Pilot scale production of a non-immunogenic soluble gluten by wheat flour transamidation with applications in food processing for celiac-susceptible people

Celiac disease (CD) is an immune-mediated disease triggered by wheat gluten and related prolamins. A lifelong gluten-free (GF) diet is mandatory to normalize the intestinal mucosa. We previously found that transamidation by microbial transglutaminase of gluten was effective in suppressing the gliadin-specific inflammatory response in CD patients without influencing the main technological properties of wheat flour or semolina. In this study, we produced on a pilot scale a soluble form of transamidated gluten (soluble protein fraction, spf), characterised by a high protein content (88 mg/ml), while native gluten was dramatically reduced (32 ± 2 ppm; R5-ELISA). Using HLA-DQ8 transgenic mice as a CD model, we found suppression of interferon-γ secretion in gliadin-specific CD4⁺ T cells challenged with spf-primed dendritic cells. In terms of functional properties, spf showed both solubility and emulsifying activity values within the range of commercial soluble glutens. Notably, dough prepared by mixing rice flour with spf could leaven. After baking, blended rice bread had a higher specific volume (2.9 ± 0.1) than control rice bread (2.0 ± 0.1) and acquired wheat-like sensory features. Taken together, our results highlighted the technological value of transamidated soluble gluten to improve both nutritional and sensory parameters of GF food.     

Sprouting as a pre-processing for producing quinoa-enriched bread

The impact of 48 h sprouted quinoa (SQ) was assessed in bread-making. Wheat flour (WF) was replaced with SQ at different levels (i.e., 10:90, 20:80 and 30:70, SQ:WF ratio). Once the optimal replacement level of SQ was identified, the bread-making performance of this ingredient was compared with those of pearled quinoa (PQ), commonly used in bread-making.Starch pasting properties and gluten aggregation behavior were not strongly affected at 20:80 level. Regardless the replacement level, SQ caused an increase in dough water absorption and in softening degree, and a decrease in stability, suggesting weakening of the gluten network. During leavening, SQ improved dough development and gas production, due to increased sugar content (i.e. maltose, sucrose and D-glucose). The best bread-making performance (highest bread specific volume and lowest crumb firmness) was obtained at 20:80 replacement level. Compared to PQ, SQ exhibited the best leavening capacity (high dough development, gas production and gas retention) and bread properties (high specific volume and low crumb firmness), likely due to its higher sugar content. Moreover, 20SQ bread was characterized by a decreased bitterness assessed by electronic-tongue. In conclusion, sprouting might be considered a valid alternative to pearling to improve the characteristics of quinoa enriched bread     

Impact of ethanol,succinic acid,and the combination thereof at levels produced during sponge fermentation on hard wheat,soft wheat,and durum wheat farinograph rheology

The sponge and dough mixing process is one of the most common in the world, yet the mechanistic understanding of this process has yet to be sufficiently explored. In this study, aqueous solutions of ethanol, succinic acid, and their combination were prepared at concentrations intended to replicate fermentation times of 3, 4 and 6 h. These solutions were added to a farinograph mixer to make dough using hard wheat, soft wheat, and durum wheat flour. The results indicate that these yeast metabolites (ethanol, succinic acid) impact the mixing resistance, peak mixing value, and dough mixing stability in each of the flour types, likely primarily affected by the ratios of gliadin to glutenin and LMW glutenin in each flour type. Results suggest a stabilizing non-covalent interaction imparted by gliadin at peak mixing time, a stabilizing effect of HMW glutenin during break down, and synergistic effects of ethanol and succinic acid that leads to a faster rate of breakdown in later stages of mixing. It also suggests an increase in mixing resistance when acidulants are added to durum wheat dough. Taken together, this study adds new insights on the sponge and dough mixing process in a way that has not previously been conducted.     

