Effects of enzymatic deamidation by protein-glutaminase on structure and functional properties of wheat gluten

Protein-glutaminase (PG) purified from Chryseobacterium proteolyticum was used to investigate its deamidation effects on wheat gluten. Water-insoluble gluten was able to be deamidated to the extent of deamidation degree (DD) 72% in 200 mM sodium phosphate buffer (pH 7) at 40 degrees C for 30 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited an upper shift of gluten bands with only deamidation for 1.5-2.0 h (DD 35-45%) compared to the bands of nondeamidated gluten. Results of Fourier transform infrared analysis revealed alterations in secondary structure of gluten by PG deamidation. The assignment within amide I region showed decreases in both inter- (around 1695 cm(-1)) and intramolecular beta-sheets (around 1680 cm(-1)) by deamidation suggesting the deterioration of the aggregation ability of gluten molecules. Solubility and emulsification properties of gluten at pH 7 were improved by deamidation, while both properties at pH 3 were deteriorated by deamidation. Enzyme-linked immunosorbent assay identified that allergenicity of deamidated gluten as compared to the nondeamidated cohorts was decreased remarkably as the deamidation time was prolonged.     

Surface-associated proteins of wheat starch granules: suitability of wheat starch for celiac patients

Wheat starch is used to make baked products for celiac patients in several European countries but is avoided in the United States because of uncertainty about the amounts of associated grain storage (gluten) proteins. People with celiac disease (CD) must avoid wheat, rye, and barley proteins and products that contain them. These proteins are capable of initiating damage to the absorptive lining of the small intestine in CD patients, apparently as a consequence of undesirable interactions with the innate and adaptive immune systems. In this study, starch surface-associated proteins were extracted from four commercial wheat starches, fractionated by high-performance liquid chromatography and gel electrophoresis, and identified by tandem mass spectrometry analysis. More than 150 proteins were identified, many of which (for example, histones, purothionins, and glutenins) had not been recognized previously as starch-associated. The commercial starches were analyzed by the R-5 enzyme-linked immunosorbent assay method to estimate the amount of harmful gluten protein present. One of these starches had a low gluten content of 7 ppm and actually fell within the range proposed as a new Codex Alimentarius Standard for naturally gluten-free foods (maximum 20 ppm). This low level of gluten indicates that the starch should be especially suitable for use by celiac patients, although wheat starches with levels up to 100 ppm are deemed safe in the proposed Codex standards.     

Glycosylation and Deamidation of Rice Endosperm Protein for Improved Solubility and Emulsifying Properties

Rice endosperm protein was prepared by alkali-extraction method and subsequently modified by controlled glycosylation (RP_Glu, RP_XG), deamidation (RP_DA), and enzymatic hydrolysis by alcalase (RP_Alc) methods. The RP_Glu and RP_XG were prepared by Maillard type glycosylation with D-glucose and xanthan gum, respectively. The glycosylation improved the emulsion activity (0.721) and stability (26.8 min) of the protein but did not show a substantial improvement in solubility (39.7%). The rice protein modified by controlled alkali-deamidation (RP_DA) showed highest solubility (68%), emulsion activity (0.776), and emulsion stability (24 min) among the three protein modification methods evaluated in this study. The alcalase treatment to 1.8% DH (RP_Alc) slightly improved solubility (33%), emulsion activity (0.468), and emulsion stability (17.5 min) compared with unmodified rice protein (RP), which had 18% solubility, 0.266 emulsion activity, and 14.7 min emulsion stability. The glycosylation and deamidation methods were more effective than the controlled enzymatic hydrolysis by alcalase in improving solubility and emulsifying properties of rice endosperm protein. Glycosylated and deamidated rice endosperm proteins can find application in enhancing emulsifying properties in suitable products.     

