Pilot scale preparation of wheat gluten protein fractions: II. Technological properties of the fractions

Fractions differing in prolamin compositions were obtained on a pilot scale when wheat gluten proteins were separated by differential extraction in dilute acetic acid. The soluble fractions were enriched in gliadins and the insoluble ones were richer in glutenins. The effects of the gluten fractions on the technological properties of wheat flours were investigated using an alveographic test and a comparison was made with gluten-added flours. Gliadin-rich fractions increased the extensibility of the dough and reduced its resistance to deformation. On the other hand, glutenin-rich fractions had an opposite effect and increased the dough resistance more than that of equally-concentrated whole gluten. The magnitude of the effects was strongly related to the gliadin and glutenin contents of the fractions. Prediction of technological effects is thus possible using composition analyses based on protein extractibility or size-exclusion chromatography. Finally, the improving effects of the gliadin-rich and of the glutenin-rich fractions were observed at different stages of the breadmaking process.     

Chemistry of gluten proteins

Gluten proteins play a key role in determining the unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and elasticity on dough. Gluten proteins can be divided into two main fractions according to their solubility in aqueous alcohols: the soluble gliadins and the insoluble glutenins. Both fractions consist of numerous, partially closely related protein components characterized by high glutamine and proline contents. Gliadins are mainly monomeric proteins with molecular weights (MWs) around 28,000-55,000 and can be classified according to their different primary structures into the alpha/beta-, gamma- and omega-type. Disulphide bonds are either absent or present as intrachain crosslinks. The glutenin fraction comprises aggregated proteins linked by interchain disulphide bonds; they have a varying size ranging from about 500,000 to more than 10 million. After reduction of disulphide bonds, the resulting glutenin subunits show a solubility in aqueous alcohols similar to gliadins. Based on primary structure, glutenin subunits have been divided into the high-molecular-weight (HMW) subunits (MW=67,000-88,000) and low-molecular-weight (LMW) subunits (MW=32,000-35,000). Each gluten protein type consists or two or three different structural domains; one of them contains unique repetitive sequences rich in glutamine and proline. Native glutenins are composed of a backbone formed by HMW subunit polymers and of LMW subunit polymers branched off from HMW subunits. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are important for the aggregation of gliadins and glutenins and implicate structure and physical properties of dough.     

The influence of nitrogen fertilisation on quantities and proportions of different protein types in wheat flour

The flours of 13 wheat varieties grown at different levels of nitrogen fertilisation were characterised by the quantitative determination of flour protein groups and gluten protein types using a combined extraction/HPLC procedure. The results demonstrate that the quantities of albumins and globulins were scarcely influenced by different nitrogen fertilisation, whereas those of gluten proteins (gliadins, glutenins) were strongly influenced. The effect on gliadins was more pronounced than on glutenins, as well as the effect on major protein types (α-gliadins, γ-gliadins, LMW subunits of glutenin) in comparison with minor types (ω-gliadins, HMW subunits of glutenin). The proportions of hydrophilic proteins (ω-gliadins, HMW subunits of glutenin) were increased by high levels of nitrogen and those of hydrophobic proteins (γ-gliadin, LMW subunits of glutenin) were decreased. The degree of the effects on both quantities and proportions of flour protein groups and gluten protein types was strongly dependent on the variety. © 1998 SCI.     

Studies on the protein composition and baking quality of einkorn lines

White flours from 23 einkorn breeding lines (assortment 1) and wholemeal flours from 24 einkorn lines (assortment 2) were investigated for their qualitative and quantitative protein compositions by means of a combined extraction/HPLC procedure. The HPLC patterns of the gliadin fractions enabled the differentiation of most einkorn samples. The absence of a group of γ-gliadins at the beginning of the γ-gliadin elution region was unique for einkorn compared to all other wheat species. Differences in the patterns of γ-gliadins allowed the classification of einkorns into four groups; a further subdivision of these groups was possible by the number of ω5-gliadins and the different patterns of α-gliadins and low-molecular-weight glutenin subunits. The total gluten protein (gliadins + glutenins) contents of einkorn flours were similar to or even higher than those of common wheat and spelt. Typical for einkorn flours was the extreme excess of gliadins over glutenins with ω5-gliadins being most abundant and high-molecular-weight glutenin subunits being extremely rare. Micro-tests on the mixing properties and baking performance of assortment 2 flours revealed remarkable differences. Dough development time was negatively correlated with the ratio of gliadins to glutenins and positively with the content of glutenins; bread volume was mainly dependent on the content of glutenins. In conclusion, the determination of the quantitative gluten protein compositions offers a reliable indication of the expected baking quality during the early stages of breeding.     