Effects of wheat tempering and stone rotational speed on particle size,dough rheology and bread characteristics for a stone-milled weak flour

The poor technological performance of weak wheat flours means that they are usually considered difficult to be transformed into satisfactory bread. During milling, there are several settings that can affect flour characteristics. In this study, we tested two operative parameters that have the potential to affect flour quality – stone rotational speed and wheat tempering. Tempering moistures were set at 11%, 13%, 15%, and 17%, while stone rotational speeds were set at 173, 260, and 346 rpm. Both factors were found to affect operative milling parameters, notably flour yield, process productivity and specific energy consumption. Grain moisture had a significant effect on both dough rheology and bread characteristics (dough stability, tenacity, and extensibility). Dough stability was maximum at 13% moisture. Dough tenacity decreased as moisture increased, while extensibility increased as moisture increased. Bread specific volume and crumb specific volume were improved at 13% and 15% moisture. In conclusion, wheat tempering can be used to improve the potential of a weak flour and bread characteristics, while stone rotational speed affects operative parameters and white flour yield.     

Effect of zein extrusion and starch type on the rheological behavior of gluten-free dough

Previous research has shown that zein, above its glass transition temperature, may adopt molecular structures that are able to form doughs with viscoelastic properties comparable to those of wheat gluten. It is hypothesized that extrusion can promote molecular changes in zein and favor interactions with starch that enhance dough viscoelasticity. Thus, the effects of extruding zein at 90–160 °C on the rheological properties of doughs prepared with potato, rice, and maize starches were determined.Formulations were optimized to provide similar mixing profiles to that of a standard wheat dough. For all zein samples, creep-recovery tests demonstrated that doughs prepared with maize and potato starches were less elastic when compared to doughs prepared with rice starch. Zein doughs produced using rice starch were comparable to wheat-dough. Extensional tests showed that zein extruded at 160 °C provided a larger increase in strain-hardening behavior, which is important for bread production. These samples also exhibited larger extensional stresses. Gel electrophoresis of zein extruded at 160 °C revealed an increase in protein aggregates and the presence of smaller peptides when compared to samples subjected at lower extrusion temperatures.Scanning electron micrographs of doughs containing zein showed starch granules embedded within an amorphous material and fibrous structures, which is attributed to elongated zein.     

Localization of amino acids in germinated rice grain: Gamma-aminobutyric acid and essential amino acids production approach

Germinated rice is popularly consumed for its high gamma-aminobutyric acid (GABA) and bioactive compounds. Supporting information on how to germinate rice with high GABA content and essential amino acids is lacking. White and colored rough rice were germinated for 0, 24, 48, 72, 96, 120 and 144 h. GABA, GABA substrates and essential amino acids were also investigated using GC-MS and MALDI-MSI. GABA was more concentrated after germination and shifted into the coleoptile. High correlation was recorded between germination time and GABA (0.79–0.83, p ≤ 0.01) and glutamic acid (0.88–0.89, p ≤ 0.01). Highest rates of GABA and glutamic production were observed within 48 h of germination in both rice varieties (26.12 and 34.28 mg/100g) and reached maximum value at 96 h (31.36 and 38.75 mg/100g). Colored rice germination showed higher GABA, GABA substrates and deficient amino acids than white rice. GABA and essential amino acids drastically increased after germination, supporting the consumption of germinated rice as a functional food.     

Mass spectrometry imaging of BARLEYmax fructooligosaccharides

BARLEYmax is a novel hull-less barley cultivar lacking activity of a key enzyme required for starch synthesis (SSIIa), resulting in a grain with reduced total starch, higher dietary fiber, and higher fructan content. In this study, we examined the quantitative differences in fructan among 2 hull-less barley lines (BARLEYmax and Mannenboshi) and 2 peeled barley lines (Glutinous barley rice and Hindmarsh). We found that BARLEYmax had a higher fructan content, particularly 2 types of fructooligosaccharides (FOS), namely kestose and nystose, the important components of fructan that have some beneficial effects for human health. We established a mass spectrometry imaging (MSI) method to further investigate the content and localization of FOS in the barley lines. We found that kestose, nystose, and fructosyl-nystose showed increased molecular ion intensities in BARLEYmax compared with those in other barley lines. These results confirmed that the loss of SSIIa activity in BARLEYmax leads to a decrease in starch in the cytoplasm and an increase in fructan in the vacuole as an energy store. Overall, these findings demonstrate that MSI analysis is helpful for understanding FOS localization in cereal grains and for visual comparison among matured grains.     

