Effects of Four Highland Barley Proteins on the Pasting Properties and Short-Term Retrogradation of Highland Barley Starch.

This study evaluated the effects of four highland barley proteins (HBPs), namely, albumin, globulin, gliadin and glutenin, on the short-term retrogradation of highland barley starch (HBS). The findings reveal that HBPs could reduce the viscosity, storage modulus and hardness of HBS, with albumin and globulin showing more prominent effects. Furthermore, with the addition of HBPs, the loss tangent (tan δ) of HBS loss increased from 0.07 to 0.10, and the enthalpy of gelatinization decreased from 8.33 to 7.23. The degree of retrogradation (DR%) of HBS was 5.57%, and the DR% decreased by 26.65%, 38.78%, 11.67% and 20.29% with the addition of albumin, globulin, gliadin and glutenin, respectively. Moreover, the relative crystallinity (RC) and the double helix structures were inhibited with the HBPs' incorporation. Meanwhile, the HBPs also could inhibit water migration and improve the structure of HBS gels. In summary, HBPs could inhibit the retrogradation behavior of HBS, which provides new theoretical insights for the production studies of highland barley foods.

Mass spectrometry-based quantification of immunostimulatory gliadin proteins and peptides in coloured wheat varieties: Implications for Celiac Disease.

Pigmented wheat varieties (Triticum aestivum spp.) are getting increasingly popular in modern nutrition and thoroughly researched for their functional and nutraceutical value. The colour of these wheat grains is caused by the expression of natural pigments, including carotenoids and anthocyanins, that can be restricted to either the endosperm, pericarp and/or aleurone layers. While contrasts in phytochemical synthesis give rise to variations among purple, blue, dark and yellow grain's antioxidant and radical scavenging capacities, little is known about their influence on gluten proteins expression, digestibility and immunogenic potential in a Celiac Disease (CD) framework. Herein, it has been found that the expression profile and immunogenic properties of gliadin proteins in pigmented wheat grains might be affected by anthocyanins and carotenoids upregulation, and that the spectra of peptide released upon simulated gastrointestinal digestion is also significantly different. Interestingly, anthocyanin accumulation, as opposed to carotenoids, correlated with a lower immunogenicity and toxicity of gliadins at both protein and peptide levels. Altogether, this study provides first-level evidence on the impact modern breeding practices, seeking higher expression levels of health promoting phytochemicals at the grain level, may have on wheat crops functionality and CD tolerability.

Formation, structural characteristics and specific peptide identification of gluten amyloid fibrils.

This research investigates the formation of amyloid fibrils using enzymatically hydrolyzed peptides from gluten, including its components glutenin and gliadin. After completing the fibrillation incubation, the gluten group demonstrated the most significant average particle size (908.67 nm) and conversion ratio (57.64 %), with a 19.21 % increase in thioflavin T fluorescence intensity due to self-assembly. The results indicated increased levels of ß-sheet structures after fibrillation. The gliadin group exhibited the highest zeta potential (∼13 mV) and surface hydrophobicity (H0 = 809.70). Around 71.15 % of predicted amyloidogenic regions within gliadin peptides showed heightened hydrophobicity. These findings emphasize the collaborative influence of both glutenin and gliadin in the formation of gluten fibrils, influenced by hydrogen bonding, hydrophobic, and electrostatic interactions. They also highlight the crucial role played by gliadin with amyloidogenic fragments such as ILQQIL and SLVLQTL, aiming to provide a theoretical basis for understanding the utilization of gluten proteins.

Investigating the interaction mechanism between gliadin and lysozyme through multispectroscopic analysis and molecular dynamic simulations.

The development of stable nanocomplexes based on gliadin and other biopolymers shows potential applications as delivery vehicles in the food industry. However, there is limited study specifically targeting the gliadin-lysozyme system, and their underlying interaction mechanism remains poorly understood. Therefore, the objective of this study was to investigate the binding mechanism between gliadin and lysozyme using a combination of multispectroscopic methods and molecular dynamic simulations. Stable gliadin-lysozyme complex nanoparticles were prepared using an anti-solvent precipitation method with a gliadin-to-lysozyme mass ratio of 2:1 and pH 4.0. The characteristic changes in the UV-visible spectrum of gliadin induced by lysozyme confirmed the complex formation. The analyses of fluorescence, FT-IR spectra, and dissociation tests demonstrated the indispensability of hydrophobic, electrostatic, and hydrogen bonding interactions in the preparation of the composites. Scanning electron microscopy revealed that the surface morphology of the nanoparticles changed from smooth and spherical to rough and irregular with the addition of lysozyme. Furthermore, molecular dynamic simulations suggested that lysozyme bound to the hydrophobic region of gliadin and hydrogen bonding was crucial for the stability of the complex. These findings contribute to the advancement of gliadin-lysozyme complex nanoparticles as an efficient delivery system for encapsulating bioactive compounds in food industry.