Immunochemical and mass spectrometry detection of residual proteins in gluten fined red wine

Recently, wheat gluten has been proposed as technological adjuvant in order to clarify wines. However, the possibility that residual gluten proteins remain in treated wines cannot be excluded, representing a hazard for wheat allergic or celiac disease patients. In this work, commercial wheat glutens, in both partially hydrolyzed (GBS-P51) and nonhydrolyzed (Gluvital 21000) forms, were used as fining agents in red wine at different concentrations. Beside immunoenzymatic analyses using anti-gliadin, anti-prolamin antibodies and pooled sera of wheat allergic patients, a method based on liquid chromatography coupled to mass spectrometry has been proposed to detect residues of gluten proteins. Residual gluten proteins were detected by anti-prolamin antibodies, anti-gliadin antibodies and sera-IgE only in the wine treated with GBS-P51 at concentration 50, 150, and 300 g/hL, respectively, whereas no residual proteins were detected by these systems in the wine treated with Gluvital 21000. In contrast liquid chromatography-mass spectrometry analyses allowed the detection of proteins in red wines fined down to 1 g/hL of Gluvital 21000 and GBS-P51. Our results indicate that MS methods are superior to immunochemical methods in detecting gluten proteins in wines and that adverse reactions against gluten treated wines cannot be excluded.     

脱酰胺与双酶协同作用提高小麦面筋蛋白酶解效率

为了探讨了不同脱酰胺处理和双酶协同作用方式对小麦面筋蛋白酶解效率及其产物抗氧化活性的影响,该文研究了小麦面筋蛋白在各种预处理方式和酶解条件下的蛋白回收率、水解度、抗氧化性能及肽分子量分布情况。结果显示,单独热处理(90℃,30 min)小麦面筋蛋白对其酶解效率无显著影响,而采用添加0.5 mol/L柠檬酸溶液进行热处理(质量分数为5%,90℃,30 min)可显著(P0.05)提高其蛋白回收率。此外,酶制剂添加顺序及双酶共同水解作用时间对酶解效率均具有较大影响:加入谷氨酰胺酶预先水解对小麦面筋蛋白的深度水解有促进作用;一定时间内的双酶协同作用有利于酶解的进行,但较长时间的双酶作用反而会抑制酶解效率。采用谷氨酰胺酶(质量分数为0.2%)对经柠檬酸加热处理的小麦面筋蛋白作用12 h后再加入胰酶(质量分数为0.6%)共同作用7 h可使蛋白回收率达70.74%,水解度达到9.88%;另外,酶解产物的自由基清除能力ABTS+(2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)+)值与氧化自由基吸收能力(ORAC,oxygen radical absorbance capacity)值分别达到478.95 mmol/g和213.85μmol/g,提示该酶解产物是一种潜在优秀食品抗氧化剂。研究结果可为拓宽小麦面筋蛋白的应用领域,以及高效制备抗氧化活性肽提供方法和理论指导。     

Improved Protocols for ELISA Determination of Gliadin in Glucose Syrups

Simple modifications of existing protocols for high‐sensitivity detection of gluten proteins by immunochemical methods allowed rapid and sensitive determination of residual gluten in highly viscous samples of glucose and maltose syrups obtained from processing wheat starch. Dilution of the original syrup to no less than 15–20% in solids allowed retention of gluten proteins in a soluble form so that ELISA determination of gliadin was possible without an extraction step in aqueous ethanol. An ultrafiltration step may be added to concentrate residual gluten proteins in the diluted syrup samples and allow a further increase in sensitivity. The results are relevant for quality assessment of wheat starch derived syrups as raw materials for use in gluten‐free foods for celiac individuals.     

Study of the temperature effect on the formation of wheat gluten network: influence on mechanical properties and protein solubility

Modifications of mechanical properties of wheat dough during thermal treatments depend mainly on the capacity of wheat gluten proteins to establish intra- and intermolecular interactions when subjected to high-temperature processing. The present study investigates the effect of thermal treatments on the mechanical properties and protein solubility of wheat gluten-based network. The increase in treatment temperatures (from 80 to 135 C) induces an increase in mechanical resistance of the gluten network (tensile strength increases from 0.26 to 2.04 MPa) and a decrease in deformability (elongation decreases from 468 to 236%). The increase in temperature (from 80 to 135 C) also induces a very strong reduction of protein solubility in 2% SDS (from 68 to 0%) that could be correlated to the mechanical changes observed. It was concluded that the modifications of the wheat gluten network properties seem to depend mainly on the temperature level, as temperatures >108-116 C allow activation of thermosetting reactions.     