Soluble arabinoxylans extracted from soft and hard wheat show a differential prebiotic effect in vitro and in vivo

Arabinoxylans (AX) are part of dietary fiber. They are currently under study due to their potential prebiotic effect. Wheat whole grain flours contain all the grain layers and, therefore, present a higher arabinoxylan content than white flour. It is known that the chemical structure of these compounds varies with the type of wheat cultivar and the tissue from which they are extracted. In this work, water soluble extractable arabinoxylans (WE-AX) from two types of wheat whole flours (hard and soft) were extracted. We characterized the molecular size distribution and the potential prebiotic effect of those extracts. The prebiotic effect was evaluated in vitro and confirmed in vivo. Bacterial group abundance (Lactobacillus, Bifidobacterium, Clostridium, Enterococcus, Bacteriodes and total bacteria) was determined by quantitative RT-PCR. The molecular size of AX from hard wheats was significantly higher than AX from soft wheats. Both extracts showed potential prebiotic activity by increasing the growth of beneficial bacteria in vitro and in vivo, decreasing the pathogens in the profile of intestinal microorganisms and increasing the amount of short chain fatty acids in the intestine. WE-AX from hard wheat showed a higher prebiotic activity. Prebiotic effect assessed in vitro and in vivo assays showed a significant correlation between both types of analysis. This finding suggests that the in vitro indices performed allow predicting the potential prebiotic effect in vivo.     

Genetic responses in milling,flour quality,and wheat sensitivity traits to grain yield improvement in U.S. hard winter wheat

A rising global population necessitates continued genetic improvement of wheat (Triticum spp.), but not without monitoring of unintended consequences to processors and consumers. Our objectives were to re-establish trends of genetic progress in agronomic and milling traits using a generational meter stick as the timeline rather than cultivar release date, and to measure correlated responses in flour quality and human wheat-sensitivity indicators. Grain yield and kernel size showed stepwise increases over cycles, whereas wheat protein content decreased by 1.1 g/100 g. Reduced protein content, however, did not result in lower dough strength pertinent to bread baking. A novel method of directly testing gluten elasticity via the compression-recovery test indicated a general increase in gluten strength, whereas the ratio of total polymeric to total monomeric proteins remained stable. Also showing no change with genetic progress in yield were flour levels of gluten epitopes within the key immunotoxic 33-mer peptide. The oligosaccharide fructan, present in milled and wholemeal flours, increased with increasing grain yield potential. While yield improvement in U.S. bread wheat was not accompanied by a decline in gluten strength or systematic shift in a key wheat sensitivity parameter, the unanticipated rise in total fructans does implicate potentially new dietary concerns.     

New angiotensin-converting enzyme inhibitory peptide from Coix prolamin and its influence on the gene expression of renin-angiotensin system in vein endothelial cells

Coix seed, which is a traditional Chinese medicine, has been used to treat hypertension for thousands of years. It has been shown that Coix prolamin peptides display high levels of angiotensin I converting enzyme (ACE) inhibitory activity. Hence, we purified the ACE inhibitory peptides from Coix prolamin hydrolysates and evaluated the influence of the most potent peptide on the renin-angiotensin system (RAS) genes expression in human umbilical vein endothelial cells (HUVECs). In this study, Coix prolamin peptides were sequentially separated by ultrafiltration, ion exchange chromatography, gel filtration chromatography and RP-HPLC, while the peptide structure was analyzed by mass spectrometry. Next, in silico proteolysis, pharmacophore and molecular docking were further applied to screen and optimize the structure of peptides. Finally, a novel ACE inhibitory peptide VDMF was obtained, in which its influence on the gene expression of RAS signaling pathway in AngⅡ-injury HUVECs was evaluated by quantitative real-time PCR. VDMF significantly down-regulated ACE, AngII type 1 receptor (AT1R) and ACE2 mRNA expression in comparation with model group, while up-regulating Mas gene expression. Hence, we obtained a novel antihypertensive candidate that was derived from the Coix peptides, which could involve a multi-modulation mechanism that regulates blood pressure.     

