Aggregation behavior of wheat gluten during carboxylic acid deamidation upon hydrothermal treatment

Changes of aggregation behavior of wheat gluten during carboxylic acid deamidation upon hydrothermal treatment were investigated to test the influences of deamidation on the aggregation extent of wheat gluten. Hydrothermal treatment induced that the size of soluble wheat gluten aggregate progressively increased by cross-linking of gliadins and slowly cleaved glutenins. But significant changes in molecular weight distribution, solubility under six denaturing agents’ treatment and Zeta potential of wheat gluten aggregates were observed at 6 min heating time and distinct shift of intra-/inter-molecular interactions of wheat gluten aggregates occurred before and after 6 min heating treatment respectively. Moreover, as heating time increased, the island-like aggregates decreased markedly and the striped aggregates increased notably. To explain the aggregation behavior in this case, we postulated that the extent of aggregation of wheat gluten depended on the balance between intra-/inter-molecular electrostatic repulsion, the non-covalent and disulfide bonds formation in the system. Hence, a scheme was drawn, which appeared to be the mechanism responsible for the aggregation of wheat gluten through thermal cross-linking and opening up of the network structure of wheat gluten aggregates by deamidation.     

Characteristics of enzymatic hydrolysis of thermal-treated wheat gluten

Effect of thermal treatment at 50–90 °C on wheat gluten hydrolysis by papain was evaluated in this study. Thermal treatment decreased the amount of sodium dodecyl sulfate (SDS) extractable protein. The treatments at 80 and 90 °C had a strong impact on protein extractability. Thermal treatment for 30 min resulted in a significant reduction in SDS extractable glutenin level in wheat gluten. A significant drop in free sulphydryl level was found in wheat gluten treated at 70 °C for 30 min. It indicated that cross-linking of glutenin through S–S occurred during thermal treatment. The treatments at 70–90 °C led to significant decreases in soluble and nitrogen level, while significant increases in peptide nitrogen amount in the hydrolysates from treated gluten were found. A time-dependent effect was observed for the changes in soluble forms of nitrogen and PN. Thermal treatment resulted in molecular mass distribution change according to gel permeation chromatography analysis. Thermal treatment significantly increased the amount of fractions with molecular mass beyond 10 K (67.2%) in the hydrolysates and greatly decreased the amounts of fractions with MM of 10–5 K and below 5 K in hydrolysates.     

Bioethanol production from sweet sorghum bagasse by Mucor hiemalis

The present work deals with production of ethanol from sweet sorghum bagasse by a zygomycetes fungus Mucor hiemalis. The bagasse was treated with phosphoric acid and sodium hydroxide, with or without ultrasonication, prior to enzymatic hydrolysis by commercial cellulase and β-glucosidase enzymes. The phosphoric acid pretreatment was performed at 50 °C for 30 min, while the alkali treatment performed with 12% NaOH at 0 °C for 3 h. The pretreatments resulted in improving the subsequent enzymatic hydrolysis to 79-92% of the theoretical yield. The best hydrolysis performance was obtained after pretreatment by NaOH assisted with ultrasonication. The fungus showed promising results in fermentation of the hydrolyzates. In the best case, the hydrolyzate of NaOH-ultrasound pretreated bagasse followed by 24 h fermentation resulted in about 81% of the corresponding theoretical ethanol yield. Furthermore, the highest volumetric ethanol productivity was observed in the hydrolyzates of NaOH pretreated bagasse, especially after ultrasonication in pretreatment stage.     

Optimization of phosphoric acid catalyzed fractionation and enzymatic digestibility of flax shives