Effect of Microbial Transglutaminase on Dough Proteins of Hard and Soft (Triticum aestivium) and Durum (Triticum durum) Wheat Cultivars

Microbial transglutaminase (MTGase), a protein‐glutamine γ‐glutamyl transferase (E.C. 2.3.2.13), catalyzes acyl transfer reactions by introducing a covalent cross‐link between l ‐lysine and l ‐glutamine residues. The use of this enzyme has been proposed as an improver to increase dough strength. The objective of this study was to assess and compare the effect of MTGase on different fractions of dough proteins found in hard, soft, and durum wheat. Three different concentrations of the MTGase (0, 5, and 10U/g of gluten) were tested. Moisture, protein, and dry gluten contents were determined for each concentration in addition to rheological measurements done with the farinograph. Following each treatment, the dough proteins were extracted and analyzed by SE‐HPLC and RP‐HPLC. Soluble polymeric protein, gliadins, albumins, and globulins were quantified in addition to the gliadin subclasses and glutenin subunit types. The combustion procedure was used to determine the amount of insoluble polymeric protein. Differences were observed in susceptibility to MTGase catalysis among the dough proteins of the cultivars studied: the cultivar Cortazar (soft wheat) was the most susceptible. The proteins of this cultivar had a characteristically higher amount of ω and α+β gliadins when compared with the other cultivars. As reported earlier, solubility of high molecular weight glutenin subunits and ω‐gliadins was reduced because of the MTGase treatment. However, all gliadin subclasses, including the γ and α+β gliadins, also participated in cross‐linking. The proteins of the cultivar Altar (durum wheat) were the least susceptible to the effects of MTGase. Albumins and globulins did not show any reduction in solubility, implying that they did not participate in cross‐linking.     

Label‐Free Proteomic Analysis of Wheat Gluten Proteins and Their Immunoreactivity to ELISA Antibodies

Enzyme‐linked immunosorbent assay (ELISA) methods are currently the most widely used for gluten quantification. However, the lack of comparable measurements among commercial kits has caused much concern. Here, we studied the immunoreactivity of five commercial ELISA kits to wheat gluten fractionated by reversed‐phase high‐performance liquid chromatography and identified the proteins and peptides in the resulting fractions by mass spectrometry to understand the extent to which these may be contributing to the lack of comparability. The investigated monoclonal antibodies clearly demonstrated divergent responses to the fractioned wheat gluten proteins and sometimes to their initial intended targets. To make comparable gluten measurements a reality, the analytical measurement community requires a set of agreed peptide markers, known conversion factors from these markers to total gluten content, and appropriately characterized (certified) reference materials representative of gluten.     

Biochemical Analysis and Rheological Properties of Gluten Modified by Transglutaminase

A transglutaminase from Streptoverticillium sp. was used to create new covalent intermolecular cross‐links between proteins in gluten. This modification induced drastic changes in its physicochemical properties as well as in its rheological behavior. To understand these changes, we characterized the gluten extractability in acetic acid and identified the proteins of supernatant and pellet by immunoblotting using antibodies specific for each prolamin class. The proportion of soluble proteins decreased drastically after transglutaminase treatment due to the formation of large insoluble polymers as shown by SDS‐PAGE. Among the constitutive proteins of gluten, the high molecular weight glutenin subunits were the most affected in the transglutaminase reaction. The rheological behavior of gluten after 18 hr of incubation with transglutaminase was studied in shear by dynamic measurements over 10^‐3 – 10¹ Hz frequency range and by creep and recovery tests. The behavior of treated glutens remained that of a transient network, but the viscoelastic response was shifted toward shorter times and the steady‐state viscosity was greatly increased. The enzymatic treatment caused a considerable reinforcement of the network. The modified glutens were also less sensitive to thermal processing than unmodified glutens, as shown by a lower amplitude of variation of storage modulus G′ with temperature after enzymatic treatment.     

