Gluten Fractions Research Articles

Impact of the preparation procedure on gliadin, glutenin and gluten contents of wheat starches determined by RP-HPLC and ELISA

Celiac disease patients rely on safe gluten-free products, and the use of purified wheat starch (WSt) as part of the diet is usually well tolerated. However, uncertainties about residual gluten amounts in WSt remain, because ELISAs target the alcohol-soluble prolamin (gliadin) fraction of gluten, but hardly detect the alcohol-insoluble glutelin (glutenin) fraction. Therefore, gliadin, glutenin and gluten contents of WSt prepared from doughs on a laboratory scale were monitored by RP-HPLC. WSt washed with water from optimally or overmixed doughs had the lowest gluten contents. Gliadins were removed more extensively than glutenins during consecutive washing steps, so that gluten contents analyzed by ELISA were lower than those by RP-HPLC in 17 out of 24 WSt samples. The inability to detect glutelins by ELISA may thus lead to an underestimation of gluten contents in WSt. These findings highlight the need for improved analytical methods capable of detecting both prolamins and glutelins in processed food samples.

Effects of inulin on the structure and emulsifying properties of protein components in dough

High-purity gliadin, glutenin and gluten fractions were extracted from wheat gluten flour. To investigate the effects of three types of inulin with different degrees of polymerization (DP) on the emulsifying properties, disulfide contents, secondary structures and microstructures of these fractions, Turbidimetry, spectrophotometer, Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) were used in this study. The results showed that the emulsifying activity of gliadin was higher than that of glutenin and gluten, but its emulsion stability was lower than that of glutenin. Adding inulin increased the emulsifying activity of the three protein fractions and emulsion stability of gliadin and gluten, but decreased the emulsion stability of glutenin and disulfide bond contents of glutenin and gluten. In the presence of inulin, the α-helical structure of the three proteins had no significant change, whereas the β-turn structure decreased and β-sheet structure increased. The SEM images showed that inulin had the most significant effect on the glutenin microstructure. In general, inulin with a higher DP had greater effects on the structure and emulsifying properties of protein components in dough.

Nitrogen fertilisation of durum wheat: a case study in Mediterranean area during transition to conservation agriculture

Nitrogen (N) nutrition plays a key role for high yields and quality in durum wheat (Triticum turgidum L. subsp. durum (Desf.) Husn); in Mediterranean environments, data regarding N fertilisation management during the transition phase to conservation agriculture (CA) are limited. The aim of this work was to study the effects of N fertiliser forms and rates on yield and some quality traits of durum wheat, during the transition period to CA in Mediterranean areas; moreover, indication on the recommendable N form/rate combinations have been given. Field trials were carried out in south of Italy, during the first two years of transition to CA (from 2010 to 2012) in a durum wheat-based rotation. Following a split-plot design arranged on a randomised complete blocks with three replications, two N forms (main plots) - urea and calcium nitrate - and four N rates (sub-plots) - 50, 100, 150 and 200 kg N ha–1 - plus an un-fertilised Control, were compared. The following parameters were analysed: grain yield, N-input efficiency, grains protein concentration (GPC), total gluten, gluten fractions and minerals concentration in kernels. Calcium nitrate gave the highest yield (4.48 t ha–1), as predicted by the quadratic model, at 146 kg N ha–1, on average. This was particularly noticeable in 2012, when the distribution of rainfall and temperatures regimes as well as residues’ status could have favoured such N-form. These results were confirmed by the observed higher values of all indices describing N-input efficiency. High GPC values (14.8%) were predicted at slightly higher N-rates (173 kg N ha–1, averaging both N forms). In particular, gluten proteins and glutenin/gliadin ratio accrued as the N doses increased, reaching the highest values at 150 kg N ha–1, also positively affecting the quality of durum wheat flour. Iron and zinc concentrations were noticeably increased (38% and 37% on average) by N supply, probably due to the enhanced water use efficiency under CA systems. Principal component analysis summarised properly the obtained results: analysing the N-rates at 150 kg N ha–1, it was confirmed that yields and quality characteristics of durum wheat were optimised in the wettest year (2011) with calcium nitrate. Moreover, the scarce amount of residues characterising the transition phase to CA, requires N application rates not lower than 150 kg ha–1 in order to ensure stable yields and important quality traits. These N rates should be modulated as the accumulation of crop residues increases over time, thanks to long-term effects of CA on soil chemical, physical and biological properties.

