Soluble Wheat Proteins Research Articles

Determination of soluble wheat protein fractions using the Bradford assay

AbstractBackground and objectivesDetermination of different grain protein fractions in wheat cultivars is an important task in analyzing bread baking quality. In many laboratories, the Bradford assay is used to determine protein concentrations in solutions. In any protein assay (including Bradford), the ideal protein to use as a standard is the purified protein being assayed. In the absence of such an absolute reference, protein another protein must be selected as a relative standard such as bovine serum albumin (BSA) which is widely used. The aim of this work was to find conversion factors for BSA to determine correct albumin–globulin, gliadin, and glutenin concentrations, because these purified wheat grain protein fractions are mostly not available to be used for calibration purposes.FindingsIn case of BSA calibration, gluten concentration was underestimated (50%–54%) compared to calibration with the respective purified wheat proteins (65%–70%) in extracts of wheat grain samples. This result is explained with the different amino acid composition of BSA and wheat protein fractions leading to a more intense signal with BSA in the Bradford assay. Calibration of the Bradford assay using BSA as well as purified wheat protein fractions allowed to calculate the conversion factors of 2.11 for BSA/albumin–globulin, 4.25 for BSA/gliadin, and 3.42 for BSA/glutenin. Application of these conversion factors proved to accurately adjust protein concentrations of wheat fractions originating from ten cultivars, determined with BSA calibration of the Bradford assay.ConclusionsBSA calibration of the Bradford assay in combination with the conversion factors can be used to determine protein concentration of wheat grain fractions.Significance and noveltyFindings of this study make a contribution toward the correction of a common method, to provide a basis for better comparability of research results from different laboratories.

Effect of soy proteins on characteristics of dough and gluten

The object of this research was to compare the influence of different soy protein products on wheat dough and its gluten characteristics, including soy protein isolate (SPI), texturized soy protein (TSP) and hydrolyzed soy proteins (SPH), all of which with similar protein content. Addition of TSP could increase dough stability and gluten content, but gluten could not be detected when flour was fortified with SPH. During mixing, SPI tended to interact with SDS soluble wheat proteins, SPH tended to interact with SDS soluble and alcohol soluble wheat proteins, and TSP tended to interact with SDS soluble wheat proteins and TSP. A new protein component was observed from TSP fortified dough by SDS-PAGE. Disulfide bonds change confirmed the new linkage formation in blend dough. CLSM micrographs revealed that effect of SPI, SPH and TSP was different, and this difference was responsible for the change of gluten and dough characteristics.

Effects of short-chain fructooligosaccharides on growth performance of preruminant veal calves

This study was conducted to evaluate the effects of 2 different daily doses of short-chain fructooligosaccharides (scFOS), a prebiotic ingredient, added to a calf milk replacer on growth performance, carcass characteristics, and fecal concentrations of short-chain fatty acids of preruminant veal calves. In total, 112 male Prim’Holstein calves, between 8 and 10d of age, were randomized in this study according to their body weight and were bred until the age of 168 d. They were fed a calf milk replacer containing 5% soluble wheat proteins as well as cereal-based pellets, the composition of which was adapted to cover the needs of the animals throughout the study. After 2wk of adaptation, the calf milk replacer was supplemented or not supplemented with a daily dose of 3 or 6g of scFOS. Growth performance of calves, as measured by body weight, cold carcass weight, feed intake, average daily gain, and feed conversion ratio, was recorded and feces samples were taken to evaluate short-chain fatty acid concentrations. The inclusion of wheat proteins in milk replacer did not negatively affect the growth performance of calves in comparison with general standards. The addition of scFOS in the milk reduced the feed conversion ratio of veal calves in a dose-dependent manner and tended to increase the carcass weight. A general trend was observed for an increased production of total short-chain fatty acids in time, but scFOS decreased acetate proportion to the benefit of butyrate proportion. These data suggest that inclusion of scFOS in the calf milk replacer allowed the growth performance of preruminant calves to be enhanced, possibly via a modification of the activities of microbial fermentation.

MALDI based identification of whey protein derived tryptic marker peptides that resist protein glycation

Cow's milk and dairy products are a common cause of food allergy especially for children. The extensive use of milk proteins, especially whey, in food products poses a serious threat to allergic consumers, therefore reliable detection methods are needed in order to ensure meticulous labeling and to protect allergic consumers. The aim of the study was to do a screening of the tryptic peptides derived from whey proteins which were still identifiable using MALDI-TOF/MS and MS/MS analysis despite severe modification through food processing simulating reactions. A bottom-up approach was therefore used. Whey protein mixtures were subjected to the Maillard reaction with glucose, both in the absence or presence of soluble wheat proteins. Despite severe changes on protein level as evidenced by a time dependent increase of protein bound carbonyls, fluorescent compounds, and aggregation as well as a parallel decrease of available lysine residues, several peptides were proven to remain stable. Using MALDI-TOF/MS and MS/MS analysis of tryptic digests of the glycated proteins, two unmodified peptides derived from β-lactoglobulin were identified (15Val–Arg40 and 41Val–Lys60). These stable peptides could serve as analytical targets for the development of new robust analytical approaches for detection of undeclared whey protein residues in foods.

Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA

Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used.

ELISA detection of hazelnut proteins: effect of protein glycation in the presence or absence of wheat proteins

Hazelnuts are widely used in the food industry, especially confectionary foods. Nevertheless, these nuts contain several allergenic proteins that may be unexpectedly present as contaminants in various foods and may pose a serious threat to allergic consumers. The enzyme-linked immunosorbent assay (ELISA) is the preferred method to assess the level of hazelnut protein contamination. It is commonly used by both the food industry and enforcement agencies. Several ELISA kits are commercially available. However, protein detectability by ELISA may be affected by severe changes that proteins undergo during processing. The aim of this study is therefore to investigate the impact of processing on the ability to detect hazelnut protein by four commercial ELISA kits. Hazelnut proteins in the presence or absence of soluble wheat proteins were modified with glucose via the Maillard reaction. Changes in hazelnut proteins, such as the formation of protein-bound carbonyls, losses of reactive lysine residues and free amino groups, and severe aggregation dramatically affected the hazelnut protein detection by the commercial kits. The observed impact was highly dependent on the type of ELISA kit used.

Influence of glycation and pepsin hydrolysis on immunoreactivity of albumin/globulin fraction of herbicide resistant wheat line

The aim of this study was to investigate the influence of non-enzymatic glycosylation on the immunogenic properties of soluble wheat proteins. Albumin/globulin fractions of herbicide resistant wheat line were non-enzymatically glycosylated using glucose for seven days at 37°C. The changes in their structures and immunoreactivity were then determined. The protein fractions were also hydrolysed with pepsin to determine the resistance to digestion. Albumin/globulin fractions before and after non-enzymatic glycosylation were analysed using <i>o</i>-phthaldialdehyde method and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The immunoreactivity of the protein fractions was determined using enzyme-linked immunosorbent assay methods, as well as affinity chromatography. The soluble wheat proteins showed smaller amounts of available α-amino groups after non-enzymatic glycosylation, and were stronger immunogens after glycation, but their antigenicity was not been affected significantly. However, pepsin hydrolysis of wheat proteins decreased their immunoreactivity.

Investigation of chloroform-methanol soluble wheat proteins and sphingolipids as potential dietary triggers of diabetes in BBdp rats

The present study is a follow-up to the study by Coleman (1990), which found that the incidence of diabetes reduced in non-obese diabetic (NOD) mice when chloroform-methanol soluble substances were removed from a cereal-based diet. We used a 2:1 chloroform–methanol (C–M) mixture to extract C–M soluble sphingolipids and proteins from wheat gluten. The C–M extract was subjected to silica gel chromatography and saponification to remove triglycerides, glycolipids and phospholipids. This sphingolipid-enriched fraction from wheat gluten was subsequently incorporated into a BioBreeding diabetes-prone (BBdp) rat diet. Five diets were fed over a 120-day period to BBdp rats: a negative control hydrolysed casein (HC) diet, a positive control National Toxicology Program 2000 (NTP 2000) diet, a wheat gluten (WG)-based diet, a WGGSLF diet containing the WG residue remaining after chloroform-methanol extraction, and a hydrolysed casein plus sphingolipid-enriched fraction (HCGSL) diet. There was a significant increase in diabetes incidence in rats fed the HC + wheat sphingolipid diet versus the HC diet at days 70 and 80 of the feeding study, but not at the end of the study (120 days). There were no significant differences in diabetes incidence in rats fed the WG diet and WG diet with sphingolipids removed. There were no significant differences in pancreatic insulitis scores or in the numbers of jejunal CD4+ and γδTCR + T cells in BBdp rats due to the dietary addition or absence of CM soluble substances from wheat gluten. Sera IFN-γ levels were significantly higher in the sphingolipid-removed WG diet group compared to the BBdp rats in the other four diet groups. Overall, the results of this study provide some evidence for a slight promotional effect of C–M soluble sphingolipids and proteins in wheat gluten on the incidence of diabetes and up-regulation of immune responses in the BBdp rat.