Comparative investigations of gluten proteins from different wheat species I. Qualitative and quantitative composition of gluten protein types

In contrast to the hexaploid common (bread) wheat, little information is available on the qualitative and quantitative compositions of gluten proteins from other cultivated wheat species. Therefore, representatives of hexaploid spelt, tetraploid durum wheat and emmer, and diploid einkorn were compared with three classes of common wheat (winter wheat, spring wheat, wheat rye hybrid). The flours were extracted to yield total endosperm proteins and the gluten protein fractions (gliadins and glutenin subunits). The extracts were characterised using sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversed-phase HPLC; both methods revealed that gluten protein groups and types known from common wheat (ω-, α-, γ-gliadins, HMW and LMW subunits of glutenin) were present in all species. The HPLC platterns of gliadins and glutenin subunits from species with the same genome composition (common wheat/spelt or durum wheat/emmer) were related, and those of einkorn quite different. According to the quantities determined by reversed-phase HPLC, α-gliadins were predominant in most cases, followed by γ-gliadins and LMW subunits; ω-gliadins and HMW subunits were generally minor components. Common wheats were characterised by the highest proportions of total glutenins and HMW subunits, which are known to be important for breadmaking quality. Moreover, the lower ratio of gliadins to glutenins was typical. Emmer had the lowest proportions of total glutenins and of HMW and LMW subunits, together with einkorn the highest proportion of α-gliadins, and, by far, the highest ratio of gliadins to glutenins. The values for spelt and durum wheat were mostly in a medium range between common wheats, emmer, and einkorn, respectively. Amongst common wheats, spring wheat was characterised by more balanced quantities of α- and γ-gliadins, and wheat rye hybrid by the highest proportions of ω-gliadins. Received: 26 November 1999     

Molecular Analysis of the Polymeric Glutenins with Gliadin‐Like Characteristics That Were Produced by Acid Dispersion of Wheat Gluten

We had earlier shown that the dispersion of wheat gluten in acetic acid solution conferred gliadin‐like characteristics to the polymeric glutenins. To elucidate the molecular behavior of its polymeric glutenins, the characteristics of gluten powder prepared from dispersions with various types of acid were investigated in this study. Mixograph measurements showed that the acid‐treated gluten powders, regardless of the type of acid, had dough properties markedly weakened in both resistance and elasticity properties, as though gliadin was supplemented. The polymeric glutenins extracted with 70% ethanol increased greatly in all acid‐treated gluten powders. Size exclusion HPLC and SDS‐PAGE indicated that the behavior of polymeric glutenins due to acid treatment was attributed to their subunit composition rich in high molecular weight glutenin subunit (HMW‐GS) and not their molecular size. The gluten prepared with the addition of NaCl in acid dispersion had properties similar to those of the control gluten. The results suggest that ionic repulsion induced by acid dispersion made the polymeric glutenins rich in HMW‐GS disaggregate, and therefore, act like gliadins.     

Effects of different <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Enterococcus</Emphasis> strains and chemical acidification regarding degradation of gluten proteins during sourdough fermentation

Dough quality and baking performance of wheat dough are significantly affected by the qualitative and quantitative composition of the gluten. Therefore, the degradation was studied of specific fractions of gluten proteins in sourdough as affected by starter cultures. Doughs were fermented for 0, 5, and 24 h at 30 °C after addition of Lactobacillus sakei, L. plantarum, L. sanfranciscensis or Enterococcus faecalis. Chemically acidified doughs were used as controls. All doughs were analyzed quantitatively for their content of albumins, globulins, gliadins, glutenins, and glutenin macropolymer by means of a combined extraction/HPLC procedure. Protein degradation during sourdough fermentation was primarily due to acidic proteases present in flour. While L. sakei, L. plantarum and L. sanfranciscensis were mostly non-proteolytic, E. faecalis clearly contributed to gluten proteolysis. Single gluten protein types were clearly different in their resistance to proteolytic activities of the dough system and E. faecalis, and, in contrast to total glutenins, the amounts of gluten macropolymer were significantly reduced already after 5 h of incubation. When longer fermentation times were applied, gluten was substantially degraded. The strongest decrease was found for the glutenin fraction leading to an increase of alcohol soluble oligomeric proteins in the gliadin fraction. The extent of the decrease of monomeric gliadins was strongest for the γ-type followed by the α- and the ω-types. This indicates that dough properties residing in specific types of gluten fractions can be influenced by the duration of fermentation and the application of proteolytic strains.     

