Prolamin hydrolysis in wheat sourdoughs with differing proteolytic activities

The prolamins of wheat, rye, and barley contain structures that are harmful to gluten-sensitive people, and an extensive degradation of these prolamins during food processing might eliminate this problem. Sourdough fermentation is a cereal food process during which some protein degradation occurs. In this study, the prolamin hydrolysis that occurred in a high-proteolytic-activity germinated-wheat sourdough (GWSD) was compared with that of wheat sourdough systems which contained moderate or no proteolytic activities. Virtually all of the wheat prolamins (gliadins and glutenins) were degraded during the GWSD fermentation. Quantification of its prolamin levels confirmed that extensive prolamin hydrolysis had occurred in the GWSD. This hydrolysis was attributed to the cysteine proteinase activities of the germinated wheat. The use of high-proteolytic sourdoughs in baking could make it possible to prepare new low-prolamin cereal-based products for use by gluten-sensitive people, who could then diversify their diets by including these whole-grain containing products into their every-day diets.     

Endogenous Action of Cysteine Endopeptidase and Three Carboxypeptidases on Triticale Prolamins

Quantitative and qualitative changes occurring in the prolamin fraction in the starchy endosperm of triticale grains were analyzed by SDS‐PAGE on consecutive days of germination. The most intensive hydrolysis of prolamins was observed after the second day of the process. The high molecular weight fractions of prolamins were degraded with the highest rate. Endopeptidase EP8 was capable of hydrolyzing all fractions of prolamins isolated from dry triticale grains, but the high molecular weight fractions were the most rapidly degraded by the enzyme. Carboxypeptidases I, II, and III isolated from triticale grains hydrolyzed prolamins proteolytically modified by endopeptidase EP8, whereas intact prolamins were degraded slightly. Differences in the activity of the studied carboxypeptidases against crude prolamins indicate that carboxypeptidase II may be involved in the initiation of the hydrolysis process and, together with carboxypeptidases I and III, participates in the later stages of degradation of prolamins to amino acids. Experiments with exogenous GA₃ demonstrated that the synthesis of EP8 is induced by this hormone and takes place in the aleurone layer. Mass spectrometry analysis showed the enzyme to be a homologue of barley endopeptidase EP‐A. Both enzymes belong to the cysteine class endopeptidases.     

Zinc Blotting Assay for Detection of Zinc‐Binding Prolamin in Barley (Hordeum vulgare) Grain

In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare), major storage proteins are alcohol‐soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. ⁶⁵Zn overlay and blotting techniques have been widely used for detecting zinc‐binding protein. However, to our knowledge so far this zinc blotting assay has never been applied to detect a prolamin fraction in barley grains. A radioactive zinc (⁶⁵ZnCl₂) blotting technique was optimized to detect zinc‐binding prolamins, followed by development of an easy‐to‐follow nonradioactive colorimetric zinc blotting method with a zinc‐sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS‐PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc‐binding specificity of certain proteins was detected either by autoradiography or color formation. The dithizone staining method gave similar reproducibility to the radioactive blotting. The detected zinc‐binding protein was identified as B‐hordein by Western blotting.     

Biochemical Characterization and Quantification of the Storage Protein (Secalin) Types in Rye Flour