The relationship between wheat baking properties, specific high molecular weight glutenin components and characteristics of varieties

The main objective of this study was to compare the aeration level of ring shaped breads, produced from flour made from different winter wheat varieties with different glutenins composition. Analysis of the qualitative and quantitative composition of low molecular weight glutenins (LMW-glutenins), high molecular weight glutenins (HMW-glutenins) and wheat gluten protein fractions α-, γ-, ω-gliadins was performed by electrophoresis in polyacrylamide gel using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). For qualitative evaluation of HMW-glutenin subunits, the obtained electrophoregrams were compared with the standard electrophoretic spectrums of the glutenin components presented in the catalogue. In particular, the indirect acoustic echolocation method can be applied in measurements of the structures of ring shaped breads. Correlations between wheat gluten fractions and some quality parameters characterizing the raw grain material show that HMW-glutenins have the strongest influence on the wheat flour baking properties, and the acoustic method allowed us to assess the quality of ring shaped breads according to the amount of water absorbed and the nature of swelling. This made it possible to classify the ring shaped breads into three quality categories: good (the amount of water absorbed from 37.5 to 50.75 relative units), medium (the amount of water absorbed from 2.54 to 6.28 and from 23.45 to 24.91 relative units), and poor (the amount of water absorbed from 3.6 to 14.7 relative units). According to the specific HMW-glutenin components, it is possible to select the best quality wheat varieties for flour production which could be recommended for ring shaped breads production.

Immunological characterization of the gluten fractions and their hydrolysates from wheat, rye and barley.

Gluten proteins in wheat, rye and barley cause celiac disease, an autoimmune disorder of the small intestine, which affects approximately 1% of the world population. Gluten is comprised of prolamin and glutelin. Since avoidance of dietary gluten is the only option for celiac patients, a sensitive gluten detection and quantitation method is warranted. Most regulatory agencies have set a threshold of 20 ppm gluten in foods labeled gluten-free, based on the currently available ELISA methods. However, these methods may exhibit differences in gluten quantitation from different gluten-containing grains. In this study, prolamin and glutelin fractions were isolated from wheat, rye, barley, oats and corn. Intact and pepsin-trypsin (PT)-digested prolamin and glutelin fractions were used to assess their immunoreactivity and gluten recovery by three sandwich and two competitive ELISA kits. The Western blots revealed varied affinity of ELISA antibodies to gluten-containing grain proteins and no reactivity to oat and corn proteins. ELISA results showed considerable variation in gluten recoveries from both intact and PT-digested gluten fractions among different kits. Prolamin fractions showed higher gluten recovery compared to their respective glutelin fractions. Among prolamins, barley exhibited higher recovery compared to wheat and rye with most of the ELISA kits used. Hydrolysis resulted in reduced gluten recovery of most gluten fractions. These results suggest that the suitability of ELISA for accurate gluten quantitation is dependent upon various factors, such as grain source, antibody specificity, gluten proteins and the level of their hydrolysis in foods.

COMPARISON OF 2-DE PROTEOME MAPS OF WHEAT, RYE AND AMARANTH

The protein maps of wheat (Triticum aestivum L.), cv. Brea; rye (Secale cereale L.), cv. Oklon and amaranth (Amaranthus sp.) cv. Plaisman were obtained by performing 2-DE over a broad pH range (pH 3-11NL) to obtain total protein profiles. Focussing on the gluten fraction, as it is the major trigger for Celiac disease, proteins were separated with a basic pI over a narrow pH range of pH 6-11. Putative identifications of proteins were determined using current literature. The overall spot pattern from wheat over pH 6-11was identified as gamma-gliadins (~ pI 8-10, Mrs 28 - 30, 000 Da), alpha/beta-gliadins (~pI 6.5-7.5, Mrs 25 - 35, 000 Da) and gluten proteins (pI 6-10, Mrs 35 - 60, 000 Da.). The rye had a higher number of abundant groups visible over a broad range (~ 6-200,000 Da, pI 4-9). Comparison of rye to wheat protein maps showed that each cereal had completely different spot patterns in the LMW region of the gels (