Chemical Modification of Wheat Protein-Based Natural Polymers: Grafting and Cross-Linking Reactions with Poly(ethylene oxide) Diglycidyl Ether and Ethyl Diamine

Mobile poly(ethylene oxide) diglycidyl ether (PEODGE) segments were chemically grafted onto a soluble wheat protein (WP), and different network structures were formed via coupling reactions with ethyl diamine (EDA) in different PEODGE/EDA (PE) ratios. When the PE ratio was 1:1, linear PEs were the predominant segments grafted onto WP chains and the whole WP-PEODGE-EDA (WPE) system was still soluble with an increased molecular weight. Reducing the amount of EDA in the systems produced insoluble cross-linked WPE networks. The broad distribution of network structures and chain mobility resulted in a broad glass transition for the WPE materials. However, the glass transition started at lower temperatures, and the materials became flexible at room temperature. The PE segments were present in all rigid, intermediate, and mobile phases in WPE networks, while the proportion of mobile WP chains was increased as a result of the plasticization effect from the mobile PE segments. The mobility of the most mobile component lipid was also restricted to some extent when forming the cross-linked WPE networks. The study demonstrated that the formation of different network structures with PE segments could significantly improve the flexibility of WP materials, vary the solubility, and modify the mechanical performance of WP-based natural polymer materials.

Luminex-based triplex immunoassay for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powder.

An automated fluorescent microsphere-based flow cytometric triplex immunoassay, using the Luminex 100 flow analyzer with MultiAnalyte Profiling (xMAP) technology, was developed for the simultaneous detection of proteins from three vegetable sources as potential fraudulent adulterants in milk powder. In the final triplex inhibition immunoassay, soluble wheat proteins (SWP) and proteins from soy and pea were coupled to three different microsphere sets. A mixture of these microsphere sets was transferred to a microtiter plate well together with the sample and a mixture of three affinity-purified polyclonal antibodies raised against the proteins and labeled with a fluorophore (Alexa 532). After incubation for 1.5 h at room temperature in the dark, the fluorescence intensities on the microspheres were directly measured (no wash procedure) in the Luminex during 10 s per well (100 microspheres per set). The sensitivities of the three assays for plant protein extracts were determined as 0.5-0.6 microg/mL at 50% inhibition. For the detection of the vegetable proteins in milk powder, the samples were dissolved in buffer (0.1 g in 10 mL) and further diluted (20 times) to create a 50% inhibition at approximately 0.5% of the vegetable proteins in the total protein content of milk powder. With the help of calibration standards, prepared under conditions comparable to those for sample materials, the triplex immunoassay proved to be quantitative above 0.1%, although concentrations in high-heated milk powders were underestimated. Due to the xMAP technology, in which 100 different microsphere sets can be distinguished, this triplex immunoassay can easily be extended to detect other possible adulterants.

IgE binding to pepsin-digested water soluble and insoluble wheat proteins.

Positive skin prick tests to wheat are a common finding among atopic patients, but only a minor fraction of these patients show immediate clinical symptoms after wheat ingestion. The aim of this study was to evaluate the allergenicity of wheat proteins after pepsin treatments. Six patients with positive specific IgE and/or skin prick test to soluble wheat proteins and to gluten were studied. Five of them had no symptoms after wheat ingestion and showed a negative oral challenge test. All sera were analysed with extracts obtained after pepsin hydrolysis during different time periods of water-soluble and insoluble wheat proteins by IgE immunoblotting. A pepsin-sensitive allergen of around 35 kDa was recognized by the five atopic negative wheat oral challenge patients in the undigested water-insoluble extract. A patient showing immediate urticaria after wheat ingestion detected a pepsin-resistant allergen with similar molecular weight. Finally, an inhalation-induced asthma patient recognized water-soluble proteins (of around 14 kDa) that could correspond to the alpha-amylase inhibitors. Our immunoblotting method shows better correlation with the clinical symptoms than skin prick test and CAP results. Pepsin resistance of the acetic acid-soluble wheat proteins may be responsible for the immediate symptoms of the patient who presented immediate urticaria.