Kernels of the rye cultivars Danko and Halo were milled into white flour and compared with flour of the wheat cultivar Rektor. Flour proteins were extracted stepwise with a salt solution (albumins‐globulins), 60% ethanol (prolamins), and 50% 2‐propanol under reducing conditions (glutelins). The quantification by reversed‐phase HPLC indicated that the extractable proteins of both rye flours consisted of ≈26% albumins‐globulins, 65% prolamins, and 9% glutelins. Compared with wheat flour, rye flours comprised significantly higher proportions of nonstorage proteins (albumins‐globulins) and lower proportions of polymerized storage proteins (glutelins). SDS‐PAGE revealed that the prolamin fractions of rye contained all four storage protein types (HMW, γ‐75k, ω, and γ‐40k secalins), whereas the glutelin fractions contained only HMW and γ‐75k secalins. The quantification of secalin types by RP‐HPLC showed a close relationship between the two cultivars.The γ‐75k secalins contributed nearly half (≈46%) of the total storage proteins, followed by γ‐40k secalins (24%) and ω secalins (17%); HMW secalins (≈7%) were minor components, and 6% of eluted proteins were not identified. The amino acid composition of γ‐40k secalins corresponded to those of γ‐gliadins of wheat, whereas γ‐75k secalins were characterized by higher contents of glutamine and proline. Matrix‐assisted laser desorption/ionization and time of flight mass spectrometry (MALDI‐TOF MS) indicated molecular masses of about 52,000 (γ‐75k) and 32,000 (γ‐40k), respectively. N‐terminal amino acid sequences were homologous with those of wheat γ‐ gliadins except for position 5 (asparagine in γ‐75k and glutamine in γ‐40k secalins) and position 12 (cysteine in γ‐75k secalins). The N‐terminal amino acid sequences of HMW and ω‐secalins were homologous with those of the corresponding protein types of wheat. Gel‐permeation HPLC of prolamin fractions revealed that rye flours contained a significantly higher proportion of ethanol‐soluble oligomeric proteins than wheat flour.     

Characterization of Kafirins in Algerian Sorghum Cultivars

The prolamins in seven Algerian Sahara sorghum cultivars of varying seed shape and color were investigated. Protein contents ranged from 12 to 16%. Prolamins were the major protein fraction. They could be separated according to degree of disulfide cross‐linking. Kafirin monomers and low molecular weight polymers could be extracted with 70% ethanol, whereas highly cross‐linked kafirins additionally needed a reducing agent to become extractable. Kafirin monomers of α‐, β‐ and γ‐type were purified and N‐terminally sequenced. For the first time, δ‐kafirin was identified at the protein level. The study clearly revealed intercultivar differences between protein levels. The joint use of SDS‐PAGE, SE‐HPLC, and RP‐HPLC allowed discriminating among cultivars based on the differences in prolamin levels and composition.     

Effect of Selected Hofmeister Anions on the Secondary Structure and Dynamics of Wheat Prolamins in Gluten

The effect of selected salts (NaCl, NaBr, NaI) with increasingly chaotropic anions on gluten prolamin secondary structure and dynamics was studied using FT‐IR and NMR spectroscopy. FT‐IR spectra indicated a small increase in intermolecular β‐sheet structures when gluten samples were incubated with low levels of NaCl and NaBr. The β‐sheet content remained constant with further NaCl addition, but was reduced again with higher NaBr concentrations. NaI addition generally reduced the amount of β‐sheet and increased the amount of β‐turns. From these data, it could be concluded that, in the presence of chaotropic anions, the proteins tended to prefer the β‐turn helix structure characteristic of glutenin subunits in solution. ¹H‐NMR relaxation showed that the molecules were more rigid in 1M NaCl than in pure water, and that the mobility of the prolamins in the presence of 1M NaCl, NaBr, and NaI increased in accordance with the position of the anions in the Hofmeister series. The increase in mobility appeared to be associated with the increase in β‐turn helical structures.     

Improvement of Sorghum-Wheat Composite Dough Rheological Properties and Breadmaking Quality Through Zein Addition

Addition of sorghum flour to wheat flour produces marked negative effects on rheological properties of dough and loaf volume. Although there are notable differences in the chemical composition of sorghum proteins (kafirins) compared with wheat gluten that might imply poor functionality in breadmaking systems, a larger constraint may be the unavailability of kafirins due to encapsulation in protein bodies. In this study, zein, the analogous maize prolamin to kafirin, was used to determine the potential effects of protein-body-free prolamins on dough rheology and baking quality of wheat-sorghum composite flour. Mixograms run at 35°C (above the glass transition temperature of zein) were significantly (P < 0.01) improved with addition of zein. Mixogram peak heights increased while mixing time decreased uniformly with addition of zein. Dough extensibility studies showed an increase in maximum tensile stress, while baking studies showed an increase in loaf volume with increasing amounts of added zein. These data are supported by a previous study showing that, in a model system, zein mixed with starch can form viscoelastic networks, and suggest that kafirin, if made available, could contribute to dough formation.     