Influence of Degree of Protein Aggregation on Mass Transport Through Wheat Gluten Membranes and Their Digestibility—An In Vitro Study

The influence of the network structure of wheat gluten on the barrier properties against enzymes was investigated in vitro. The changes in the network structure were introduced by different temperature treatments. The modifications were assessed with solubility studies of wheat gluten proteins in sodium dodecyl sulfate (SDS). The physical barrier properties of wheat gluten membranes were investigated with transport studies examining the transfer of a model protein with no enzymatic activity (BSA) through gluten membranes. The protein network was an effective barrier for BSA, although lightly cross‐linked films were mechanically instable. Membrane breaks occurred in function of the cross‐linking density (percentage of SDS‐insoluble proteins) after only 24 hr for lightly cross‐linked films (≈30% SDS‐insoluble proteins), while highly cross‐linked membranes (≈80% SDS‐insoluble protein) were tight up to more than 33 days. The digestion experiments of the gluten films with pepsin showed that the hydrolysis of wheat gluten films with >72% of SDS‐insoluble protein was significantly retarded. In conclusion, technological treatments to increase the cross‐linking density of gluten have the potential to slow the digestion of cereal‐based foodstuff and to reduce the degradation rate of composite biomaterials.     

Compliance with Gluten‐Free Labelling Regulation in the Brazilian Food Industry

Gluten is an important protein complex for baking products found in wheat, rye, barley, and some oat varieties. However, some people need to avoid these grains and their products because they result in gluten‐related disorders. The only treatment for these individuals is to engage in a gluten‐free diet. The objective of this work was to verify if the gluten content of several commercial food products sold in Brazil complied with their labeling. The Méndez ELISA R5 sandwich method was used to analyze 437 samples, and of these, 70% were labeled as gluten‐free, 26% as containing gluten, and 4% not labeled in relation to gluten. The results indicated that 89% of the products labeled as gluten‐free were correctly labeled and 11% were not, which represented a risk for celiac people.     

喷射蒸煮技术在制备易消化玉米浓缩蛋白中的应用

为了提供一种具有良好消化性的玉米蛋白,该研究以玉米黄粉(corn gluten meal,CGM)为原料,利用亚临界脱脂及酶解超滤技术制备了一种蛋白纯度较高的玉米浓缩蛋白(corn protein concentrates,CPC),并重点考察了制备过程中喷射蒸煮对玉米浓缩蛋白功能性质及消化性的影响。研究结果表明,经喷射蒸煮处理后的玉米浓缩蛋白(jet cooking corn protein concentrates,JC-CPC),普通高温处理的玉米浓缩蛋白(heat treatment corn protein concentrates,HT-CPC)以及未经高温处理的玉米浓缩蛋白(CPC),三者在蛋白质质量分数及氨基酸组成上无明显差异(P0.05)。但JC-CPC的溶解性及功能性质(持水性、起泡性及泡沫稳定性)均显著高于CPC和HT-CPC(P0.05)。体外模拟消化试验结果表明,JC-CPC的水解度(24.02%±0.49%)明显高于CPC和HT-CPC(分别为9.23%±0.45%和14.52%±1.26%)(P0.05),其可溶性氮释放量(62.05%±0.75%)亦高于HT-CPC(40.25%±0.19%)、JC-CPC(21.02%±0.72%)(P0.05)。同时,JC-CPC消化产物的抗氧化试验结果表明,其消化产物具有较高的还原力及1,1-二苯基-2-苦基肼(1,1-Diphenyl-2-picrylhydrazyl,DPPH)自由基的清除能力。因此,利用喷射蒸煮技术,结合亚临界及超滤除杂技术能够为食品工业提供一种具有良好消化性的玉米浓缩蛋白,有望为玉米黄粉的利用提供一条有效途径。     

