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.     

Modification of protein structure and dough rheological properties of wheat flour through superheated steam treatment

Native (NF, 13.5% w.b) and moistened (MF, 27% w.b) wheat flours were treated with superheated steam (SS) at 170 °C for 1, 2 and 4 min, and their protein structure as well as dough rheological properties were analyzed. Confocal laser scanning microscopy (CLSM) and SDS-PAGE patterns indicated the formation of protein aggregates with reduced SDS extractability after treatment. Farinograph and dynamic rheometry measurements showed that the strength as well as elastic and viscous moduli of the dough made from SS-treated flours progressively increased with SS treatment time. And both the improvements were more pronounced for superheated steam-treated moistened flours (SS-MF) than for superheated steam-treated native flours (SS-NF). Size-exclusion high performance liquid chromatography (SE-HPLC) analysis demonstrated that dough rheological parameters have positive correlations with SDS unextractable polymeric proteins (UPP) contents. SS treatment on flours led to a transition of protein secondary structures to more ordered form (α-helix and β-sheet). Additionally, free sulfhydryl (SH) contents decreased after treatment, which implied that disulfide bonds accounted for protein extractability loss and dough rheological properties improvement. Elevated moisture level promoted the modification of both protein structure and dough behaviors of flours during SS treatment.     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

Impact of lipases with different substrate specificity in wheat flour separation on the properties of the resultant gluten

Wheat gluten was isolated in a laboratory dough-batter flour separation process in the presence or absence of lipases differing in hydrolysis specificity. The obtained gluten was blended with wheat starch to obtain gluten-starch (GS) blends of which the water and oil binding capacities were investigated. Furthermore, GS blends were mixed into dough and processed into model breads, of which dough extensibility and loaf volume were measured, respectively. In comparison to GS blends prepared with control gluten, oil binding capacity was higher when GS blends contained gluten isolated with Lecitase Ultra (at 5.0 mg enzyme protein/kg flour), a lipase hydrolyzing both non-polar and polar lipids. Additionally, dough extensibility and total work needed for fracture were lower for dough prepared from GS blends containing gluten isolated with Lipolase (at 5.0 mg enzyme protein/kg flour), a lipase selectively degrading non-polar lipids. In GS blend bread making, this resulted in inferior loaf volumes. Comparable GS blend properties were measured when using control gluten and gluten isolated with YieldMAX, a lipase mainly degrading N-acyl phosphatidylethanolamine. In conclusion, properties of GS blend model systems are altered when gluten prepared in the presence of lipases is used to a degree which depends on lipase specificity and concentration.     

“Antioxidant activity and characterization of protein fractions and hydrolysates from normal and quality protein maize kernels”

Maize is the main crop cultivated worldwide with more than 1 billion metric tons produced annually and is one of the most relevant sources of protein for human consumption in developing countries. Proteins and peptides isolated from maize exert relevant antioxidant activity which is increased by enzymatic hydrolysis. However, there is limited information about the antioxidant potential of proteins isolated from Quality Protein Maize (QPM) varieties and their hydrolysates. The aim of this research was to determine the differences in protein profile and antioxidant activity of protein fractions and hydrolysates between a hybrid white maize (Asgrow 773) and a QPM variety (CML-502). The biophysical evaluation and the total protein quantification by Kjeldahl and fractions by ninhydrin were consistent with the changes due to the breeding process of the QPM material. The antioxidant potential of the hydrolysates obtained from albumins and globulins had a 3-fold increase in both maize varieties. The prolamins hydrolysates presented an increase of 7-fold for the normal variety and 2-fold for the QPM variety. The results of this research allow indicate that the QPM varieties are a source of antioxidant peptides and promising candidates in the search for proteins and peptides with other bioactivities.     

The impact of redox agents on further dough development,relaxation and elastic recoil during lamination and fermentation of multi-layered pastry dough

The role of gluten proteins during lamination and fermentation of multi-layered wheat flour pastry dough was examined by including oxidizing or reducing agents in the recipe to respectively strengthen or weaken the gluten protein network. Pastry burst rig textural measurements showed that dough strength increases during lamination up to 16 fat layers. However, further lamination up to 64 and 128 fat layers decreases the dough strength, most likely due to destruction of layer integrity. Redox agents strongly affect dough strength. Furthermore, fermentation and spread tests showed that they strongly influence elastic recoil immediately after lamination and during relaxation. Moreover, elastic recoil consistently occurs to a greater extent in the final direction of sheeting. None of the observed changes in dough strength and relaxation behaviour could be linked to changes in the levels of protein extractable in sodium dodecyl sulfate containing medium (SDS-EP). This suggests that changes occur preferentially either within the SDS-extractable or within the non-SDS-EP fraction and that they do not render non-extractable protein fractions extractable or vice versa. Furthermore, elastic recoil is most likely caused by reformation of inter- and intramolecular hydrogen bonds and hydrophobic interactions.     