Celiac Disease: An Immune Dysregulation Syndrome

Celiac disease is a chronic immune-mediated condition that develops in genetically predisposed individuals. It is characterized by the presence of circulating auto-antibodies in addition to an enteropathy and at times, other extra-intestinal manifestations triggered by exposure to the gliadin fraction of gluten, a family of proteins found in wheat, barley, and rye. There seems to be a rise in reported adverse reactions to gluten, an entity currently termed non-celiac gluten (or perhaps more accurately, wheat) sensitivity, where neither the enteropathy nor the auto-antibodies are present. Celiac disease has protean extra-intestinal manifestations, and an accurate diagnosis should be sought in people suffering from seemingly unrelated complaints, such as fatigue, anorexia, delayed puberty, short stature, decreased bone density, unusual skin rashes, unexplained iron deficiency, and infertility. The presence of an enteropathy, in conjunction with the positive serology, is considered the diagnostic gold standard for making the diagnosis of celiac disease. It is important to stress that the elimination of gluten, even in asymptomatic patients, brings about health benefits, particularly in relation to bone health, as well as a decrease in the incidence of small bowel malignancy, especially lymphoma. Better understanding of the pathophysiology of celiac disease and the molecular mechanisms involved in antigen recognition and processing has provided the impetus for the development of pharmacologic agents that might block the recognition of gluten and its conversion to a toxic antigenic target. Inhibition of tight junction dysregulation could also prevent or minimize the damage triggered by gluten. Work on genetically modified wheat cultivars has progressed, and the possibility of a vaccine to block the immune mediated trigger is being actively investigated. Education and guidance by a knowledgeable nutritionist or registered dietitian can go a long way in minimizing the stress and facilitating the acceptance of the diet and the life-style changes that it represents.

Electrostatic Separation of Peeling and Gluten from Finely Ground Wheat Grains

Sieving and air classification are not efficient enough for the extraction of the high-nutritional-value constituents of wheat. The aim of this article is to validate a simple electrostatic separation method of peeling and gluten, which are two such nutriments contained in finely ground wheat grains. The electrostatic separator is composed of metallic grounded belt conveyer and a rotating roll electrode connected to a high voltage supply. The electrostatic behavior of peeling and gluten powders was characterized using surface potential decay and direct charge measurements. These first set of experiments pointed out the conductive behavior of these powders: in contact with a grounded electrode, they lose their charge in less than 10 s. In a second set of experiments, mixtures of 50% peeling and 50% gluten powders were processed by electrostatic separation. Experimental design methodology was used to model the outcome of the separation process as function of two control variables: the high-voltage applied to the roll electrode and the speed of the belt conveyor electrode. In this way, it was possible to determine the optimal operational conditions for the recovery of high-purity peeling and gluten fractions.

Development, characterization and chemometric analysis of a gluten-free food bar containing whole flour from a new cultivar of amaranth

Food bars are consumed heavily, especially because of their practicality; however they cannot be ingested by celiac patients and present low contents of essential nutrients. The goal of this study was the development and physical-chemical, nutritional and sensory evaluation of a gluten-free food bar containing amaranth and linseed. Gluten fractions were not detected in the food bar formulations. Crude protein and total lipid contents ranged from 68.32 to 76.60 and 74.56 to 82.06 g kg-1 of food, respectively. The polyunsaturated/saturated and n-6:n-3 fatty acid ratios ranged from 0.45:1 to 0.55:1 and 1.44:1 to 2.50:1, respectively. Calcium, magnesium, copper, iron, manganese and zinc were the principal minerals. Application of multivariate analysis enabled sample B to be distinguished according to its mineral and alfa-linolenic acid content. All food bar formulations had good sensory acceptance and high purchase intent.

The role of gluten proteins in production and quality of a yeast leavened sugar and fat rich wheat based food model system

Little if any literature is available on the functionality of gluten proteins in yeast leavened developed dough systems containing high sugar and fat levels. We here studied whether and to what extent gluten proteins impact the different phases of the production of such a system (which contains about 35 parts invert syrup and 70 parts margarine per 100 parts flour) and how these proteins affect its quality. In a first approach, we used model systems based on gluten–starch blends as this allowed specifically altering the gluten levels without altering the gluten quality and/or levels of other wheat flour components. In a second approach flour was heat treated upfront to alter its gluten proteins. Despite the large amounts of sugars and fat, the presence of a developed gluten network in these systems allowed dough pieces to retain gas during fermentation. Also, dough pieces with a developed gluten network could expand faster during subsequent baking than dough pieces without a developed gluten network (i.e. ca. 3.6 vs. 1.9cm/min, respectively). Remarkably, at a given moment during baking (i.e. between 65 and 105sec of baking), dough pieces started to shrink in all directions. This was related to the (elastic) protein network present in the system. However, the time at which shrinking occurred was influenced by factors other than the gluten network. Besides the level, the quality of the gluten fraction determined gluten development during mixing of dough systems containing high levels of sugars and fat, and thus, the spread behavior during fermentation and baking and the final product's dimensions.

