的效果,应用SDS-PAGE和A-PAGE电泳分析了离子注入介导大豆DNA转入普通小麦后代中高蛋白含量植株的麦谷蛋白和醇溶蛋白。SDS-PAGE电泳结果表明,与对照相比有8个高蛋白含量植株的HMW-GS谱带数目发生了变化或着色增强。A-PAGE电泳结果表明,与对照相比有9个高蛋白含量植株的醇溶蛋白谱带数目及着色强度发生了变化。说明离子束介导大豆DNA转入普通小麦后代中的高蛋白含量植株在醇溶蛋白和麦谷蛋白基因位点上可能出现了变异。     

小麦高压诱变后代醇溶蛋白变异分析及其对品质性状的影响

为了验证高压诱变对小麦品质改良的有效性,利用酸性聚丙烯酰胺凝胶电泳(A-PAGE)分析了39份小麦高压诱变后代的醇溶蛋白遗传变异情况,并测试了小麦高压诱变后代的理化品质和面团流变学特性.结果表明,39份高压诱变材料出现24条迁移率不同的电泳谱带,与对照相比共有6条迁移率不同的谱带;发生的变异可分为三大类,共37种,变异率达94.8％,变异主要发生在ω和γ区,6条变化的谱带均与8项品质性状相关显著或极显著.总体趋势是当经过高压诱变之后,醇溶蛋白谱带数总量减少,但是部分面团相关品质提高.     

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.