In vitro degradation of wheat gluten fractions by <Emphasis Type="Italic">Fusarium graminearum</Emphasis> proteases

Fusarium spp. infection of cereal grain is a common problem, which leads to a dramatic loss of grain quality. The aim of the present study was to investigate the effect of Fusarium infection on the wheat storage protein gluten and its fractions, the gliadins and glutenins, in an in vitro model system. Gluten proteins were digested by F. graminearum proteases for 2, 4, 8 and 24 h, separated by Osborne fractionation and characterised by chromatographic (RP-HPLC) and electrophoretic analysis (SDS-Page). Gluten digestion by F. graminearum proteases showed in comparison with gliadins a preference for the glutenins whereas the HMW subfraction was at most affected. In comparison with a untreated control, the HMW subfraction was degraded of about 97% after 4 h incubation with Fusarium proteases. Separate digestion of gliadin and glutenin underlined the preference for HMW-GS. Analogue to the observed change in the gluten composition, the yield of the proteins extracted changed. A higher amount of glutenin fragments was found in the gliadin extraction solution after digestion and could mask a gliadin destruction at the same time. This observation can contribute to explain the frequently reported reduced glutenin amount parallel to an increase in gliadin quantity after Fusarium infection in grains.     

Protein–lipid interactions in gluten elucidated using acetic acid fractionation

Protein–lipid interactions in dough have an important impact on the quality of bakery products. Understanding of protein–lipid interactions in gluten can enhance the development of technological solutions to improve the breadmaking quality of flour as well as the functional properties of gluten. In this study, acetic acid at two different concentrations was used for treating and fractionating gluten. The impact of these procedures on the distribution of lipid components was measured. Acetic acid was able to dissociate non-polar lipids from the gluten protein matrix. Upon fractionation monomeric proteins (predominantly gliadins) and phospholipids were high in the 0.01 M acetic acid soluble fraction. The subsequent fractionation step using 0.1 M acetic acid resulted in an increased amount of high-molecular-weight glutenin subunits (HMW-GS) in the soluble fraction, along with more non-polar lipids and glycolipids in both the free and bound lipid extracts. The distribution of lipid classes demonstrates that non-polar lipids are either associated with the glutenin polymeric network through hydrophobic interactions or entrapped within the gluten matrix. The results also indicate that in gluten, glycolipids are likely to be associated with glutenins through both hydrophobic interactions and hydrogen bonds whilst phospholipids preferentially interact with gliadins and lipid binding proteins.     

Emulsifying and Surface Properties of Wheat Gluten under Acidic Conditions

The solubility of wheat gluten was greatly improved at pH 4 or lower where it showed good emulsifying activity. This might be due to its high surface activity in the acidic pH range and the formation of a stable protein film surrounding the oil droplets. Among the major gluten proteins, gliadins showed higher surface activity than glutenins. The content of glutenins in the adsorbed protein film was higher than that of gliadins, and glutenins are likely to have been adsorbed more tightly than gliadins. These results suggest that gluten proteins exhibit complex behavior, such as adsorption/desorption/displacement/rearrangement during the adsorption process in a gluten‐stabilized emulsion.     

The influence of changes in gluten complex structure on technological quality of wheat (Triticum aestivum L.)

The influence of changes in glutenin–gliadin complex of grain on technological quality of the wheat variety (Triticum aestivum L.) was studied. It was shown that wheat-bug attack caused differences in electrophoregram pattern of glutenins and gliadins concerning their number, intensities and molecular weights. The environmental influence had detrimental effect on rheological properties of dough. Expected heat-stress effect – the increase of gliadin–glutenin ratio was not detected. The modified method for gluten index was introduced and it was proven as superior to the standard method in predicting technological quality of wheat.     