Preparation and Functional Properties of Gluten Hydrolysates with Wheat‐Bug (Eurygaster spp.) Protease

Suni‐bug (Eurygaster spp.) enzyme was partially purified from bug‐damaged wheat and used to prepare gluten hydrolysates at 3% and 5% degree of hydrolysis (DH). Functional properties of gluten and gluten hydrolysates were determined at 0.2% (w/v) protein concentration and pH 2–10. Gluten solubility after enzymatic hydrolysis increased significantly (P < 0.05) up to 89.1, 89.6, and 95.0% at pH 7, 8, and 10, respectively. Emulsion stability (ES) of gluten hydrolysates improved at neutral and alkaline pH (P < 0.05) and emulsifying capacity (EC) increased significantly (P < 0.05) except at pH 10. Foaming capacity (FC) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6, 7, 8; foam stability (FS) values of gluten hydrolysates were significantly higher (P < 0.05) at pH 6 and 7. Enzymatic modification of gluten by wheat‐bug enzyme resulted in hydrolysates with higher antioxidant activity compared to gluten. Significant correlations (P < 0.001) were found between solubility and EC, ES, FC, and FS values of gluten and its hydrolysates with 3% and 5% DH.     

Optimization of the enzymatic deamidation of soy protein by protein-glutaminase and its effect on the functional properties of the protein

The effects of enzymatic deamidation by protein-glutaminase (PG) on the functional properties of soy protein isolate (SPI) were studied. Conditions for the deamidation were evaluated by means of response surface methodology (RSM). Optimal conditions based on achieving a high degree of deamidation (DD) with a concurrently low degree of hydrolysis (DH) were 44 °C, enzyme:substrate ratio (E/S) of 40 U/g protein and pH 7.0. Under optimal conditions, both DD and DH increased over time. SDS-PAGE results indicated that lower molecular mass subunits were produced with increasing DD. Far-UV circular dichroism spectra revealed that the α-helix structure decreased with higher DD, while the β-sheet structure increased until 15 min of deamidation (32.9% DD), but then decreased at higher DD. The solubility of deamidated SPI was enhanced under both acidic and neutral conditions. SPI with higher DD showed better emulsifying properties and greater foaming capacity than SPI, while foaming stability was decreased. It is possible to modify and potentially improve the functional properties of SPI by enzymatic deamidation using PG.     

Impact of protein size distribution on gluten thermal reactivity and functional properties

Wheat gluten structure was modified in different ways: Disulfide bonds were reduced by sulfitolysis, or protein chains were enzymatically hydrolyzed at three different degrees of proteolysis. A kinetic study of the thermal reactivity of the modified glutens showed that gluten aggregation kinetic was slowed in consequence to the shift of gluten size distribution toward smaller proteins. In contrary to sulfitolysis, proteolysis also affected the gluten reactivity potential because of the formation of numerous nonreactive species. Moreover, the thermally induced browning reaction was greatly enhanced by proteolysis, which increased the amount of free amine residues, substrates of the Maillard reaction. On the contrary, a whitening effect was observed for reduced gluten with bisulfite. Proteolysis was also found to decrease plasticized gluten viscosity, to increase gluten-based materials water solubility, and to enhance gluten adhesiveness properties but to reduce its mechanical performance. Sulfitolysis was considered as a possible way of extending gluten processability by extrusion or injection molding, whereas proteolysis was found to confer enhanced gluten stickiness that suggests new potential end uses of gluten in the pressure sensitive adhesives domain.     