Impact of pre-germination on amylopectin molecular structures,crystallinity, and thermal properties of pre-germinated brown rice starches

Ungerminated brown rice (UGBR) and pre-germinated brown rice (PGBR) obtained from different pre-germination durations were studied to investigate the changes in total starch contents of flour, amylopectin molecular structures, crystallinity, and thermal properties of starches as affected by pre-germination. Each paddy of three rice cultivars with different amylose contents (RD6, waxy; KDML105, low amylose; and RD31, high amylose) was soaked in water at 30°C for 12 h and incubated over different periods until the three stages of embryonic growth length (EGL) were achieved. The total starch contents of three-stage PGBR flour from all rice cultivars decreased when pre-germination durations were increased. The three-stage PGBR starches from the three rice cultivars had lower weight-average molecular weight (Mw) and number-average molecular weight (Mn) than UGBR starches. All starches from the three rice cultivars displayed an A-type X-ray diffraction pattern (XRD). Isolated UGBR starch from RD6 had the highest (31.33%) relative crystallinity (RC), while RD31 showed the lowest RC (26.79%). The slight increases in the RC of three-stage PGBR starches from three rice cultivars were found after pre-germination. Isolated PGBR starches from the three rice cultivars had higher gelatinization temperatures and enthalpy, but lower retrogradation enthalpy and %retrogradation than UGBR starches.     

Kernel vitreousness and protein content: Relationship,interaction and synergistic effects on durum wheat quality

Four sets of durum samples were used in this study to further understand the interrelationships among hard vitreous kernels (HVK), protein content, and pigment concentration, with a focus on the interaction and synergistic effects of protein content and vitreousness on durum quality. HVK level increases with higher protein content in the range of 9.5–12.5%, but this relationship is less evident in durum samples with high protein content (12.5–14.5%). Both protein content and kernel vitreousness can significantly affect durum milling quality. White starchy kernels (WSK) in low protein durum have a very detrimental impact on milling and pasta processing quality, but high protein content can mitigate the adverse impact of WSK on durum quality. Although protein content plays a dominant role, higher HVK might contribute positively to pasta firmness. There was no significant difference in yellow pigment content between HVK and WSK. However, pigment loss from semolina to dough was higher for WSK than HVK. Despite the difference in protein content, HVK and WSK have little difference in gluten strength. The monomeric protein was preferentially accumulated in HVK. The glutenin proteins of HVK and WSK were similar in the ratios of 1Bx/1By and HMW/LMW-GS.     

Dough rheological properties and noodle-making performance of non-waxy and waxy whole-wheat flour blends

Dough rheological properties and noodle-making performance of non-waxy whole-wheat flour (WWF) with partial- or full-waxy (PW- or FW-) WWF substitution were studied. The substitution levels were 0, 250, 500, 750, and 1000 g/kg, respectively. FW-WWF reduced the peak viscosity and pasting temperature of WWF blends as its substitution level was increased due to its higher proportions of B-type starch granules and short amylopectin chains, while PW-WWF increased peak viscosity with the increasing substitution level because of its higher amylopectin content. As demonstrated by farinograph and rheometer measurements, FW-WWF interfered with gluten development because of the increased competition for water by arabinoxylans and amylopectin; however, PW-WWF enhanced dough strength due primarily to its increased protein content. Consequently, FW-WWF showed a detrimental effect on cooked noodle texture as the cooked noodle hardness was reduced by 50% at the 1000 g/kg substitution level. In contrast, PW-WWF enhanced noodle integrity and elasticity by increasing cooked noodle cohesiveness and resilience by 10.1% and 14.8%, respectively, at the 1000 g/kg substitution level. The results suggest that with waxy WWF substitution, the changes in starch composition, arabinoxylans, and protein content could modify the interactions among flour components and influence the quality characteristics of noodle products.     