The polypeptide composition,structural properties and antioxidant capacity of gluten proteins of diverse bread and durum wheat varieties,and their relationship to the rheological performance of dough

The aim of this study was to compare five bread and five durum wheat genotypes for gliadins and glutenins profiles, the concentration of free sulphhydryl groups and disulphide bonds, antioxidant capacity of gluten proteins and their bread‐making performance. On average, bread wheat had significantly higher concentration of total sulphur‐rich (S‐rich) and sulphur‐poor (S‐poor) subunits of gliadins, as well as total low molecular weight (LMW) and high molecular weight (HMW) subunits of glutenins than durum wheat. However, durum wheat had higher concentration of S‐rich γ‐gliadins and S‐poor D‐LMW‐glutenins, but did not possess S‐poor ω‐gliadins. The concentration of disulphide bonds and total cysteine was higher in the durum gluten than that in the bread gluten, as well as antioxidant capacity (on average 90.6 vs. 85.9 mmol Trolox Eq kg^?1, respectively). In contrast to the bread wheat, the concentration of HMW‐glutenins was negatively associated with extensibility, as well as resistance to extension in durum wheat flour dough.     

Dispersion in the Presence of Acetic Acid or Ammonia Confers Gliadin‐Like Characteristics to the Glutenin in Wheat Gluten

Spray‐dried gluten has unique properties and is commercially available in the food industry worldwide. In this study, we examined the viscoelastic properties of gluten powder prepared by dispersion in the presence of acetic acid or an ammonia solvent and then followed by lyophilization instead of a spray drying. Mixograph measurements showed that the acid‐ and ammonia‐treated gluten powders had marked decreases in the time to peak dough resistance when compared with the control gluten powder. The integrals of the dough resistance and bandwidth for 3 min after peak dough resistance decreased in both treated gluten powders. Similar phenomena were observed when gliadin was supplemented to gluten powders. Basic and acidic conditions were applied to the acid‐ and ammonia‐treated gluten powders, respectively, and the viscoelastic behaviors were found to depend on the pH in the gluten dispersion just before lyophilization. These behaviors suggest that gluten may assume a reversible change in viscoelasticity by a fluctuation in pH during gluten dispersion. SDS‐PAGE showed that the extractable proteins substantially increased in some polymeric glutenins including the low molecular weight‐glutenin subunit (LMW‐GS) when the ammonia‐treated gluten powder was extracted with 70% ethanol. In contrast, the extractable proteins markedly increased in many polymeric glutenins including the high molecular weight‐glutenin subunit and/or the LMW‐GS when the acid‐treated gluten powder was extracted with 70% ethanol. It thus follows that the extractability of polymeric glutenin to ethanol increases similarly to gliadin when gluten is exposed to an acidic or a basic pH condition; therefore, glutenin adopts gliadin‐like characteristics.     

Extraction and fractionation of wheat flour proteins

With the aid of a 1.5% sodium dodecyl sulphate solution (SDS) the proteins of two Dutch wheat flours were separated into an SDS-soluble and an SDS-insoluble fraction. The SDS-soluble fraction was fractionated with the aid of ethanol precipitation and gel filtration chromatography into albumins, globulins, gliadins, glutenins II and III, and glutelins I, II, III and IV. The SDS-insoluble proteinaceous material was separated into glutenins I and glycoproteins. The protein fractions were identified with the aid of SDS-polyacrylamide gel electrophoresis and amino acid analysis. The glutenins I and II consist of A, B and C subunits. Their molecular weights ranged from about 600 000 to 10 million. The glutenins III consist only of B and C subunits and their molecular weights ranged from 300 000 to 600 000. A hypothesis explains how the glutenins III are the precursors for glutenins I and II and how they contribute to gluten's structure. The glutelins I consist of α β and γ subunits and their molecular weights ranged from about 600 000 to several millions. The glutelins II consist also of α β and γ subunits with a molecular weight of about 300 000 to 600 000, while the molecular weights of the glutelins III and IV ranged from 10 000 to 200 000. All glutelins are insoluble in 70 % aqueous ethanol and 5 M urea, but soluble in SDS and 0.1M NaOH. Only the glutelins III and IV are soluble in 0.1M HAc. The globulins consist of a heterogeneous fraction of components, their molecular weights arranged from 98 000 to 10 000. The gliadins form a heterogeneous but homologous group of polypeptides with an average molecular weight of 35 000. The albumins consist of components with molecular weights lower than 15 000.     