Complex bio-nanoparticles assembled by a pH-driven method: environmental stress stability and oil-water interfacial behavior.

BACKGROUND: Protein-based nanoparticles have gained considerable interest in recent years due to their biodegradability, biocompatibility, and functional properties. However, nanoparticles formed from hydrophobic proteins are prone to instability under environmental stress, which restricts their potential applications. It is therefore of great importance to develop green approaches for the fabrication of hydrophobic protein-based nanoparticles and to improve their physicochemical performance. RESULTS: Gliadin/shellac complex nanoparticles (168.87 ~ 403.67 nm) with various gliadin/shellac mass ratios (10:0 ~ 5:5) were prepared using a pH-driven approach. In comparison with gliadin nanoparticles, complex nanoparticles have shown enhanced stability against neutral pH, ions, and boiling. They remained stable under neutral conditions at NaCl concentrations ranging from 0 to 100 mmol L-1 and even when boiled at 100 °C for 90 min. These nanoparticles were capable of effectively reducing oil-water interfacial tension (5 ~ 11 mNm-1 ) but a higher amount of shellac in the nanoparticles compromised their ability to lower interfacial tension. Moreover, the wettability of the nanoparticles changed as the gliadin/shellac mass ratio changed, leading to a range of three-phase contact angles from 52.41° to 84.85°. Notably, complex nanoparticles with a gliadin/shellac mass ratio of 8:2 (G/S 8:2) showed a contact angle of 84.85°, which is considered suitable for the Pickering stabilization mechanism. Moreover, these nanoparticles exhibited the highest emulsifying activity of 52.42 m2 g-1 and emulsifying stability of 65.33%. CONCLUSIONS: The findings of the study revealed that gliadin/shellac complex nanoparticles exhibited excellent resistance to environmental stress and demonstrated superior oil-water interfacial behavior. They have strong potential for further development as food emulsifiers or as nano-delivery systems for nutraceuticals. © 2023 Society of Chemical Industry.

Insight into Organization of Gliadin and Glutenin Extracted from Gluten Modified by Phenolic Acids.

The changes in the secondary structure of individual gluten protein fractions (gliadin and glutenin) caused by the supplementation of model dough with eight phenolic acids were analysed. Gliadins and glutenins were extracted from gluten samples obtained from overmixed dough. The changes in the gliadin secondary structure depended on the amount of phenolic acid added to the dough. Higher acid concentrations (0.1% and 0.2%) led to a significant reduction in the amount of α-helices and to the formation of aggregates, non-ordered secondary structures, and antiparallel ß-sheets. After the addition of acids at a lower concentration (0.05%), the disaggregation of pseudo-ß-sheet structures and the formation of ß-turns, hydrogen-bonded ß-turns, and antiparallel ß-sheets were detected. In the case of glutenin, most of the phenolic acids induced the formation of intermolecular hydrogen bonds between the polypeptide chains, leading to glutenin aggregation. When phenolic acids were added at a concentration of 0.05%, the process of protein folding and regular secondary structure formation was also observed. In this system, antiparallel ß-sheets and ß-turns were created at the expense of pseudo-ß-sheets.

Agro-Industrial Protein Waste and Co-Products Valorization for the Development of Bioplastics: Thermoprocessing and Characterization of Feather Keratin/Gliadin Blends.

Biopolymers based on plant and animal proteins are interesting alternatives in the development of films with future prospects as food packaging. Considering that in recent years there has been an increasing interest in the valorization of agro-industrial residues and by-products and that the blending of polymers can lead to materials with improved properties, in this work, keratin-rich feather fibers and gliadins were blended at different ratios in order to develop sustainable and biodegradable films. Control gliadin G100, feather F100 films, and their blends at 3:1 (G75F25), 2:2 (G50F50), and 1:3 (G25F75) ratios were successfully developed through thermoprocessing. The physical properties were differentiated as a function of the concentration of both polymeric matrices. Although gliadins showed higher hydrophilicity as confirmed by their highest swelling degree, films with high gliadin ratios exhibited lower water vapor permeability values at low and medium relative humidities. On the other hand, the feather fiber-based films displayed the highest Young's modulus values and provided an oxygen barrier to the blends, principally at the highest relative humidity. In conclusion, the blend of these protein-based polymers at different ratio resulted in interesting composites whose physical properties could be adjusted.