Investigating the impact of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough fermentation and bread sugar levels

Wheat flour is generally supplemented with α-amylases to increase maltose levels in bread dough and increase loaf volume. While the preference of yeast for glucose and fructose over maltose as substrate for fermentation is well documented, the impact of maltose versus glucose producing enzymes on bread dough fermentation kinetics and bread sugar levels is ill documented. Hence the impact of α-amylase, α-glucosidase and glucoamylase action on both aspects was investigated. Glucoamylase and α-amylase increase the total fermentable sugar content of dough, while α-glucosidase only affects the glucose/maltose ratio. Due to their effect on total fermentable sugar levels, addition of α-amylase or glucoamylase prolongs the total productive fermentation time, while this is not the case for α-glucosidase. In contrast to α-amylase, both glucoamylase and α-glucosidase supplementation leads to higher CO₂ production rates during the initial stages of fermentation. In the final bread product, different sugar levels are observed depending on the dosage and type of starch-degrading enzyme. The results of this study imply that long and short fermentation processes benefit from α-amylase and α-glucosidase addition, respectively, while glucoamylase supplementation is suitable for both long and short fermentation times.     

Characterizing the pigmented traditional rice cultivars grown in temperate regions of Kashmir (India) for free and bound phenolics compounds and in vitro antioxidant properties

The purpose of the research was to identify the phenolic and flavonoid compounds of seven different traditional pigmented whole rice cultivars grown in the temperate regions of Kashmir so as to study their relationship with in vitro antioxidant capacities. The completely pigmented rice cultivars were found to have higher phenolic, flavonoid, anthocyanin contents and exhibited higher antioxidant capacities than the light colored and sparely colored rice cultivars. A total of 40 compounds had been identified in the analyzed rice cultivars that were found to be distributed in 6 major categories with 6-phenolics, 6-flavonoids, 11-hydroxycinnamic acid derivatives, 7-hydroxybenzoic acid derivatives, 3-anthocyanins and 7-flavonoid glucosides of different flavonoid compounds. Among the free and bound fractions for each cultivars the light and sparsely colored depicted higher content of phenolics and in vitro antioxidant properties in bound faction, while the completely pigmented cultivars showed higher antioxidant properties in free fractions. The anthocyanins quercetin-3-O-galactoside, cyanidin-3-O-rutinoside and pelargonidin-3-O-diglucoside had been identified by LC-MS existing in the free fractions of the analyzed rice cultivars whereas, the free fraction of acetone + H₂O possessed higher percentage of phenolic compounds as compared to methanolic extracts and bound fractions. The black colored cultivars possessed higher DPPH scavenging activity and lipid peroxidation inhibition.     

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     

A rapid extensigraph protocol for measuring dough viscoelasticity and mixing requirement

The extensigraph is particularly useful in characterizing dough viscoelastic properties; however, testing throughput for standard method is low due to the prerequisite for farinograph water absorption, long dough resting and milling to prepare large amounts of flour. Therefore, a rapid extensigraph method was developed that reduced sample size (165 g wheat) for milling and more than tripled throughput. Wheat is milled in Quadrumat Junior mill with a modified sieving system. The resulting flour (100 g) was mixed with a pin mixer at constant water absorption to allow the evaluation of wheat genotypes at the absorption level they are expected to perform. Dough was subsequently stretched by an extensigraph after 15 min of floor time and 30 min resting. Strong correlations for extensigram R_max (r > 0.93), extensibility (r > 0.64) and area (r > 0.88) were found for the proposed method compared to the standard method. Mixing parameters (time and energy) obtained during dough preparation provided further information about dough strength and mixing requirement. By significantly reducing sample size requirement and increasing testing throughput, this rapid extensigraph method can be widely adopted in milling and baking industry and meets the need for a fast evaluation of dough strength in breeding trials.     

Study of the mechanism of improvement due to waxy wheat flour addition on the quality of frozen dough bread

Waxy wheat flour (WWF) was substituted for 10% regular wheat flour (RWF) in frozen doughs and the physicochemical properties of starch and protein isolated from the frozen doughs stored for different time intervals (0, 1, 2, 4 and 8 weeks) were determined to establish the underlying reasons leading to the effects observed in WWF addition on frozen dough quality. Using Nuclear Magnetic Resonance (NMR), Differential Scanning Calorimeter (DSC) and X-ray Diffraction (XRD) among others, the gluten content, water molecular state, glutenin macropolymer content, damaged starch content, starch swelling power, gelatinization properties, starch crystallinity and bread specific volume were measured. Compared to RWF dough at the same frozen storage condition, 10% WWF addition decreased dry gluten and glutenin macropolymer contents and T₂₃ proton density of frozen dough, but increased the wet gluten content, T₂₁ and T₂₂ proton density. 10% WWF addition also decreased damaged starch content, but increased starch swelling power, gelatinization temperature and enthalpy, crystallinity of starch and bread specific volume of frozen dough. Results in the present study showed that the improvement observed due to WWF addition in frozen dough bread quality might be attributed to its inhibition of redistribution of water molecules bound to proteins, increase in damaged starch content and decrease in starch swelling power.     