Interleukin-10 gene promoter polymorphisms in celiac patients from north-eastern Italy

Celiac disease is a complex chronic intestinal disorder driven by an immune response against the gliadin fraction of gluten: many factors are involved in the pathogenesis of the disease, and among these Interleukin-10 could play an important role. In the present study, the −1082A>G, −819T>C and −592A>C IL10 functional polymorphisms were analyzed in 565 celiac patients and 576 healthy controls from north-eastern Italy, stratified for HLA class II celiac disease risk haplotypes. No significant differences were observed for the three IL10 polymorphisms distribution between celiac patients and controls with the exception of a slightly increased risk for the −1082A allele in HLA-DQ8 male individuals. Although our findings suggest that the IL10 genetic variants analyzed do not have a major role in the susceptibility to the development of celiac disease in north-eastern Italian patients, we think that the possible involvement of IL10 gene in CD should deserve further investigation and that large-scale studies are recommended to confirm our findings.

<b>Development, characterization and chemometric analysis of gluten-free granolas containing whole flour of pseudo-cereals new cultivars</b> - doi: 10.4025/actascitechnol.v36i1.19195

The goal of this study was the development, quimiometric analysis, physical-chemical, microbiological, nutritional, and sensory evaluation of gluten-free granolas containing quinoa, amaranth and linseed. Gluten fractions were not detected in the granola formulations prepared. The crude protein and total lipids contents ranged from 86.72 to 97.49 and 97.84 to 134.03 g kg -1 of food, respectively. The polyunsaturated/saturated and n-6: n-3 fatty acid ratios were 3:1. Calcium was the major mineral and the contents of trace minerals copper, iron, magnesium, manganese and zinc were over 10% of the dietary reference intake values. The granola color tended to light brown. The absence of Bacillus cereus , thermotolerant coliforms, coagulase positive staphylococcus, and Salmonella sp . ensured the product safety. All the granola formulations had good sensory acceptance and high purchase intent. The gluten-free granola formulations had good physical-chemical, sensory and nutritional quality. The use of chemometric analysis enabled to distinguish the samples with respect to their fatty acid composition, minerals content and sensory aspects.

Impact of Puroindolines on Semisweet Biscuit Quality: A Fractionation–Reconstitution Approach

ABSTRACTPuroindoline (PIN) proteins are a factor determining wheat kernel endosperm texture. Biscuits are preferably made from flour from soft wheat (Triticum aestivum L.). Such wheat contains relatively high levels of wild‐type PINs, the impact of which on biscuit quality is unclear. We here studied the impact of PINs on biscuit texture using model flour samples reconstituted from starch and gluten fractions with varying PIN levels. These were obtained by fractionating flour from soft or durum wheat containing either wild‐type or no PINs, respectively. This approach allowed largely retaining the interaction between PINs and either starch or gluten, such as it exists in flour. High PIN levels enhanced air incorporation during dough preparation, increased dough (lateral) expansion, and yielded larger biscuits with higher porosity, which was mainly because of the larger pores. Biscuit fracture stress negatively correlated with PIN level. Porosity contributed to biscuit mechanical properties, but PINs also affected biscuit matrix strength, which in turn affected fracture stress. PINs seem to exert their softening effect when present above a threshold level, and then they have a stronger impact on biscuit fracture stress than the wheat endogenous lipids.

Effect of composition of gluten proteins and dough rheological properties on the cookie‐making quality

PurposeThe purpose of the paper is to study the effect of the gliadin and glutenin fractions of gluten on the cookie quality.Design/methodology/approachTen different wheat varieties were analyzed for the flour physicochemical characteristics, gluten composition and rheological properties. The different flours were baked into cookies. The cookies were analyzed for spread ratio and texture. Relationships of various flour parameters, gluten composition and cookie characteristics were determined.FindingsGluten subfractions, damaged starch, protein, AWRC values were significantly correlated to the cookie spread and texture. Damaged starch (r=−0.638), protein (r=−0.508) and AWRC (r=−0.844) had a negative relationship with the cookie spread. The flours with higher SDS sedimentation value also produced cookies with lower spread ratio and harder texture. The study clearly demonstrated a positive correlation between Gli:Glu ratio (r=0.765) and spread ratio and a negative correlation (r=−0.528) with hardness of the cookies measured in terms of breaking force. Flours with lower dough development time and dough stability performed better as cookie flours.Originality/valueThe gliadins and Gli/Glu ratio had a significant effect on the cookie spread and texture and the prediction equation developed during the study could accurately predict the cookie spread from the Gli/Glu ratio and AWRC values.