Organic acids in bread-making affecting gluten structure and digestibility.

Although wheat gluten has remarkable technological properties, it can induce adverse immune reactions in susceptible individuals, such as wheat allergy and celiac disease. Technological processing and some additives on bread formulation can modify gluten physicochemical structure, but the knowledge about the impacts on the digestibility and immunogenicity of gluten is limited. The present study aimed to study the effect of adding organic acids (acetic or ascorbic) on dough rheological properties and bread technological characteristics. In addition, breads were subjected to in vitro digestion and the digesta were analyzed by confocal microscopy, SDS-PAGE and ELISA immunoassay. Acetic acid resulted in a decrease in dough development time up to 44 % and a reduction in stability up to 20 %. Ascorbic acid, present in vinegar, on the other hand, increased elastic modulus (G') and resistance to extension of dough. After the in vitro digestion, SDS-PAGE indicated that protein degradation started in the gastric phase, with the generation of low molecular weight peptides. Accordingly, ELISA immunoassay suggested a great reduction in immunogenic gliadin content from oral to gastric phase. At the end of the intestinal phase, samples with ascorbic acid did not differ from the control, while vinegar addition indicated a reduction in gluten immunogenicity with a reduction of about 44 % in immunogenic gliadin content compared to the control. Results show a window of opportunity in the modulation of wheat bread formulation with reduced allergenicity, while maintaining the technofunctional properties.

Evidence of increased gluten-induced perturbations in the nucleophilic tone and detoxifying defences of intestinal epithelial cells impaired by gastric disfunction.

It has been increasingly demonstrated over the past few years that some proteolytically resistant gluten peptides may directly affect intestinal cell structure and functions by modulating pro-inflammatory gene expression and oxidative stress. The relationship between oxidative cell damage and Celiac Disease (CD) is supported by several studies on human intestinal epithelial cell lines, such as the Caco-2 cell model, already shown to be particularly sensitive to the pro-oxidative and pro-apoptotic properties of gluten protein digests. Through providing valuable evidence concerning some of the pathophysiological mechanisms that may be at play in gluten-related disorders, most of these in vitro studies have been employing simplified digestion schemes and intestinal cell systems that do not fully resemble mature enterocytes in terms of their characteristic tight junctions, microvilli and membrane transporters. Herein the peptide profile and pro-oxidative effect of two different gastrointestinal gliadin digestions was thoroughly characterized and comprehensively compared: one following the complete INFOGEST workflow and a second one by-passing gastric processing, to assess the dependence of gliadin-triggered downstream cell effects on pepsin activity. In both matrices, gluten-derived immunogenic peptide sequences were identified by non-targeted LC-MS/MS. Altogether, this study provides first-hand data concerning the still unexplored peptide composition, gastric-dependence and immunogenicity of physiologically representative gliadin protein digests as well as foundational clues stressing the need for more complex and integrated in vitro cell systems when modelling and exploiting gluten-induced perturbations in the nucleophilic tone and inflammatory status of intestinal epithelial cells.

Effect of different enzymes on thermal and structural properties of gluten, gliadin, and glutenin in triticale whole-wheat dough.

Three enzymes promoted the development of the gluten network in triticale whole-wheat noodles (TWWN). To further understand the mechanism of gluten enhancement, the effects of three enzymes on the structure of gluten and its fractions (gliadin and glutenin) were evaluated. The results showed that glucose oxidase (GOD), xylanase (XYL), and laccase (LAC) decreased the content of sodium dodecyl sulfate (SDS) extractable proteins. The content of glutenin subunits was reduced by 17.25 %, 30.60 %, and 20.09 % with the addition of GOD, XYL, and LAC, respectively. Furthermore, GOD and LAC increased the content of glutenin macropolymer (GMP) by 2.64 % and 7.71 %, respectively, suggesting the promotion of glutenin aggregation. The addition of three enzymes decreased the weight loss and increased the degradation temperature of the gluten and its fractions. GOD and XYL decreased the fluorescence intensity of gluten and its fractions, except for XYL which increased the fluorescence intensity of glutenin by 10.50 %. Intermolecular interactions and surface hydrophobicity were enhanced by XYL in gluten and its fractions. GOD and LAC decreased the free sulfhydryl content and increased the ß-sheet content, suggesting that the covalent interaction between gluten fractions was enhanced. Therefore, this research can enrich the theoretical study of enzymatic cross-linking.