Influence of Rice Proteins on Eating Quality of Cooked Rice and on Aroma and Flavor of Sake

An improved method for the extraction of storage proteins from rice endosperm under conditions safe for producing food was developed. The contribution of the protein extracts to the eating quality of cooked rice and to the aroma and flavor of sake was examined. Sensory analysis was performed to evaluate the eating quality of cooked rice enriched with the protein extracts. Prolamin‐enrichment increased the hardness of cooked rice, and glutelin‐enrichment degraded the appearance of cooked rice. Physical analyses confirmed that prolamin‐enrichment changed, whereas the glutelin‐enrichment did not change the physical properties of cooked rice. Light and scanning electron microscopy of rice protein extracts revealed small particles of the prolamin extract and larger aggregated particles of the glutelin extract; these features remained after heating. The aroma and flavor of sake were negatively affected by the addition of the protein extracts. Especially, addition of prolamin significantly lowered the evaluation score of the aroma obtained by sensory analysis.     

Influences of Acid and Alkali on Mechanical Properties of Compression‐Molded Gluten Bioplastics

The present work was focused on the effects of acid and alkali on the uniaxial extensional deformation of gluten bioplastics compression‐molded at 80 and 110°C. Water absorption and morphology observation were also performed to characterize the properties and morphology of the gluten bioplastics. The results show that HCl did not show catalysis function for cross‐linking proteins at 80°C and even hindered the cross‐linking reaction at 110°C. On the other hand, NaOH generally accelerated the cross‐linking reaction during compression‐molding, thus significantly improving Young's modulus, tensile strength, and strain at break. However, an NaOH content >0.5 wt% could cause adverse effects at molding temperatures as high as 110°C.     

基于两维图论聚类的辽宁省土地利用多功能性分区

辽宁省土地利用功能间的矛盾相对突出,明晰区域土地利用多功能的现状、进行土地利用多功能性分区可为促进区域土地利用功能的协调发展提供依据。选取2017年辽宁省县域土地利用生活、生产、生态方面22个典型指标构建区域土地利用多功能性评价指标体系,运用熵权法和两维图论聚类法进行土地利用多功能性评价及分区研究。研究发现:1)研究区生活功能高值区于中部呈斜"X"状分布,生产功能值主要呈现以沈阳为中心的"环状"递减蔓延结构,生态功能值由外至内逐渐减少。2)区域县域生产功能和生活功能呈显著正相关,生产功能和生态功能呈显著负相关。3)根据区域土地利用多功能值和两维图论聚类法可将研究区划分为辽东生态功能优势区、辽北功能欠协调区、辽南功能相对均衡区以及辽西功能欠发展区。研究结果可为县域尺度国土空间规划的制定与实施等提供技术支撑和科学依据。     

REVIEW: Developments in Our Understanding of Sorghum Polysaccharides and Their Health Benefits

The composition and structure of sorghum polysaccharides are remarkably similar to those in maize. Sorghum grain is rich in starch, cellulosic and noncellulosic polysaccharides (mainly glucuronoarabinoxylans [GAX]). Sorghum starch is similar to maize starch in terms of amylopectin, but the amylose may be more branched. This may account for sorghum starch having a generally slightly higher gelatinization temperature. The GAX in sorghum are highly substituted with glucuronic acid and arabinose, but the degree of these substitutions is lower when compared with maize GAX. Sorghum polysaccharides themselves are not sufficiently functional to allow the production of high‐quality baked goods. Sorghum has generally lower starch digestibility than maize. This is primarily due to the endosperm protein matrix, cell wall material, and tannins (if present) inhibiting enzymatic hydrolysis of the starch. Protein disulfide bond cross‐linking involving the kafirin prolamins in the protein matrix around the starch granules seems to be of major importance in reducing starch digestibility. It does not seem that sorghum polysaccharides, per se, have any unique health‐promoting effects. Any health‐promoting effects related to sorghum polysaccharides seem to be due to interactions between the polysaccharides and the endosperm matrix protein and phenolics.     