Responses of glutamine synthetase activity and gene expression to nitrogen levels in winter wheat cultivars with different grain protein content

Glutamine synthetase (GS) plays a central role in plant nitrogen (N) metabolism, which improves crops grain protein content. A pot experiment in field condition was carried out to evaluate GS expression and activity, and grain protein content in high (Wanmai16) and low grain protein (Loumai24) wheat cultivars under two N levels (0.05 and 0.15 g N kg⁻¹ soil). High nitrogen (HN) resulted in significant increases in GS1 and GS2 expression at 10 days after anthesis (DAA), and higher GS activity during the entire grain filling stage. HN also significantly increased yield, grain protein content and protein fraction (except for glutenin of Luomai24) in two wheat cultivars, which indicated that it increased grain yield and protein content by improving nitrogen metabolism. Wanmai16 showed higher grain protein content, gliadin and glutenin content, and had higher expression level of GS2 both in flag leaves and grains at early grain filling stage. However, Luomai24 had greater yield and higher expression level of GS1. The difference expression of GS2 and GS1 genes indicates they had various contributions to the accumulation of protein and starch in wheat grains, respectively. The results suggest that GS2 would be serving as a potential breeding target for improving wheat quality.     

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.     

A new lean no time test baking method with improved discriminating power

In response to customer concerns related to gluten strength in commercial baking, the Canadian Grain Commission assessed whether the Canadian Short Process (CSP) test bake method was generating useful data related to intrinsic strength of wheat varieties. Assessment of CSP loaf volume data for Canadian variety trials spanning 2003 to 2013 showed very little correlation with dough strength parameters as measured by farinograph and extensigraph. A lean no time (LNT) test baking method was developed that can better discriminate genotypes and provide objective indicators of the effect of intrinsic dough strength on baking quality. From early method development, through method validation and verification using diverse sets of samples targeting different Canadian wheat classes and grown in three different crop years, results showed the LNT method to be more discriminating and easily adopted by other laboratories. In 2015, the LNT method was adopted as the method of choice in future Canadian variety registration trials. The LNT method is fast, simple and well-suited to high throughput test baking conditions encountered in the evaluation of large numbers of breeder lines. A new objective parameter, loaf top ratio, was also introduced and found to correlate well with dough strength and dough handling properties.     

Effect of physicochemical and empirical rheological wheat flour properties on quality parameters of bread made from pre-fermented frozen dough

The objective of this study was to examine the influence of flour quality on the properties of bread made from pre-fermented frozen dough. The physicochemical parameters of 8 different wheat flours were determined, especially the protein quality was analysed in detail by a RP-HPLC procedure. A standardized baking experiment was performed with frozen storage periods from 1 to 168 days. Baked bread was characterised for specific loaf volume, crumb firmness and crumb elasticity. The results were compared to none frozen control breads. Duration of frozen storage significantly affected specific loaf volume and crumb firmness. The reduction of specific loaf volume was different among the used flours and its behaviour and intensity was highly influenced by flour properties. For control breads wet gluten, flourgraph E7 maximum resistance and RVA peak viscosity were positively correlated with specific loaf volume. However, after 1–28 days of frozen storage, wet gluten content was not significantly influencing specific loaf volume, while other parameters were still significantly correlated with the final bread properties. After 168 days of frozen storage all breads showed low volume and high crumb firmness, thus no significant correlations between flour properties and bread quality were found. Findings suggest that flours with strong gluten networks, which show high resistance to extension, are most suitable for frozen dough production. Furthermore, starch pasting characteristics were also affecting bread quality in pre-fermented frozen dough.     

A new lean no time test baking method with improved discriminating power

In response to customer concerns related to gluten strength in commercial baking, the Canadian Grain Commission assessed whether the Canadian Short Process (CSP) test bake method was generating useful data related to intrinsic strength of wheat varieties. Assessment of CSP loaf volume data for Canadian variety trials spanning 2003 to 2013 showed very little correlation with dough strength parameters as measured by farinograph and extensigraph. A lean no time (LNT) test baking method was developed that can better discriminate genotypes and provide objective indicators of the effect of intrinsic dough strength on baking quality. From early method development, through method validation and verification using diverse sets of samples targeting different Canadian wheat classes and grown in three different crop years, results showed the LNT method to be more discriminating and easily adopted by other laboratories. In 2015, the LNT method was adopted as the method of choice in future Canadian variety registration trials. The LNT method is fast, simple and well-suited to high throughput test baking conditions encountered in the evaluation of large numbers of breeder lines. A new objective parameter, loaf top ratio, was also introduced and found to correlate well with dough strength and dough handling properties.     

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.