Flax shives are the woody residue left over from processing flax straw into fiber, and are an abundant renewable lignocellulosic material with a potential for the conversion into bioethanol and other value added products. In this study, prior to enzymatic hydrolysis for the liberation of fermentable sugars, such as glucose and xylose, flax shives were treated with concentrated phosphoric acid. In order to optimize the phosphoric acid pretreatment and enzymatic hydrolysis steps, the effects of three process variables on the fractionation of flax shives, and enzymatic digestibility of pretreated flax shives were evaluated. The optimization process employed a central composite design (CCD), where the variables selected were concentration of phosphoric acid (40.8–86.2%), pretreatment time (9.5–110.5 min), and cellulase loading (13.1–71.9 FPU/g cellulose). Using three-variable and five-level CCD, all tested independent variables were identified to have significant effects (P < 0.05) on the digestibility of pretreated flax shives. It was found that the level of phosphoric acid (P < 0.0001) affects the digestibility most significantly when compared with other variables. When the optimization was conducted under a constrain of minimum cellulase loading, the maximum digestibility of 94.8% was predicted when the phosphoric acid concentration, pretreatment time, and cellulase loading were 86.2%, 110.5 min, and 13.1 FPU/g cellulose at 50 °C and 120 h, respectively. Under these conditions, digestibility of pretreated flax shives in the validation study reached a maximum of 93% at 120 h of incubation, showing good agreement with the values from the validation experiment of 93.4%, indicating high accuracy of the CCD procedure. When triticale straw, pine wood, and poplar wood were pretreated and hydrolyzed under optimum conditions obtained from the flax shives experiment, the digestibility reached 98.2, 74.8, and 95.7%, respectively, suggesting that the modest pretreatment process using phosphoric acid is an effective method for perennial plants as well as hard wood.     

Influence of high solid concentrations on enzymatic wheat gluten hydrolysis and resulting functional properties

Enzymatic hydrolysis at increased solid concentrations is beneficial with regard to energy and water consumption. This study examines the influence of the solid concentration on the enzymatic hydrolysis of wheat gluten and the resulting functional properties of the hydrolysate. Wheat gluten was mildly hydrolyzed at a solid concentration varying from 10% to 60% to degrees of hydrolysis (DH%) ranging from 3.2% to 10.2%. The gluten was susceptible to hydrolysis at all solid concentrations but the hydrolysis rate was influenced by increasing solid concentrations. Size-exclusion high-performance liquid chromatography revealed an increase in the ratio of peptides with a molecular mass >25 kDa for solid concentrations of 40% and 60%. The water solubility increased on hydrolysis and was independent of the solid concentration during proteolysis. The foam stability was not influenced by the solid concentration at low DH%. At DH% higher than 8%, high solid concentrations increased the foam stability, which might be related to the presence of more peptides with a molecular mass >25 kDa. In addition, we found increased reactor productivity. The results show the potential of hydrolyzing wheat gluten at high solid concentrations, which could lead to large savings for water and energy when applied industrially.     

Optimization of formic/acetic acid delignification of Miscanthus × giganteus for enzymatic hydrolysis using response surface methodology

A Box-Behnken experimental design and response surface methodology were employed to optimize the pretreatment parameters of a formic/acetic acid delignification treatment of Miscanthus × giganteus for enzymatic hydrolysis. The effects of three independent variables, namely cooking time (1, 2 and 3 h), formic acid/acetic acid/water ratio (20/60/20, 30/50/20 and 40/40/20) and temperature (80, 90 and 107 °C) on pulp yield, residual Klason lignin content, concentration of degradation products (furfural and hydroxymethylfurfural) in the black liquor, and enzymatic digestibility of the pulps were investigated. The major parameter influencing was the temperature for pulp yield, delignification degree, furfural production and enzymatic digestibility. According to the response surface analysis the optimum conditions predicted for a maximum enzymatic digestibility of the glucan (75.3%) would be obtained using a cooking time of 3 h, at 107 °C and with a formic acid/acetic acid/water ratio of 40/40/20%. Glucan digestibility was highly dependent on the delignification degree.     

Deamidation of gluten proteins and peptides decreases the antibody affinity in gluten analysis assays

Wheat gluten is a widely used ingredient in the food industry due to its unique properties and relatively low price. Modification of wheat gluten makes it a versatile ingredient and, thus, increases its applicability in foods. Therefore, gluten proteins can be found in unexpected sources, and this makes the gluten-free diet challenging to follow. Deamidation is one way to modify protein structure. It increases solubility and surface activity of gluten improving its functionality, but consequently, also influencing the accuracy of quantification by immunoassays. In this study, the effect of deamidation on the antibody recognition with gluten analysis methods based on monoclonal R5, omega-gliadin or G12 antibodies was studied. Random deamidation decreased the intensities to 13–54% of the intensity obtained for the intact peptides. Deamidation representing the transglutaminase deamidation decreased the intensities to 4–8%. Deamidation of gluten proteins abolished the recognition by omega-gliadin and G12 antibodies and decreased the recognition of R5 by 600 times when analyzed by the sandwich method and 125 times by the competitive method. In conclusion, with all of the investigated gluten-specific antibodies, deamidation decreased the affinity of antibodies to gluten peptides and proteins, which needs to be considered when assays and regulations are developed for gluten-free products.     