Clay mineralogy differs qualitatively in aggregate‐size classes: clay‐mineral‐based evidence for aggregate hierarchy in temperate soils

Clay minerals have a major role in soil aggregation because of their large specific surface area and surface charges, which stimulate interactions with other mineral particles and organic matter. Soils usually contain a mixture of clay minerals with contrasting surface properties. Although these differences should result in different abilities of clay minerals regarding aggregate formation and stabilization, the role of different clay minerals in aggregation has been seldom evaluated. In this study, we took advantage of the intrinsic mineral heterogeneity of a temperate Luvisol to compare the role of clay minerals in aggregation. First, grassland and tilled soil samples were separated in water into aggregate‐size classes based on the aggregate hierarchy model. Then, clay mineralogy and organic C in the aggregate‐size classes were analysed. Interstratified minerals containing swelling phases accumulated in aggregated fractions compared with free clay fractions under the two land‐uses. The accumulation increased with decreasing aggregate size from large macroaggregates (> 500 µm) to microaggregates (50–250 µm). Carbon content and carbon‐to‐nitrogen ratio followed the opposite trend. This fully supports the aggregate hierarchy model, which postulates an increasing importance of mineral reactivity in smaller aggregates than in larger aggregates in which the cohesion relies mostly on physical enmeshment by fungal hyphae or small roots. Consequently, differences in the proportion of the different 2:1 clay minerals in soils can influence their structure development. Further research on the links between clay mineralogy and aggregation can improve our understanding of mechanisms of soil resistance to erosion and organic matter stabilization.     

Temperature‐Induced Changes in the Dynamic Rheological Behavior and Size Distribution of Polymeric Proteins for Glutens from Wheat Near‐Isogenic Lines Differing in HMW Glutenin Subunit Composition

Viscoelasticity of hydrated gluten depends on composition of HMW gluten subunits (GS), size distribution of glutenin polymers, and proteinprotein interactions. Glutens extracted from four near‐isogenic lines with differing HMW‐GS were analyzed. Rheological properties were studied by dynamic assay in shear. Size distribution of prolamins was determined by sequential extraction and size‐exclusion HPLC. Assays performed at 20°C confirmed that viscoelasticity was determined by large glutenin polymers. The abundance of large glutenin polymers depended on the HMW‐GS composition of the lines. Difference of functionality linked to subunit structure was highlighted by comparing the behaviors of the 1A/1B null and 1A/1D null lines. Glutens were submitted to heating and cooling cycles, with or without an SH‐blocking agent (N‐ethylmaleimide [NEMI]). At 20–40°C, no irreversible changes of the mechanical properties occurred. Thermal treatment affected chain mobility, and possibly H bonds, but not the chemical structure of the network. At >40°C, irreversible rheological changes were observed without NEMI. Irreversibility was mainly due to chemical modifications affecting the polymer size distribution through SH‐SS exchange reactions. The sensitivity of gluten to temperature depended on subunit composition.     