Gelation properties of salt soluble meat protein and soluble wheat protein mixtures

Salt soluble meat proteins (SSMP) and commercially available soluble wheat proteins (SWP) were characterised by SDS-polyacrylamide gel electrophoresis, differential scanning calorimetry (DSC) and small and large deformation testing. DSC scans indicated transitions similar to those of native actomyosin for the salt soluble meat extract whereas SWP did not indicate any transitions between 20 and 120 °C. Small deformation tests on SWP indicated a G′/ G″ crossover gelation temperature of 90 °C and weak gels as judged by frequency sweeps. In contrast, SSMP gelled at 40 °C and formed strong gels on heating to 90 °C. However, on autoclaving at 120 °C, 20% SWP in distilled water produced strong elastic gels with little syneresis, compared with the more brittle gels produced with 20% (w/w) SSMP as indicated by large deformation testing. Mixtures of the two proteins in the ratio SSMP/SWP (15:5) gave strong elastic gels similar to the SWP gels. Even the presence of very small amounts of SWP in the mixture, e.g. SSMP/SWP 20:1 trebled the elastic modulus compared with a SSMP gel and reduced syneresis. This was probably due to the close association of SWP with actomyosin strands as viewed by transmission electron microscopy. However, increased levels of SWP in the mixture, for example SSMP/SWP 10:10 ratio, resulted in the separation of the two protein phases as shown by phase contrast microscopy, and consequently led to lower G′ values in the mixed gels. The addition of 20 mM chloride salts showed that potassium reduced the shear modulus, sodium had no effect and calcium enhanced the shear modulus for SWP gels formed at 120 °C. In contrast, SSMP gels were stronger in the presence of potassium, followed by sodium and calcium.

Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder.

The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders. Affinity-purified polyclonal antibodies raised against the three protein sources were immobilized in different flow channels (Fcs) on the biosensor chip (CM5). Dissolved milk powders were injected (20 microL injections at 20 microL min(-1)) through the serially connected Fcs, and the antibody-bound plant proteins were detected directly. The total run time between samples, including a regeneration step with 5 microL of 10 mM HCl, was 5 min. The limits of detection in milk powder were below 0.1% of plant protein in the total milk protein content. The antibodies also recognized some proteins from other plant sources, which made this BIA even more suitable as a broad screening assay for nonmilk proteins.

The thermostability of spray dried imitation coffee whiteners

Imitation creamer formulations were spray dried and agglomerated on a pilot scale tall‐form drier in order to evaluate the stability of the resulting powders when added to hot aqueous coffee solutions. The study explored the effects of different protein ingredients (sodium caseinate; milk protein concentrate; whey protein concentrate; milk proteinate; soluble wheat protein) in combination with non‐protein emulsifiers and disodium hydrogen orthophosphate. Adaptation of coffee stability test methodology was necessary to take account of the presence of significantly more ‘floaters’ in the case of imitation coffee whiteners which did not sediment during centrifugation. A new non‐dairy protein, soluble wheat protein, proved to have exceptional stabilizing ability compared to all other protein ingredients evaluated. Sodium caseinate performed the best out of the dairy proteins, while formulations incorporating milk protein concentrate tended to be the least stable. When working with whey protein concentrate as the principal ingredient source, an emulsifier system based on mono/diglycerides was inadequate, and it was necessary to use a combination of polysorbate and sodium stearoyl lactylate in its place.

Interaction of deamidated soluble wheat protein with sodium alginate

The viscous behaviour of mixtures of deamidated gluten or soluble wheat protein (SWP) (7.5–13.5%, w/v) and sodium alginate (0.5–1.5%, w/v) in distilled water pH 6.5 were investigated. Mixtures of SWP and sodium alginate mixed under varying conditions indicated greater than expected viscosity values at higher biopolymer concentrations. Measurements made when the biopolymers were mixed at room temperature followed by heating at 60°C showed maximum viscosity at a ratio of 50 parts SWP to 50 parts sodium alginate. When the biopolymers were heated separately and then mixed, the maximum viscosity occurred at a ratio of 80 parts SWP to 20 parts sodium alginate. SWP spontaneously aggregated on hydration. Heating at 60°C caused fragmentation or disaggregation of the SWP leading to a reduction in viscosity and an increase in hydrophobicity ( Friedli, & Howell, (1996) Food Hydrocolloids, 10, 255–261). Sodium alginate inhibited the breakdown of SWP aggregates at 60°C, when both polymers were mixed prior to heating, which was confirmed by phase contrast microscopy. Although the mixture did not appear to separate visually, microphase separation was evident by microscopy. The phase separation may be due to repulsion between the negatively charged sodium alginate and SWP which resulted in bicontinuous and more concentrated networks of the two polymers. The addition of SWP (1–8%) to a 1% (w/w) sodium alginate solution increased the gelation time from 30 to 90 min and resulted in soft opaque creamy gels with lower G′ values.