Low temperature increased the biosynthesis of 2-AP,cooked rice elongation percentage and amylose content percentage in rice

Rice fragrance, cooked rice elongation and amylose content are some of the traits used in determining rice grain quality. 2-acetyl-1-pyrroline (2-AP) is the major compound that is responsible for fragrance in rice.This experiment was conducted with two indica rice cultivars: Meixiangzhan 2 and Xiangyaxiangzhan. Three temperature-controlled chambers: High (32 °C from 06:00 to 17:59 and 26 °C from 18:00 to 05:59), medium (27 °C from 06:00 to 17:59 and 21 °C from 18:00 to 05:59) and low (22 °C from 06:00 to 17:59 and 16 °C from 18:00 to 05:59) were used for the study. At the onset of heading, both cultivars were transferred to the three temperature-controlled chambers. Our results showed that in both cultivars, low temperature growth had the highest 2-AP content, while high temperature growth recorded the lowest 2-AP content. Principal component analysis showed remarkable differences in the metabolomes of rice grown in high, medium and low regimes. In the grains of both cultivars, we observed significant reduction in 2-AP content after 6 and 9 months of storage, however; 2-AP retention differed greatly in both cultivars. We also reported that low temperature growth increased cooked rice elongation percentage and the biosynthesis of amylose in rice. In conclusion, our findings will provide more information to breeders working on the improvement of rice grain quality.     

Development of a limited-water soaking method on the fortification of rice with calcium and iron by parboiling

Fortifying rice using parboiling has been shown to have a higher retention rate of nutrients and acceptable sensory characteristics. However, the conventional soaking step employs excess water and therefore generates large amounts of wastewater containing nutrients that, when discarded without treatment, can lead to nutrient overload in soil. A limited-water soaking method to fortify rice with calcium and iron by parboiling was developed and evaluated. Fortified rice quality attributes, mineral contents, the amount of wastewater, total solids in wastewater, washing-retention and bioavailability of calcium and iron were determined and compared with properties of rice that was fortified in excess-water soaking. The limited-water soaking method on average reduced the amount of effluent by 89% and the amount of total solids in wastewater by up to 85%. Rice fortified by the limited-water soaking method showed similar color and head rice yield, and slightly lower mineral uptakes compared to conventional soaking. Rice fortified simultaneously with calcium and iron in limited-water soaking contained about 999 mg Ca and 33 mg Fe per kg milled rice, and approximately 66% of calcium and 71% of iron remained in the rice after simulated washing. Calcium and iron dilute HCl-solubility was not affected by the soaking condition. Therefore, the limited-water soaking method can reduce the cost of fortification and wastewater treatment without affecting rice quality attributes.     

Spatial distribution of functional components in the starchy endosperm of wheat grains

The starchy endosperm of the mature wheat grain comprises three major cell types, namely sub-aleurone cells, prismatic cells and central cells, which differ in their contents of functional components: gluten proteins, starch, cell wall polysaccharides (dietary fibre) and lipids. Gradients are established during grain development but may be modified during grain maturation and are affected by plant nutrition, particularly nitrogen application, and environmental factors. Although the molecular controls of their formation are unknown, the high content of protein and low content of starch of sub-aleurone cells, compared to the other starchy endosperm cells types, may result from differences in developmental programming related to the cells having a separate origin (from anticlinal division of the aleurone cells). The gradients within the grain may be reflected in differences in the compositions of mill streams, particularly those streams enriched in the central and outer cells of the starchy endosperm, respectively, allowing the production of specialist flours for specific end uses.     