Extraction, identification and application of gliadin from gluten: Impact of pH on physicochemical properties of unloaded- and lutein-loaded gliadin nanoparticles.

In the present study, high purity gliadin was extracted from gluten by the marginally modified Osborne method and the effect of different pHs in the aqueous ethanol on the physicochemical properties of unloaded gliadin nanoparticles (UGNs) and lutein-loaded gliadin nanoparticles (LGNs) was investigated. The results revealed that the formation of UGNs and LGNs at diverse pHs was driven by a conjunction of hydrogen bonding, electrostatic interactions and hydrophobic effects, but their dominant roles varied at different pHs. pH also significantly impacted the surface hydrophobicity, secondary structure and aromatic amino acid microenvironment of UGNs and LGNs. LGNs at pH 5.0 and at pH 9.0 exhibited better loading capacity and could reach 9.7884 ± 0.0006 % and 9.7360 ± 0.0017 %, respectively. These two samples also had greater photostability and thermal stability. Half-lives of LGNs at pH 5.0 were 2.185 h and 54.579 h, respectively. Half-lives of LGNs at pH 9.0 were 2.937 h and 49.159 h, respectively. LGNs at pH 5.0 and LGNs at pH 9.0 also had higher bioaccessibility of lutein, with 15.98 ± 0.04 % and 15.27 ± 0.03 %, respectively. These findings yielded precious inspirations for designing innovative lutein delivery system.

Proteomic Characterization of Wheat Protein Fractions Taken at Different Baking Conditions.

Food processing conditions affect the structure, solubility, and therefore accurate detection of gluten proteins. We investigated the influence of dough, bread, and pretzel making on the composition of different wheat protein fractions obtained by Osborne fractionation. The albumin/globulin, gliadin, and glutenin fractions from flour, dough, crispbread, bread, and pretzel were analyzed using RP-HPLC, SDS-PAGE, and untargeted nanoLC-MS/MS. This approach enabled an in-depth profiling of the fractionated proteomes and related compositional changes to processing conditions (mixing, heat, and alkali treatment). Overall, heat treatment demonstrated the most pronounced effect. Label-free quantitation revealed significant changes in the relative abundances of 82 proteins within the fractions of bread crumb and crust in comparison to flour. Certain gluten proteins showed shifts or reductions in particular fractions, indicating their incorporation into the gluten network through SS and non-SS cross-links. Other gluten proteins were enriched, suggesting their limited involvement in the gluten network formation.

Gliadin nanoparticles for oral administration of bioactives: Ex vivo and in vivo investigations.

This study aims to provide a thorough characterization of Brij O2-stabilized gliadin nanoparticles to be used for the potential oral administration of various compounds. Different techniques were used in order to evaluate their physico-chemical features and then in vivo studies in rats were performed for the investigation of their biodistribution and gastrointestinal transit profiles. The results showed that the gliadin nanoparticles accumulated in the mucus layer of the bowel mucosa and evidenced their ability to move along the digestive systems of the animals. The incubation of the nanosystems with Caenorhabditis elegans, used as an additional in vivo model, confirmed the intake of the particles and evidenced their presence along the entire gastrointestinal tract of these nematodes. The gliadin nanoparticles influenced neither the egg-laying activity of the worms nor their metabolism of lipids up to 10 µg/mL of nanoformulation. The systems decreased the content of the age-related lipofuscin pigment in the nematodes in a dose-dependent manner, demonstrating a certain antioxidant activity. Lastly, dihydroethidium staining showed the absence of oxidative stress upon incubation of the worms together with the formulations, confirming their safe profile. This data paves the way for the future application of the proposed nanosystems regarding the oral delivery of various bioactives.

One-Step Generation of O/W/O Double Pickering Emulsions Utilizing Biocompatible Gliadin/Ethyl Cellulose Complex Particles as the Exclusive Stabilizer.