Physicochemical and Thermal Properties of Wheat Gluten Fractions from Hard Red Winter Wheat Cultivar

Enriched gliadins, HMW‐GS and LMW‐GS were obtained by differential solvent fractionation from flour using a modification of the method described by Verbruggen et al (1998). Briefly, flour was defatted with chloroform (CHCl3) followed by gliadins extraction with 50% (v/v) n‐Propanol (n‐PrOH), glutenin subunits were extracted from the residue at 60°C by adding 50% n‐PrOH containing 1% (w/v) dithiothreitol (DTT), and the low molecular weight glutenins were precipitated by 85% n‐PrOH. The surface hydrophobicity, charge‐to‐mass ratio, and thermal properties of the three fractions were investigated using reverse‐phaseHPLC, capillary electrophoresis (CE), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Gliadin and LMW‐GS fractions resolved in peaks with different surface hydrophobicity and a relatively wide range of charge‐to‐mass ratio. Samples were pre‐heated to remove residual solvent using TGA. The DSC profile of the pre‐heated gliadin and LMW‐GS‐rich fractions revealed similar glass transition onset, midpoint and conclusion temperatures, averaging 134.9, 143.0 and 151.3°C, respectively, and 0.255 J/g/°C. While the glass transition for the HMW‐GS rich fraction showed higher trend averaging 137.5, 144.3 and 153.1°C for onset, midpoint and conclusion temperature, respectively, and 0.318 J/g/°C.

Osmotic Dehydration of Mango Cubes: Effect of Novel Gluten-Based Coating

Edible coating before osmotic dehydration (OD) reduces solute uptake, which can deteriorate product quality. Polysaccharide-based coatings have been reported for this purpose with limited success. Gluten, due to its inherent film formation property, has the potential to be used for this purpose. In this study, mango cubes of different maturity were osmotically dehydrated with and without gluten-based coating. An edible coating was prepared through fractionation of gluten. Osmotic dehydration was performed at 50°C for 2 h with osmotic solutions (OSs) at various concentrations (45, 55, 65 °Brix). Coating improved the dehydration efficiency index (DEI) due to a substantial decrease in solute gain (SG) and slight increase in water loss (WL). The coating modified the physicochemical properties and this effect was significantly observed at 55 °Brix. Coated mango cubes were harder and shrank more than uncoated mango. The physicochemical properties after OD were also found to be influenced by the maturity of raw mangoes.

Integration of promoters, inverted repeat sequences and proteomic data into a model for high silencing efficiency of coeliac disease related gliadins in bread wheat

BackgroundWheat gluten has unique nutritional and technological characteristics, but is also a major trigger of allergies and intolerances. One of the most severe diseases caused by gluten is coeliac disease. The peptides produced in the digestive tract by the incomplete digestion of gluten proteins trigger the disease. The majority of the epitopes responsible reside in the gliadin fraction of gluten. The location of the multiple gliadin genes in blocks has to date complicated their elimination by classical breeding techniques or by the use of biotechnological tools.As an approach to silence multiple gliadin genes we have produced 38 transgenic lines of bread wheat containing combinations of two endosperm-specific promoters and three different inverted repeat sequences to silence three fractions of gliadins by RNA interference.ResultsThe effects of the RNA interference constructs on the content of the gluten proteins, total protein and starch, thousand seed weights and SDSS quality tests of flour were analyzed in these transgenic lines in two consecutive years. The characteristics of the inverted repeat sequences were the main factor that determined the efficiency of silencing. The promoter used had less influence on silencing, although a synergy in silencing efficiency was observed when the two promoters were used simultaneously. Genotype and the environment also influenced silencing efficiency.ConclusionsWe conclude that to obtain wheat lines with an optimum reduction of toxic gluten epitopes one needs to take into account the factors of inverted repeat sequences design, promoter choice and also the wheat background used.