Effects of supplemental‐nitrogen on the quality of rice proteins

A study was conducted on the effect of supplemental nitrogen (N) (20 hg/ha) applied as a foliar spray or to the soil on seed production, protein percentage, and protein fractions of rice. Plants were grown in a greenhouse over two different periods of time, i.e., August 1988 to January 1989 (Period I), and December 1988 to April 1989 (Period II). Nitrogen was applied to the leaves 10 and 20 days after anthesis (DAA), and to the soil at anthesis and at 15 DAA. Average temperature was 28.7°C during Period I and 32°C during Period II, corresponding to 18.7 and 22.0 growing degree‐day/day (GDD/day), respectively. The difference in GDD/day reduced the plant cycle from 130 days during Period I to 109 days during Period II. Plants grown during Period II had larger numbers of spikelets, a higher percentage of “full grown grains”;, and higher grain weight. Although percentage crude protein was about the same for the two periods, prolamin content was increased and the albumin+globulin fraction was decreased during Period II, but with no difference in glutelin content. The increase in number of spikelets, percent full grains, and grain weight appeared to result in a greater energy demand for plants grown during Period II. This may explain the increase in prolamins, since prolamin synthesis requires less energy than globulin or albumin synthesis. There was a simultaneous decrease in albumin and globulin synthesis during Period II. The content of glutelins, which represent the major reserve proteins in rice grains, was constant during both periods.     

Understanding mechanisms of soil biota involvement in soil aggregation: A way forward with saprobic fungi?

Soil aggregation is a key ecosystem process that strongly affects soil structure. Soil structure is the three dimensional arrangement of primary particles, organic matter, soil aggregates and associated pores. As such, soil aggregation influences the organization of soil biodiversity and soil-borne biogeochemical processes. Saprobic fungi (SF) have promising but largely untapped potential to offer new perspectives and insights into mechanisms of soil aggregation. The study of SF permits identification of traits that may predict soil aggregation component processes: formation, stabilization and disintegration. The measurement of fungal key traits in experiments aimed at soil aggregation effects will generate data necessary for mechanistic understanding. When such efforts are combined with collecting such information across a range of systems in curated databases this can, by channeling efforts, lead to a step change in our understanding and modeling of organism-mediated soil aggregation mechanisms and changes in functional diversity due to global change.     

Identification of dehydro-ferulic acid-tyrosine in rye and wheat: evidence for a covalent cross-link between arabinoxylans and proteins

To monitor chemical reactions between ferulate and proteins during breadmaking, 8-(14)C-(E)-ferulic acid-(d-galactopyranose-6'-yl)ester was synthesized as a radiotracer and added to wheat and rye flour prior to breadmaking. Breads were lyophilized, extracted by means of a modified Osborne fractionation, and the radioactivity of the fractions was determined by scintillation analysis. The major portion of the radioactivity remained in the water-soluble fraction. However, a significant enrichment of the tracer was also detected in the prolamin and glutelin fractions in comparison to the control experiment. Separation of the prolamin fraction by RP-HPLC and scintillation measurement of the fractions gave evidence for a chemical modification of the tracer. To determine the structure of the reaction product, the prolamin fractions were completely hydrolyzed to free amino acids by means of an enzyme cocktail, and the digests were studied by LC-MS. In one fraction, a newly formed compound was detected. Comparison of its chromatographic and mass spectrometric behavior with a synthetic reference compound gave evidence that the newly identified compound was a dehydroferulic acid-tyrosine cross-link. It is likely that this cross-link represented a covalent linkage between arabinoxylans and cereal proteins. The newly identified cross-link was also identified in wheat and rye flour doughs, which had been prepared without addition of the ferulate tracer. The relative concentration of the dehydroferulic acid-tyrosine cross-link increased during wheat dough preparation.     

Effect of Transglutaminase,Citrate Buffer,and Temperature on a Soft Wheat Flour Dough System

Transglutaminase (TGase) can improve the functional characteristics of proteins by introducing covalent bonds inter‐ or intrachains. Temperature and pH interfere with the protein structure and the catalytic activity of enzymes. Because these three factors can act synergistically, TGase, citrate buffer, and temperature were evaluated for their effects on the rheological and chemical changes in low‐protein wheat flour dough. Dough strength, measured by microextension test, significantly increased with increasing levels of TGase (8 U/g of protein), with changes in pH of the citrate buffer (pH 6.5), and by the effect of interaction between these factors. The same trend was observed in the size‐exclusion HPLC measurements, indicating that these two parameters have the effect of increasing gluten protein aggregation. Temperature had a significant effect on dough extension, measured by microextension test. The changes in secondary structure of gluten protein were investigated by FTIR second‐derivative spectra (amide I region, 1,600–1,700 cm⁻¹) and showed an increase in β‐sheet structures initiated by TGase, citrate buffer pH, and their interaction.     