Double emulsions hold great potential for various applications due to their compartmentalized internal structures. However, achieving their long-term physical stability remains a challenging task. Here, we present a simple one-step method for producing stable oil-in-water-in-oil (O/W/O) double emulsions using biocompatible gliadin/ethyl cellulose complex particles as the sole stabilizer. The resulting O/W/O systems serve as effective platforms for encapsulating enzymes and as templates for synthesizing porous microspheres. We investigated the impact of particle concentration and water fraction on the properties of Pickering O/W/O emulsions. Our results demonstrate that the number and volume of inner oil droplets increased proportionally with both the water fraction and particle concentration after a 60-day storage period. Moreover, the catalytic reaction rate of the encapsulated lipase within the double emulsion exhibited a significant acceleration, achieving a substrate conversion of 80.9% within 15 min. Remarkably, the encapsulated enzyme showed excellent recyclability, enabling up to 10 cycles of reuse. Additionally, by utilizing the O/W/O systems as templates, we successfully obtained porous microspheres whose size can be controlled by the outer water droplet. These findings have significant implications for the future design of Pickering complex emulsion-based systems, opening avenues for extensive applications in pharmaceuticals, food, cosmetics, material synthesis, and (bio)catalysis.

Effect of gliadin from Psathrostachys huashanica on dough rheological properties and biscuit quality.

Psathrostachys huashanica (P. huashanica), a wild relative of common wheat, is widely used in wheat variety improvement because of its many beneficial properties. In this study, we carried out preliminary analysis on the grain and flour quality of wheat-P. huashanica addition line 7182-6Ns and its wheat parents 7182, and found that 7182-6Ns had a higher protein content and great dough rheological characteristics and investigated the reasons for the changes. The results indicated that 7182-6Ns contained exogenous gliadin, which changed the gliadin composition and increased the ratio of gliadin in total gluten proteins, rebuilt gluten microstructure and thus optimized dough extensibility. As the addition of 7182-6Ns gliadin gradually increased to wheat flour, the diameter, crispness and spread rate of biscuit increased, the thickness and hardness decreased, and the colour improved. The current research provides a basis for understanding the introduction of exogenic gliadin to improve biscuit wheat varieties.

Specific Genetic Polymorphisms Contributing in Differential Binding of Gliadin Peptides to HLA-DQ and TCR to Elicit Immunogenicity in Celiac Disease.

Immunogenicity of gliadin peptides in celiac disease (CD) is majorly determined by the pattern of molecular interactions with HLA-DQ and T-cell receptors (TCR). Investigation of the interactions between immune-dominant gliadin peptides, DQ protein, and TCR are warranted to unravel the basis of immunogenicity and variability contributed by the genetic polymorphisms. Homology modeling of HLA and TCR done using Swiss Model and iTASSER, respectively. Molecular interactions of eight common deamidated immune-dominant gliadin with HLA-DQ allotypes and specific TCR gene pairs were evaluated. Docking of the three structures was performed with ClusPro2.0 and ProDiGY was used to predict binding energies. Effects of known allelic polymorphisms and reported susceptibility SNPs were predicted on protein-protein interactions. CD susceptible allele, HLA-DQ2.5 was shown to have considerable binding affinity to 33-mer gliadin (ΔG = - 13.9; Kd = 1.5E - 10) in the presence of TRAV26/TRBV7. Higher binding affinity was predicted (ΔG = - 14.3, Kd = 8.9E - 11) when TRBV28 was replaced with TRBV20 paired with TRAV4 suggesting its role in CD predisposition. SNP rs12722069 at HLA-DQ8 that codes Arg76α forms three H-bonds with Glu12 and two H-bonds with Asn13 of DQ2 restricted gliadin in the presence of TRAV8-3/TRBV6. None of the HLA-DQ polymorphisms was found to be in linkage disequilibrium with reported CD susceptibility markers. Haplotypic presentations of rs12722069-G, rs1130392-C, rs3188043-C and rs4193-A with CD reported SNPs were observed in sub-ethnic groups. Highly polymorphic sites of HLA alleles and TCR variable regions could be utilized for better risk prediction models in CD. Therapeutic strategies by identifying inhibitors or blockers targeting specific gliadin:HLA-DQ:TCR binding sites could be investigated.

Functional properties and formation mechanisms of dialdehyde polysaccharides crosslinked gliadin-films enforced by alkaline condition.