Identification of the major water/salt insoluble wheat proteins involved in cereal hypersensitivity

Several studies have investigated water/salt soluble proteins which comprise 50% of the proteins in wheat. The remaining 50% of wheat proteins, are water/salt insoluble proteins of which there is limited information on their role in cereal hypersensitivity. To investigate the allergenicity of the water/salt insoluble gliadin and glutenin proteins (prolamins). RAST, electrophoresis and Western blotting were used to identify water/salt insoluble wheat allergens. Competitive RAST inhibition was conducted to investigate cross-reactivity between prolamins and water/salt soluble wheat proteins. Specific IgE to alpha-gliadin and to total glutenins were detected in all sera. IgE to beta-, gamma-, fast omega-, and slow omega-gliadin were present in lower numbers of sera. Prolamin allergens of 90-11 kDa were identified by immunoblotting. Water/salt soluble proteins crossreacted with alpha-gliadin and total glutenins. Individuals who are hypersensitive to water/salt soluble wheat proteins produce specific IgE to water/salt insoluble wheat proteins. Western blotting has shown that gliadins, glutenins and proteins with similar molecular weights as the endogenous water/salt soluble wheat enzyme inhibitors are important allergens. Alpha and fast omega- are the most allergenic gliadins. The water/salt insoluble proteins share cross-reacting epitopes with water/salt soluble proteins. These data show that the numbers of proteins involved in the development of cereal hypersensitivity is greater than previously believed and that the development of specific IgE to alpha-gliadin may in part depend on the presence of cross-reacting antibodies to water/salt soluble flour allergens.

Gelation properties of deamidated soluble wheat proteins

Compared with native wheat gluten proteins, chemically modified deamidated gluten proteins are more readily dispersed in aqueous solution. In this paper the gelation behaviour of commercial deamidated gluten, commonly referred to as soluble wheat protein (SWP) on heating as well as its interaction with calcium and basic proteins was investigated. Soluble wheat protein decreased in viscosity on heating at 60°C due to the breakdown of protein aggregates which was observed by phase contrast microscopy. Heating to 60°C was accompanied by an increase in hydrophobicity without conformational transitions as observed by differential scanning calorimetry. In aqueous solutions at concentrations greater than 17% wlw and after autoclaving at 120°C, SWP produced firm gels which were examined by large deformation testing. In the presence of calcium strong but coarse gels were formed which exhibited syneresis. The addition of 0.1–0.5% wlw of basic lysozyme or clupeine to the highly negatively charged SWP induced gelation at the lower temperature of 80°C which was detected by small deformation rheological testing and microscopy using basic and acidic stains. The structure and mechanism of lysozyme-induced gelation of SWP is different from the high thermal (>100°C) gelation of SWP alone. In the lysozyme-induced gelation non-covalent mainly electrostatic interactions occur between the positively charged lysozyme and the negatively charged soluble wheat proteins with the absence of conformational changes in the SWP. In contrast, in the heat gelation of SWP proteins at 120°C it is proposed that the intra-disulphide bonds are broken leading to conformational changes and subsequent association and gelation. This mechanism is supported by the fact that gelation occurred spontaneously in the presence of urea and β-mercaptoethanol indicating that in SWP the disulphide bonds normally prevent gelation unless exposed to very high heat (120°C) or disulphide reducing reagents.

Fuel alcohol production: Assessment of selected commercial proteases for very high gravity wheat mash fermentation

Seven commercial proteolytic enzymes were studied for use in very high gravity (VHG) fermentation. Wheat mashes containing 35 g dissolved solids per 100 ml were prepared and fermented at 20°C with active dry Saccharomyces cerevisiae. Proteases were assessed for their ability to hydrolyze soluble wheat proteins to free amino nitrogen and to reduce the viscosity of VHG wheat mashes. It was found that the increased levels of yeast-assimilable nitrogen and reduced mash viscosity stimulated the rate of VHG fermentation. Ethanol yield was not appreciably affected, nor did the addition of proteolytic enzymes adversely affect saccharification by the glucoamylase enzyme. Therefore, under VHG conditions, utilization of proteases could eliminate the need for supplementing wheat mash with assimilable nitrogenous yeast foods. These data further contribute to the industrial assessment of VHG fermentation technology for the manufacture of fuel alcohol from wheat.

Effects of wheat proteins on the viscoelastic properties of starch gels

AbstractThe interactions between starch and wheat proteins were studied in oscillatory rheological measurements. The starch varieties examined were maize, barley, wheat, triticale, rye and potato. The storage modulus, G′, and the phase angle, δ, were found to be most sensitive to the composition of the starch gel. When starch was gelatinised in presence of gluten it was found that G′ increased for wheat and rye starches, whereas the presence of gluten proteins caused the opposite effect to maize starch. The viscoelastic behaviour of the other starches was not affected by the presence of gluten. A water soluble wheat protein fraction had the same effect on the viscoelastic behaviour of wheat starch as gluten, whereas ligolin behaved like a polar lipid.