Physical properties of soils in Rostov agglomeration

Physical properties of natural and anthropogenically transformed soils of Rostov agglomeration were examined. The data obtained by conventional methods and new approaches to the study of soil physical properties (in particular, tomographic study of soil monoliths) were used for comparing the soils of different functional zones of the urban area. For urban territories in the steppe zone, a comparison of humus-accumulative horizons (А, Asod, Ap, and buried [A] horizons) made it possible to trace tendencies of changes in surface soils under different anthropogenic impacts and in the buried and sealed soils. The microtomographic study demonstrated differences in the bulk density and aggregation of urban soils from different functional zones. The A horizon in the forest-park zone is characterized by good aggregation and high porosity, whereas buried humus-accumulative horizons of anthropogenically transformed soils are characterized by poor aggregation and low porosity. The traditional parameters of soil structure and texture also proved to be informative for the identification of urban pedogenesis.     

The effect of biochar with biogas digestate or mineral fertilizer on fertility,aggregation and organic carbon content of a sandy soil: Results of a temperate field experiment

Substitution of mineral fertilizers with organic soil amendments is postulated to improve productivity‐relevant soil properties such as aggregation and organic matter (OM) content. However, there is a lack of studies analyzing the effects of biochar and biogas digestate versus mineral fertilizer on soil aggregation and OM dynamics under temperate field conditions. To address this research gap, a field experiment was sampled four years after establishment on a sandy Cambisol in Germany where mineral fertilizer or liquid biogas digestate was applied with or without 3 or 40 Mg biochar ha^?1 (produced at 650°C). Soil samples were analyzed for soil organic carbon (SOC) content, pH, cation exchange capacity, bulk density, water‐holding capacity, microbial biomass, aggregate size class distribution, and the SOC content associated with these size classes. 40 Mg biochar ha^?1 significantly increased SOC content in all fractions, especially free particulate OM and the 2–0.25 mm fraction. The yield of small macroaggregates (2–0.25 mm) was increased by biochar, but cation exchange capacity, water‐holding capacity, and pH were not consistently improved. Thus, high‐temperature biochar applied to a sandy soil under temperate conditions is primarily recommended to increase SOC content, which could contribute to climate change mitigation if this C remains sequestered over the long‐term. Fertilizer type did not significantly affect SOC content or other measured properties of the sandy Cambisol, suggesting that replacement of mineral fertilizer with digestate has a neutral effect on soil fertility. Co‐application of biochar with digestate provided no advantages for soil properties compared to co‐application with mineral fertilizer. Thus, independent utilization of these organic amendments is equally suitable.     

水稻籽粒灌浆的发育遗传与分子生态特性研究

本文从发育遗传生理和分子生态学角度, 综述了近年来国内外研究水稻籽粒灌浆的现状与存在的问题。提出了水稻籽粒发育与物质充实涉及信号传递与多基因调控及其与环境互作的分子生态学过程。强、弱势粒由于基因时空表达模式和功能蛋白作用方式的不同, 导致不同粒位胚乳细胞发育程序、籽粒灌浆速率和干物质重的明显差异。已有研究结果表明, 强势粒发育灌浆早、充实快, 具有明显的遗传稳定性; 而弱势粒发育灌浆起步晚, 充实度较差, 易受环境调控。通过分子遗传改良和分子生态调节是改善弱势粒胚乳细胞健康顺利发育, 实时启动灌浆充实, 提高结实率和千粒重, 最终实现高产的两条有效途径, 已成为世界各国优先研究的领域。本文认为籽粒灌浆为何存在一个滞育期(stagnant phase), 强弱势粒在这一发育阶段基因表达和功能作用为何存在差异是值得深入研究和回答的科学问题。