Large deformation stress relaxation and compression-recovery of gluten representing different wheat classes

Despite the great variety of physicochemical and rheological tests available for measuring wheat flour, dough and gluten quality, the US wheat marketing system still relies primarily on wheat kernel hardness and growing season to categorize cultivars. To better understand and differentiate wheat cultivars of the same class, the tensile strength, and stress relaxation behavior of gluten from 15 wheat cultivars was measured and compared to other available physicochemical parameters, including but not limited to protein content, glutenin macropolymer content (GMP) and bread loaf volume. In addition, a novel gluten compression–relaxation (Gluten CORE) instrument was used to measure the degree of elastic recovery of gluten for 15 common US wheat cultivars. Gluten strength ranged from 0.04 to 0.43 N at 500% extension, while the degree of recovery ranged from 5 to 78%. Measuring gluten strength clearly differentiated cultivars within a wheat class; nonetheless it was not a good predictor of baking quality on its own in terms of bread volume. Gluten strength was highly correlated with mixograph mixing times (r = 0.879) and degree of recovery (r = 0.855), suggesting that dough development time was influenced by gluten strength and that the CORE instrument was a suitable alternative to tensile testing, since it is less time intensive and less laborious to use.     

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.     

Effect of native aggregation state of soluble wheat gluten on deamidation behavior in a carboxylic acid/heat water solution

To understand the role of native aggregation state (NAS) of soluble wheat gluten and fractions during deamidation in a carboxylic acid/heat water solution, changes in conformation and deamidation behavior as function of protein concentration from dilute to semi-concentrated regimes to control NAS were investigated by physicochemical properties, SDS-PAGE, molecular force change, intrinsic fluorescence emission spectroscopy (IFES) and FTIR. Our data show that, in this solution, the deamidated proteins displayed features characteristic of more scattered and flexible polymer structure in dilute concentration than concentrated ones. Degree of deamidation (DD), HD and Zeta potential exhibited strongly oppositely with the decreasing concentration. HWM-GS, ω-gliadins and LWM-GS degraded into smaller peptides with decreasing of NAS. FTIR and IFES displayed that improved molecular flexibility with decreasing of concentration as detected by the increasing content of β-turn and β-sheet, as well as the red-shift of wheat gluten and gliadins at the expense of α-helix. Hydrophobic and hydrogen bond increased gradually and were dominant in inter-molecule as function of increasing concentration. The above information demonstrated that NAS of soluble wheat gluten dominated changes of deamidation behavior and conformation in a carboxylic acid/heat water solution.     

Sunflower Protein Hydrolysates Reduce Cholesterol Micellar Solubility

Plant protein hydrolysates are a source of bioactive peptides. There are peptides that decrease the micellar cholesterol solubility from bile acids and therefore may reduce in vivo cholesterol absorption. The presence of these peptides in sunflower protein hydrolysates has been studied. Sunflower protein hydrolysates produced with alcalase plus flavourzyme or with pepsin plus pancreatin inhibited in some degree the cholesterol incorporation to micelles. Protein hydrolysates generated after 30 min of hydrolysis with alcalase, and after 30 min of hydrolysis with pepsin, were the inhibitoriest of the cholesterol incorporation to micelles. The average amino acid hydrophobicity of inhibitory peptides in cholesterol micelles was higher than the observed in the corresponding protein hydrolysates. This high hydrophobicity probably favours their inclusion in the lipid micelles. In vivo, this inhibition may translate in a decrease of cholesterol absorption. Reported results show that a combination of different characteristics such as peptide size or hydrophobicity may be responsible of the inhibitory activity of generated peptides.     

Effects of vacuum mixing and mixing time on the processing quality of noodle dough with high oat flour content

The effects of different mixing parameters (vacuum mixing and mixing time) on oat (70% oat flour) and wheat noodle dough were investigated on the basis of textural properties and gluten formation. The results showed that at a vacuum degree of −0.06 MPa and mixing time of 10 min, oat and wheat dough sheets exhibited the highest resistance to extension and glutenin macropolymer (GMP) content, and had the most compact and uniform gluten network. Compared with wheat noodle dough, oat dough had lower resistance to extension, lower tightly bound water content, and higher GMP content. Microstructural examination showed that oat noodle dough had a more aggregated distribution of gluten protein compared with wheat noodle dough under the optimum mixing parameters. Furthermore, the poor binding ability of vital wheat gluten with water molecules caused the indexes of oat noodle dough to be more strongly affected by the changes in mixing parameters than wheat noodle dough.     