Variation in physicochemical properties and nutritional quality in chalky mutants derived from an indica rice

The physicochemical and nutritional quality of four chalky mutants (JM1-4) rice and their parent 93-11 were investigated. JM1 and JM4 are white-belly mutants, and JM2 is white-core mutant, while JM3 is floury mutant. JM2 is also a high amylose mutant with AAC of 29%. JM3 had the lipid content nearly three times of the parent, so it is also a high lipid mutant. JM1 had similar pasting viscosities with the parent, while JM2 and JM3 had smaller breakdown and setback than 93-11. JM2 had lower enthalpy of gelatinization, but higher enthalpy of retrogradation, so it had highest retrogradation percentage. JM3 had the highest accumulation of amino acids, fatty acids, and some major mineral elements such as P, K, Mg and Ca. The amino acids and fatty acids had negative correlation with PV, HPV and CPV. The pleiotropic effects caused by the mutations on starch and lipid metabolism deserve further investigation.     

Variation in avenanthramide content in spring oat over multiple environments

Avenanthramides (AVNs), a group of phytochemicals which are unique to oats, provide health benefits through antioxidant activity and other bioactivities. In this study, we explored genotype-by-environment interactions and heritability for AVN concentrations in oats. Avenanthramide concentrations were quantified for 100 breeding lines and cultivars at three locations over two years. While year and environment had an influence on AVN concentrations, with the influence of year being more apparent than that of environment within a year, genotype had the largest impact on AVN concentration. All three major AVNs were found to be heritable. Two methods of calculating heritability on a line mean basis were used. The statistical method yielded heritability estimates of 0.34, 0.39, and 0.41 for AVN 2c, AVN 2p, and AVN 2f, respectively. By comparison the traditional plant breeding method yielded heritability estimates of 0.82, 0.88, and 0.89 for AVN 2c, AVN 2p, and AVN 2f, respectively, indicating that expected gains will be dependent on the scale of the breeding program, the number of target environments, and climate variability. The estimated heritabilities and the 11-fold range in AVN concentrations in the 100 genotypes studied provided evidence that variability for AVN concentration should allow breeding progress for higher AVN concentration.     

Gluten proteome comparison among durum wheat genotypes with different release date

In order to deepen insight into durum wheat prolamin composition in relation to both end use quality and gluten related disorders, a phenotyping of four genotypes of different release date and technological performance was carried out. A proteomic approach integrated with the evaluation of protein aggregation level, amino acid composition and reactivity to G12 monoclonal antibody, was adopted. The degree of polymerization, estimated as unextractable polymeric proteins (UPP), was positively influenced by Cys-rich proteins (Glu-B3 LMW-GS), with Saragolla showing up to 40% higher values than other genotypes. The proteomic assessment allowed to identify proteins involved in gluten related disorders. In particular, ω5-gliadin involved in wheat allergy (WA), resulted markedly over-expressed in the old landrace Dauno III, up to four-fold than in modern Saragolla. A marked influence of genotype on the expression level of gliadins containing toxic epitopes (TECP) was observed with the more recent genotypes showing lower values (−53%). Also, reactivity to G12 moAb resulted higher in the two old genotypes consistently to their higher celiac disease (CD) toxicity evaluated by the proteomic approach. In conclusion a better gluten composition for processing seems to be associated to a lower expression level of CD toxic peptides and Tri a 19.     

Variability and cluster analysis of arabinoxylan content and its molecular profile in crossed wheat lines

Non-starch polysaccharides (NSP) in cereals, after starch and gluten proteins, determine the technological and nutritional properties of flour, dough and end product. From NSPs, the arabinoxylans are the most important components studied in wheat nowadays. The novelty of our study is the investigation of the variability and clustering ability of the wheat lines based on different quantitative and qualitative traits of arabinoxylans. The quantitative properties, the total and the water-extractable arabinoxylan content of wheat flours were measured separately by gas chromatography. The qualitative property referring to the structure of the molecule was the molecular weight distribution measured by size exclusion liquid chromatography. According to the results the variability of arabinoxylan properties in the breeding lines varied between wider ranges then the values measured for the parental varieties. The correlation between the quantitative parameters, and a trend between quantitative and qualitative parameters were described. During the cluster analysis, the parents were well separated into different groups. The parameters that played an important role in the clustering were the ratio of the probability of occurrence of molecular size ranges along with the quantitative traits of AX. The selected samples can be used for further targeted breeding while the methodology was used in this work can be suitable for selecting lines/varieties for special food or other industrial applications.     