Impact of a treatment with phospholipase A2 on the physicochemical properties of hen egg yolk

Changes in physicochemical properties of egg yolk were investigated after a treatment with phospholipase A 2 (PLA 2), where phospholipids are converted in lyso-phospholipids. Protein solubility and protein denaturation before and after modification by PLA 2 was monitored as well as the functionality of egg yolk by means of interfacial tension. Enzymatic treatment showed a significant impact on the properties of egg yolk with regard to protein solubility and denaturation behavior. To gain a closer insight, egg yolk was separated in its water-soluble fraction called plasma and the insoluble granules. Both fractions were separately modified by PLA 2. The granule fraction shows a higher protein solubility, and the plasma proteins show very high heat stability after modification by PLA 2. Hypotheses regarding related changes in the low-density lipoprotein (LDL) particles are discussed. Results suggest that significant differences in the functional properties of untreated and PLA 2-modified egg yolk do not primarily result from the existence of lyso-phospholipids but from structural changes in egg yolk granules and LDL particles.     

Effects of Micronization on Protein and Rheological Properties of Spring Wheat

This research investigated the effects of micronization, at different moisture levels, on the chemical and rheological properties of wheat. A set of tests designed to analyze protein fraction characteristics and rheological behaviors were conducted on samples from four wheat cultivars (AC Karma, AC Barrie, Glenlea, and Kanata). After being subjected to infrared radiation at three moisture levels (as‐is, 16%, and 22%), the seeds were milled to produce straight‐grade flour. The protein fractionation test revealed significant decreases (P ≤ 0.01) in both monomeric proteins (from 54% of total protein in the control to 37% in the tempered micronized sample) and soluble glutenins (9.4–2.5%). There was a strong negative correlation (r = ‐0.98) between the percentages of monomeric proteins and insoluble glutenins. Total extractable proteins of micronized samples tempered to 22% moisture decreased 43.5% when compared with nonmicronized control samples using size‐exclusion HPLC (SE‐HPLC). Micronization had a significant effect on gluten properties, as seen from a decrease in water absorption (P ≤ 0.01) and dough development time (P ≤ 0.01). Results showed that micronization at 100 ± 5°C had detrimental effects on wheat flour gluten functionality, including a decrease in protein solubility and impairment of rheological properties. These phenomena could be due to the formation of both hydrophobic and disulfide bonds in wheat during micronization.     

Properties of chemically and physically treated wheat gluten films

Chemical (vapors of formaldehyde), physical (temperature, UV and gamma radiation), and aging treatments were applied to wheat gluten films. Changes in film mechanical properties, water vapor permeability, solubility, and color coordinates were investigated. An aging of 360 h led to a 75 and 314% increase in tensile strength and Young's modulus, respectively, and a 36% decrease in elongation. Severe thermal (above 110 degrees C, 15 min) and formaldehyde treatments highly improved the mechanical resistance of the films. Under these conditions, up to 376 and 654% increase in tensile strength and Young's modulus and up to 66% decrease in elongation have been observed. Water solubility was only slightly modified, whereas water vapor permeability was not affected. Color coordinates of films heated above 95 degrees C changed to a great extent. An almost total insolubilization of proteins in sodium dodecyl sulfate occurred for heat- and formaldehyde-treated films, due to the modification of protein network leading to changes in properties of the films.     

Wheat Flour Proteins as Affected by Transglutaminase and Glucose Oxidase

Enzymes are good tool to modify wheat proteins by creating new bonds between the protein chains. In this study, the effect of the addition of glucose oxidase (GO) and transglutaminase (TG) on the wheat flour proteins is presented. The modification of wheat proteins was determined by analyzing the changes in gluten quality, alveograph parameters, and protein modifications. The amount of wet gluten increased with the addition of GO and TG, but the gluten quality was not improved in any case. Regarding the alveograph parameters, the effect of GO was readily evident obtaining wheat dough with higher tenacity and lower extensibility than the control, while TG led to doughs with lower tenacity and that were also less extensible. The protein modifications were characterized by free‐zone capillary electrophoresis (FZCE). FZCE data indicated that TG polymerizes mainly glutenins and, of those, the high molecular weight glutenin subunits were the most affected.