Multifunctional bioactive peptides derived from quinoa protein hydrolysates: Inhibition of α-glucosidase,dipeptidyl peptidase-IV and angiotensin I converting enzymes

The study aimed to characterize and identify anti-diabetic and anti-hypertensive bioactive peptides generated upon enzymatic hydrolysis of quinoa protein isolates. Different quinoa protein hydrolysates (QPHs) were produced using food grade enzymes like Bromelain, chymotrypsin and Pronase E at a hydrolysis interval of 2 h up to 6 h. QPHs were characterized for their physicochemical properties using degree of hydrolysis, SDS-PAGE, and their anti-diabetic properties via inhibition of dipeptidyl peptidase-IV (DPP-IV) and α-glucosidase (AG), and anti-hypertensive property via inhibition of angiotensin converting enzyme (ACE) were explored. IC₅₀ for DPP-IV, AG and ACE inhibitory activities of QPHs were in the range of 0.72–1.12, 1.00–1.86 and 0.18–0.31 mg/mL, respectively. The chymotrypsin derived 6 h hydrolysate (QC6) was sequenced for peptides identification and 136 peptides were identified among which 35 peptides were predicted as potential bio-active peptides (BAPs) based on their Peptide Ranker score. Results showed that identified peptides were predicted to possess high potential in inhibiting the DPP-IV, AG and ACE. In particular, QHPHGLGALCAAPPST was found to bind to the highest number of active hotspots of the target enzymes that are involved in their enzymatic activities. In conclusion, quinoa protein hydrolysates were identified as potential sources of BAPs with inhibitory properties towards key enzymes involved in the control of type 2 diabetes and hypertension.     

Effect of steam explosion on enzymatic hydrolysis and baking quality of wheat bran

In this study, the effect of steam explosion (SE) treatment on microstructure, enzymatic hydrolysis and baking quality of wheat bran was investigated. Coarse and fine bran were treated at different steam temperatures (120–160 °C) and residence times (5 or 10 min) and then hydrolysed with carbohydrase enzymes. The SE treatment increased water extractable arabinoxylan (WEAX) content from 0.75 to 2.06% and reducing sugars from 0.92 to 2.41% for fine bran. The effect was more pronounced with increased SE temperature and residence time. The highest carbohydrate solubilisation was observed in fine bran at SE treatment of 160 °C, 5 min. WEAX content increased to 3.13% when this bran was incubated without enzyme, while WEAX content increased to 9.14% with enzyme addition. Microscopic analysis indicated that cell wall structure of wheat bran was disrupted by severe SE conditions. Supplementation of SE treated (150 °C, 10 min) bran at 20% replacement level decreased the baking quality of bread. However SE followed by enzymatic hydrolysis increased specific volume and decreased crumb hardness (on the day of baking and after three days of storage). Phytic acid content of bread supplemented with SE treated bran was lower than the one supplemented with untreated bran.     

Foaming and Emulsifying Properties of Fractions of Gluten Peptides Obtained by Limited Enzymatic Hydrolysis and Ultrafiltration

Gluten hydrolysates were prepared by limited enzymatic hydrolysis with a protease having a chymotryptic activity in the presence or in the absence of cysteine during the dispersion phase of the process. The hydrolysates were fractionated by ultrafiltration, using two inorganic membranes with different molecular weight cut-off (MWCO), 50 kg/mol and 150 kg/mol. The retentates were enriched in hydrophobic peptides and the permeates were enriched in hydrophilic peptides. The foaming and emulsifying properties of hydrolysates, retentates and permeates were analysed at two pHs (4 and 6·5) and two salt concentrations (0·2 and 2% NaCl). Hydrolysates displayed a foaming capacity, but the foams were not stable. Permeates generated foams at pH 6·5 only, and these foams had a very short life-time. Permeates displayed no emulsifying properties. Retentates yielded foams with a good stability and were more efficient than whole hydrolysates to stabilise emulsions. They provided a strong resistance to coalescence. The functional properties of retentates were only sightly influenced by pH and ionic strength. Neither cysteine addition, which helps gluten dispersion and increases the yield of soluble hydrolysate, nor the MWCO of the ultrafiltration membranes influenced the functionality of the hydrolysate fractions.     