Studies on the distribution and binding of endogenous glutathione in wheat dough and gluten. II. Binding sites of endogenous glutathione in glutenins

For the identification of the binding sites of glutathione (GS) in glutenins, flour of the wheat cultivar " Canadian Western Red Spring " was mixed with water containing ³⁵S-labelled reduced GS as a tracer. The resulting dough was washed in a Glutomatic, and, in order to remove gliadins, the gluten obtained was extracted with 70% aqueous ethanol adjusted to pH 5.5 with acetic acid. The residual proteins (glutenins) were hydrolyzed with thermolysin, and the hydrolysate was separated by gel permeation chromatography on Sephadex G25 and by several steps of reversed-phase HPLC on C₁₈ silica gel. The major radioactive disulphide peptides identified by scintillation analysis were collected and analysed for their amino acid sequences. Twenty-five peptides linked to GS could be assigned to known sequences of gluten proteins. Most peptides (16) were derived from low molecular weight (LMW) subunits of glutenin. Among these, 13 peptides contained the cysteine residue C^b*, which is present in the repetitive sequence region of LMW subunits and which has been postulated to form intermolecular disulphide bonds. This peptide type represented 45% of the total radioactivity of isolated peptides. Three further peptides from LMW subunits representing 46% of radioactivity included cysteine C^x, which has also been proposed to form intermolecular disulphide bonds. Four peptides with 3.2% of radioactivity could be assigned to high molecular weight subunits (cysteines C^b, C^d, C^e, C^y) and four peptides (3.0% of radioactivity) to glutenin-bound %-gliadins (C^b*, C^w, C^z). One peptide (3.3% of radioactivity) corresponded to cysteine C^c from %-gliadins or LMW subunits. Altogether the cysteine residues in glutenins, which are usually linked by intermolecular disulphide bonds, contributed up to 95% of total radioactivity. The results obtained are in accordance with the effect of reduced GS on the rheological properties of dough, namely the weakening of dough by depolymerization of glutenin polymers via specific cleavage of intermolecular disulphide bonds.     

Multivariate optimisation of a capillary electrophoretic method for the separation of glutenins. Application to quantitative analysis of the endosperm storage proteins in wheat

A capillary electrophoretic method has been designed to allow separation of glutenins with high resolution. Several factors, such as buffer composition, running voltage and capillary temperature were optimised using factorial design and response surface methodology. On the other hand, quantification of content of glutenins and gliadins of different wheat varieties were achieved for the first time using a glutenin extract of wheat gluten and a gliadin extract as external standards, respectively, and using the lys-tyr-lys tripeptide as internal standard. The optimised method and an early reported method for the gliadin separation were validated by evaluating linearity, sensitivity, detection and quantitation limits, repeatability and precision.     

The influence of 1B/1R chromosome translocation on gluten protein composition and technological properties of bread wheat

The Austrian bread wheat Amadeus without and with 1BL/1RS translocation and three further translocation genotypes with known HMW subunit compositions were grown under the same environmental conditions. Their flours were characterised by the determination of crude protein content and, partly, by the determination of glutathione and cysteine. Furthermore, the qualitative and quantitative composition of gluten protein types was analysed by a combined extraction and reversed phase HPLC procedure. Dough development time, maximum resistance and extensibility of dough and gluten, and bread volume were determined by means of microscale methods. Protein, glutathione and cysteine contents of flours were only slightly influenced by translocation. The HPLC patterns of gliadins and glutenin subunits showed that translocation caused characteristic changes concerning ω‐gliadins, γ‐gliadins and LMW subunits of glutenin. The amount of ω 1,2‐gliadins was significantly increased and that of LMW subunits decreased. The effect of translocation on the rheological properties of dough and gluten was characterised by a strongly reduced dough development time, reduced maximum resistance and increased extensibility. Bread volume was decreased by about 10%. The amount of glutenin subunits was correlated with dough development time, resistance of dough and gluten, and bread volume to a higher extent (r = 0.79–0.91) than the amount of gliadins (r = 0.52–0.80). Correlation coefficients for LMW subunits were higher (r = 0.82–0.88) than those for HMW subunits (r = 0.35–0.61) when all five wheats were included. Instead, when only translocation lines were considered, HMW subunits (r = 0.89–0.98) were more important than LMW subunits (r = 0.64–0.86). Altogether, the results demonstrate that translocation causes important quantitative as well as qualitative changes in gluten protein composition which can be efficiently determined by reversed phase HPLC. © 2000 Society of Chemical Industry     