Immunochemical analysis of oat avenins in an oat cultivar and landrace collection

Oats have been found to be tolerated by most celiac disease patients, and oats are generally considered a good and safe addition to the gluten-free diet. There have been claims that some individual oat cultivars are harmful or immunogenic for celiac disease patients. In this study, we investigated 26 oat cultivars and landraces from the current breeding market and literature. Their total protein content ranged from 15.3% to 23.1% of which avenins ranged from 6.8% to 10.9%. Immunological activities of avenins were evaluated using immunochemical analyses using monoclonal antibodies (mAb) R5 and G12. No immunological activity of the oat cultivars was observed by mAb R5 either in immunoblotting or enzyme-linked immunosorbent assay (ELISA). mAB G12 showed no activity in immunoblotting, but gave responses between 13 and 53 mg/kg in ELISA for total avenin extract. To understand the varying G12 activity, avenins were further fractionated. One avenin fraction showed a higher G12 response than the other fractions. Protein sequence comparison suggests that there is no direct binding to avenin-specific T-cell epitopes but the differences in repetitive regions in avenins may contribute to varying results in G12 ELISA.     

Contributions of individual and combined Glu-B1x and Glu-B1y high-molecular-weight glutenin subunits to semolina functionality and pasta quality

Durum wheat is an important food crop used primarily for pasta production. High-molecular-weight glutenin subunits (HMW-GS) encoded by the closely linked genes Glu-B1x and Glu-B1y are known for their combined effects on pasta quality, but their individual contributions and interactions remain poorly understood. In this study, we show that individual loss-of-function mutants of Glu-B1x (ΔBx6) and Glu-B1y (ΔBy8) were associated with significant reductions in gluten strength compared to the wildtype, with stronger effects in the ΔBxy double mutant. Reductions in gluten strength were reflected in reduced mixograph and alveograph parameters, gluten index, faster extrusion flow rates and increased cooking loss. Interestingly, the Glu-B1x mutation was also associated with significant increases in grain and semolina protein content, increased pasta firmness, reduced starch viscosity and increased amylose in ΔBx6 and ΔBxy. In general, the ΔBx6 mutation had stronger effects than the ΔBy8 mutation, and significant interactions between the two genes were frequent. In addition to the basic knowledge gained on the individual effects of the Bx6 and By8 subunits and their interactions, the genetic stocks developed in this study provide useful tools to study the effects of natural or synthetic HMW-GS on pasta quality parameters in a background lacking endogenous HMW-GS.     

Effect of high temperature stress during ripening on the accumulation of key storage compounds among Japanese highly palatable rice cultivars

High temperature stress during ripening increases the frequency of chalky grains, resulting in a lower market value for rice (Oryza sativa L.). Changes in starch properties and the accumulation pattern of storage proteins are proposed to be related to the occurrence of chalky grains. This study investigated changes in the accumulation of key storage compounds in the grains of Japanese highly palatable rice cultivars, subjected to high temperature stress when grown in a growth chamber and the field. The 13 kDa prolamin content was significantly reduced in a highly heat-sensitive cultivar, Tsukushiroman, whereas the 13 kDa prolamin content was not affected in a heat-tolerant cultivar, Genkitsukushi, even in a high temperature chamber condition (31/26 °C day/night), when compared with the control chamber condition (26/21 °C day/night) for both genotypes. In addition, grains grown in field conditions revealed that severely chalky grains had less 13 kDa prolamin than perfect grains in all five genotypes. Changes in amylose content and the distribution of amylopectin chain lengths did not explain the difference in grain appearance both for chamber and field experiments. These results strongly suggest that physiological processes linked with the synthesis of 13 kDa prolamin are associated with grain appearance in Japanese highly palatable under high temperature stress.