Production of Xylose from Sorghum Straw Using Hydrochloric Acid

Xylose is a hemicellulosic sugar mainly used for its bioconversion to xylitol. Sorghum straw is a raw material for xylose production that has been studied scarcely. The objective of this work was to study the xylose production by hydrolysis of sorghum straw with hydrochloric acid at 122 °C. Several concentrations of HCl (2–6%) and reaction time (0–300 min) were evaluated. Kinetic parameters of mathematical models for predicting the concentration of xylose, glucose, acetic acid and furfural in the hydrolysates were found. Optimal conditions for hydrolysis were 6% HCl at 122 °C for 70 min, which yielded a solution with 16·2 g xylose/L, 3·8 g glucose/L, 2·0 g furfural/L and 1·9 g acetic acid/L.     

Manufacture and characterization of pasta made with wheat flour rendered gluten-free using fungal proteases and selected sourdough lactic acid bacteria

Wheat flour, which was rendered gluten-free by sourdough lactic acid bacteria fermentation and fungal proteases, was used for manufacturing experimental gluten-free pasta (E-GFp), according to a traditional process with low temperature drying cycle. Chemical, technological, structural, nutritional and sensory features were characterized and compared with those of commercial gluten-free (C-GFp) and durum wheat pasta (C-DWp). As shown through immunological analyses, the residual concentration of gluten of the hydrolyzed wheat flour was below 10 ppm. E-GFp showed rapid water uptake and shorter optimal cooking time compared to the other pastas. Despite the absence of the gluten network, the supplementation with pre-gelatinized rice flour allowed structural properties of E-GFp, which were comparable to those of C-GFp. The in vitro protein digestibility of E-GFp resulted the highest. Probably due to proteolysis during sourdough fermentation; chemical scores, essential amino acid profile, biological value and nutritional index of E-GFp were higher than those of C-DWp. The hydrolysis index (HI) of E-GFp was ca. 30% lower than that found for C-GFp. As shown by sensory analysis, the characteristic of E-GFp were acceptable. The manufacture of E-GFp should be promising to expand the choice of gluten-free foods, which combine sensory and nutritional properties.     

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.     

Preparation and properties of wheat gluten based bioplastics with fish scale

The objective of this study was to prepare the wheat gluten based bioplastics with fish scale (FS) through compression molding. The tensile strength of the wheat gluten/FS composites (the range of 6.5–7.5 MPa) was higher than that of the neat wheat gluten-based bioplastic (3.40 MPa). There was a good dispersion of the fish scale powder embedded within the wheat gluten matrix. Dynamic mechanical analysis results showed that the tan delta max peak height and storage modulus of the wheat gluten-based bioplasic was reduced by adding the fish scale. Moreover, the addition of the fish scale caused a weight loss and the surface of the wheat gluten based bioplastic after 120 h of accelerated weathering were differed from the neat wheat gluten based bioplastic. These results may help to find a new applications for fish scale waste to control the degradation rate of a wheat gluten based bioplastic in the agricultural field.     

Rheological properties of wheat flour processed at low levels of hydration: Influence of starch and gluten

The rheological properties of wheat flour under processing such as extrusion (with 28% moisture content, wet basis) are influenced by the molecular changes its components undergo during processing. But, there was no simple relationship between the wheat-flour characteristics and their rheological properties. In order to investigate the quantitative and qualitative effects of the individual flour components on rheological properties, model blends of wheat starch and wheat gluten with different starch/gluten ratios were studied. The effects of gluten and starch quality were also investigated by using different gluten types and by modifying the amylose content of starch, respectively. The shear viscosity of the blends, determined by capillary rheometry under controlled conditions (35% moisture content, 140 °C), was observed to be modified by both gluten and amylose content. The changes undergone by wheat gluten under these conditions were analysed by HPLC, to determine the levels of unextractable polymeric proteins, and by Lab-on-a-Chip analysis of protein composition, to follow the polymerisation of protein under processing. This study indicated that in low hydrated products in the molten state, shear viscosity is affected by the structure of the blends as determined by fluorescence microscopy and by the molecular changes occurring during processing.