BACKGROUND: The usage of natural polysaccharides is attractive to researchers around the world. At the same time, non-/low-toxic crosslinkers prepared by polysaccharides are expected to fabricate protein-based films in many fields. Herein, different dialdehyde polysaccharides (DPs) were successfully synthesized and applied to prepare gliadin-films under alkaline conditions. The functional properties and formation mechanisms of the films were fully investigated. RESULTS: The results showed that the mechanical properties, water-resistant properties, thermal stability, and antibacterial properties of the gliadin-films were improved by DPs and alkali treatment. Particularly dialdehyde dextrin (DAD) crosslinked gliadin-films showed the highest tensile strength, but no additional effect on their elongation, or advancement on the other functional properties. The film-forming mechanisms indicated that Schiff base bonds, hydrophobic interactions, electrostatic interactions, and hydrogen bonds were the main forces in the films, supporting their improvement in functional properties. CONCLUSION: DPs, especially DAD, can be a promising crosslinker in fabricating gliadin-films. These findings have shown great promise to seek an effective crosslinker for fabricating gliadin/protein-based packaging. © 2023 Society of Chemical Industry.

Effects of glutenin and gliadin on the surface tackiness of frozen cooked noodles.

The mechanism of glutenin and gliadin on the surface tackiness of recooked frozen cooked noodles (FCNs) is unclear. In this study, the effects of glutenin and gliadin addition on the surface tackiness of FCNs were investigated. The addition of glutenin and gliadin reduced the surface tackiness (3.60 and 3.50 N) of recooked FCNs stored for 0 min. The addition of glutenin increased the rigidity of the gluten network and the compactness of FCNs and made the FCNs have a moisture-distribution with multilayers. The addition of gliadin increased the tensile distance of FCNs, restricted water migration during frozen storage, and increased the membranous structure of the gluten network to wrap starch particles. Glutenin had a stronger effect on reducing the surface tackiness of FCNs than gliadin. In the future, the synergistic effects of different proportions of glutenin and gliadin on the gluten network of FCNs could be further studied.

IDP Force Fields Applied to Model PPII-Rich 33-mer Gliadin Peptides.

The 33-mer gliadin peptide and its deamidated metabolite, 33-mer DGP, are the immunodominant peptides responsible for the adaptive immune response in celiac disease (CD). CD is a complex autoimmune chronic disorder triggered by gluten ingestion that affects the small intestine and affects ∼1% of the global population. The 33-mers are polyproline II-rich (PPII) and intrinsically disordered peptides (IDPs), whose structures remain elusive. We sampled the conformational ensembles of both 33-mer peptides via molecular dynamics simulations employing two force fields (FFs) (Amber ff03ws and Amber ff99SB-disp) specifically validated for other IDPs. Our results show that both FFs allow the extensive exploration of the conformational landscape, which was not possible with the standard FF GROMOS53A6 reported before. Clustering analysis of the trajectories showed that the five largest clusters (78-88% of the total structures) present elongated, semielongated, and curved conformations in both FFs. Large average radius of gyration and solvent-exposed surfaces characterized these structures. While the structures sampled are similar, the Amber ff99SB-disp trajectories explored folded conformations with a higher probability. In addition, PPII secondary structure was preserved throughout the trajectories (58-73%) together with a non-negligible content of ß structures (11-23%), in agreement with previous experimental results. This work represents the initial step in studying further the interaction of these peptides with other biologically relevant molecules, which could lead to finally disclose the molecular events that lead to CD.

Functional and structural properties of gliadin as influenced by pH, extraction protocols, and wheat cultivars.

Gliadin, owing to its low cost, ease to extract, high foaming capacity, easily available and high surface hydrophobicity, has found a wide range of applications both in the food and pharmaceutical sectors. The functional and structural characteristics of gliadin extracted with four extraction protocols from six wheat cultivars were investigated in this study. The surface-active properties of gliadin protein as a function of pH, extraction protocols, and wheat cultivars were compared, including solubility, zeta-potential, foaming properties, emulsion properties, surface hydrophobicity and secondary structure. Overall gliadin extracted using different extraction protocols and from different wheat cultivars was found to be higher in ß-turns (24.88-37.91 %), followed by ß-sheet (12.81-22.37 %), α-helix (15.13-20.70 %) and lower in random coil (6.53-9.08 %). Varied pH ranges, wheat cultivars, and different extraction protocols were found to have a substantial impact on solubility, zeta potential, foaming stability, emulsion capacity and surface hydrophobicity. The foaming capacity was observed to be more influenced by extraction protocols than wheat cultivars. Emulsion stability showed statistically significant (p ≤ 0.05) influence between the wheat cultivars, and a non-significant (p ≥ 0.05) difference among extraction protocols. The functional properties of freeze-dried gliadin extracted using different protocols were found to be pH-dependent. A comprehensive understanding of how the structural, surface active and functional properties of gliadin are influenced by the extraction protocols and wheat cultivars will enable us to understand the gliadin better and broaden its use for both food and non-food applications.