Quantitative and qualitative differences in celiac disease epitopes among durum wheat varieties identified through deep RNA-amplicon sequencing

BackgroundWheat gluten is important for the industrial quality of bread wheat (Triticum aestivum L.) and durum wheat (T. turgidum L.). Gluten proteins are also the source of immunogenic peptides that can trigger a T cell reaction in celiac disease (CD) patients, leading to inflammatory responses in the small intestine. Various peptides with three major T cell epitopes involved in CD are derived from alpha-gliadin fraction of gluten. Alpha-gliadins are encoded by a large multigene family and amino acid variation in the CD epitopes is known to influence the immunogenicity of individual gene family members. Current commercial methods of gluten detection are unable to distinguish between immunogenic and non-immunogenic CD epitope variants and thus to accurately quantify the overall CD epitope load of a given wheat variety. Such quantification is indispensable for correct selection of wheat varieties with low potential to cause CD.ResultsA 454 RNA-amplicon sequencing method was developed for alpha-gliadin transcripts encompassing the three major CD epitopes and their variants. The method was used to screen developing grains on plants of 61 different durum wheat cultivars and accessions. A dedicated sequence analysis pipeline returned a total of 304 unique alpha-gliadin transcripts, corresponding to a total of 171 ‘unique deduced protein fragments’ of alpha-gliadins. The numbers of these fragments obtained in each plant were used to calculate quantitative and quantitative differences between the CD epitopes expressed in the endosperm of these wheat plants. A few plants showed a lower fraction of CD epitope-encoding alpha-gliadin transcripts, but none were free of CD epitopes.ConclusionsThe dedicated 454 RNA-amplicon sequencing method enables 1) the grouping of wheat plants according to the genetic variation in alpha-gliadin transcripts, and 2) the screening for plants which are potentially less CD-immunogenic. The resulting alpha-gliadin sequence database will be useful as a reference in proteomics analysis regarding the immunogenic potential of mature wheat grains.

Using the Gluten Peak Tester as a tool to measure physical properties of gluten

The functional properties of wheat are largely dictated by composition and interactions of the gluten proteins. All flours contain gliadin and glutenin, but produce baked products of varying quality, which provides evidence that gluten proteins from different wheats possess different properties. A common method to study differences in gluten properties, which is utilized in this study, is fractionation/reconstitution experiments to understand how various gliadin to glutenin ratios and how fractions from different wheat sources affect gluten aggregation properties. Gliadin and glutenin from a vital wheat gluten were fractionated with 70% ethanol and reconstituted at various gliadin to glutenin ratios. Gliadin and glutenin from a Canadian eastern soft, eastern hard and western hard wheat (14% moisture) were fractionated and substituted between flours at the native gliadin to glutenin ratio. Gluten combinations were evaluated with a Gluten Peak Tester at constant temperature and mixing. Varying gliadin to glutenin ratio showed that 50:50 is optimal for fast gluten aggregation while amount of glutenin dictates strength. Substitution experiments showed that replacing good quality gluten fractions with those from a lower quality wheat decreases gluten quality, and vice versa. Data also showed that cultivar specific differences in gliadin and glutenin are more important in dictating gluten strength (torque), while gliadin to glutenin ratio dictates aggregation time (PMT) independent of the source of fractions. The study demonstrated the ability of the improved method to evaluate gluten aggregation by controlling for all variables except the one being tested. The data also revealed information about gluten aggregation properties never before seen.

Effect of Frozen Storage on Molecular Weight, Size Distribution and Conformation of Gluten by SAXS and SEC-MALLS

In this study, the effects of frozen (−18 °C) storage time on molecular weight, size distribution, conformation, free amino groups and free sulfhydryl groups of gluten were studied by small-angle X-ray scattering (SAXS), multi-angle laser light scattering (MALLS) in conjunction with a size exclusion chromatography (SEC) and spectrophotometrically. The results showed that the gluten dissolved in 50 mM acetic acid appeared to be similar to quasi-spherical of the chain conformation and the slope of theconformation plot decreased during the storage. Both the molecular weight and radius of gyration of the frozen gluten decreased with the storage time showing a depolymerization in the high molecular weight fraction of gluten (105 Da ~ 109 Da). Therefore, at constant molecular weight the change of the chain conformation did not show a clear correlation with the storage time. The free amino groups content changed little and the free sulfhydryl groups content of the gluten increased from 9.8 μmol/g for the control to 12.87 μmol/g for 120-day-stored gluten, indicating that the water redistribution and ice recrystallization lead to the breakage of the disulphide bonds and may be one of the reasons for the depolymerization of gluten polymer.