Amino acid analyses of glutenins and gliadins

The amino acid compositions of glutenins and gliadins from two strong and two weak wheats have been compared. Glutenin appears to have an amino acid analysis generally similar to that of gliadin but there are individual differences. Glutenin possesses much higher proportions of lysine, glycine and tryptophan and somewhat higher proportions of arginine, tyrosine, threonine, (aspartic acid + asparagine), serine and alanine. Gliadin is richer in proline, (glutamic acid + glutamine), cystine, phenylalanine, amide nitrogen and isoleucine. No definite differences were observed in contents of methionine, valine, leucine or histidine. The possible significance of these results in explaining the properties of glutenin and gliadin is discussed, and the results are compared on a diagram with some analyses in the literature. No significant differences in amino acid composition from variety, or from strong wheats as against weak, were observed among glutenins or gliadins. The calculated factor for converting weight of nitrogen to glutenin or gliadin is 5.6. The mean percentage of (glutamic + aspartic) acids in the amide form is 82 for glutenin and 92 for gliadin.     

Analysis of wheat storage proteins by exhaustive sequential extraction followed by RP-HPLC and nitrogen determination

Wheat storage proteins are responsible for the viscoelastic properties of dough. Their effect on dough rheology depends on the glutenin components and the proportion of gliadins, high and low molecular weight glutenin subunits (HMW-GS, LMW-GS). A prediction of dough behaviour is possible when the concentration of each prolamin group is known. A method of sequential extraction and quantification of wheat flour proteins was developed. Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to quantify gliadins and HMW-GS and LMW-GS. Non-prolamin proteins and lipids were extracted with phosphate buffer and phosphate buffer containing Triton X114 respectively and 70% (v/v) ethanol was used to extract gliadins. Several solvent systems were tested to exhaustively extract glutenins and a buffer containing 0·05 M tetraborate pH 8·5, 2% (v/v) 2-mercaptoethanol, 1 g litre⁻¹ glycine, and 8 M urea was selected. It facilitated the most complete extraction and was compatible with RP-HPLC analysis of extracts. The quantities of extracted prolamins were estimated from RP-HPLC profiles using peak area determination. The concentration of different classes of prolamins obtained by RP-HPLC was compared with their determination by nitrogen (N) content. The average yield of extractions performed on 45 cultivars was 0·99 (coefficient of variation (CV)=7·6%) for gliadins and 0·89 (CV=9·1%) for glutenins. Quantifications of proteins by N determination and RP-HPLC were strongly correlated: regression coefficients were 0·86 for total prolamins and gliadins, and 0·71 for glutenins with the gradient of regression of approximately 1. Therefore, RP-HPLC could be used to quantify prolamin groups without using N determination. © 1998 SCI.     

Glucose oxidase effect on dough rheology and bread quality: A study from macroscopic to molecular level

Enzymes are used in baking to improve dough handling properties and the quality of baked products. Glucose oxidase (GO) is an enzyme with oxidizing effect due to the hydrogen peroxide released from its catalytic reaction. In this study, the macroscopic effect of increasing glucose oxidase concentrations on wheat dough rheology, fresh bread characteristics and its shelf life during storage was determined. A reinforcement or strengthening of wheat dough and an improvement of bread quality can be obtained with the addition of GO, although inverse effects were obtained when excessive enzyme levels were added. The analysis of the gluten proteins at molecular level by high performance capillary electrophoresis and at supramolecular level by cryo-scanning electron microscopy revealed that the GO treatment modified gluten proteins (gliadins and glutenins) through the formation of disulfide and non-disulfide crosslinks. The high molecular weight glutenin subunits showed to be the most susceptible glutenin fraction to the oxidation action of GO. Excessive addition of GO produced an excessive crosslinking in the gluten network, responsible of the negative effect on the breadmaking properties.