Title: Characterization of gliadin, secalin and hordein using advance analytical techniques

Prolamins extracted from wheat, rye and barley cultivars were analysed for colour characteristics, SDS-PAGE, Amino Acid, Dynamic Light Scattering (DLS), Zeta Potential, Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX), Transmission Electron Microscopy (TEM) and X-ray Diffraction (XRD) to elucidate the structure. Amino acid analysis showed signicant variation among the prolamins and the predominant essential amino acids were found to be leucine, phenylalanine and valine whereas predominant non-essential amino acids were glutamic acid, arginine and aspartic acid. All the prolamins exhibited positive zeta-potential, however gliadin had a signicantly higher zeta potential value. TEM studies of prolamins revealed the compact globular structure but gliadin also had rod-shaped structure. Morphology by SEM illustrated the globular particle arrangement in gliadin and sheet like arrangement in secalin and hordein. XRD pattern of prolamin showed the ordered crystalline domain of prolamin at 44.1°, 37.8° and 10.4°. The d-spacing obtained from XRD and TEM analysis also supports the crystalline domain of prolamin apart from amorphous domain. characterization of secalin via electrophoresis, chromatography, amino acid composition/sequencing and compared it with the reported values of gliadin and hordein. Ribeiro et al., 13 studied the polymorphism among storage protein of Portuguese rye cultivars and postulated that rye cultivars were good genetic resource for breeding purposes due to their effective gene traits such as nutritional prociency (especially dietary bre), disease tolerance, effective production with minimum pesticide and fertilizers application. The morphology and biochemical characterization of secalin and hordein have not been studied extensively. Recent advances have improved the molecular basis for better understanding of protein functionality. So, an attempt has been made to achieve structural, chemical and molecular characterization of prolamins by using a multi-disciplinary approach via protein peptide proling (SDS-PAGE), protein hydrodynamic size (DLS), net surface charge (Zeta-potential), structural morphology (SEM and TEM), elemental status (EDX) and internal molecular structure (XRD and TEM). were prepared in 50% acetonitrile containing 0.1% formic acid solvent and a drop of the diluted protein suspension solutions (15μl) was poured on a polycarbon film supported on a copper grid and dried at ambient temperature. The samples were examined on TEM at an accelerating voltage of 200 kV. The sizes of the protein nano-structures were obtained using iTEM software (Olympus, Münster, Germany). The radius of the diffraction rings in SAED pattern is the inverse of the d-spacing of the associated lattice planes and radii of the rings were determined using iTEM software. (9.84 – 14.05) indicates a profound tinge of its intrinsic color as compared to both secalin (6.95 – 10.33) and hordein (7.46 – 9.63). The analysis of data revealed that the C* values of different cereals prolamin varied signicantly (p ≤ 0.05) at inter and intra-cultivar levels except for hordein where the difference was non-signicant (p ≥ 0.05). 6.67 10.86 supports our ndings. The HMW group of prolamin is characterized glutamine, amino 33 . elasticity exibility of polypeptide is primarily HMW presence of cystine (involving disulphide linked aggregates) and glycine confers the greater conformational space in reference to its bond angle. HMW-group is accountable for unique dough elastic property which is favourable for superior baking performance 34,35 68.33 their from 99.60 99.90 and 0.40 in were in secalin from MCTLG-3 from MCTLG-4 of in peak and respectively. The PDI of to except shares S (0.12 – 0.26 %), P (0.37 – 0.61 %), Na (0.28 – 0.75 %) and I (0.25 – 0.54 %) expressed in atomic percentage. Similarly, the EDX analysis expressed in weight percentage showed the major elements as C (43.16 – 50.71 %), O (30.99 – 38.94 %) and N (9.46 – 14.80 %), while minor elements as S (0.25 – 0.63 %), P (0.84 – 1.31 %), Na (0.42 – 1.18 %) and I (1.97 – 4.56 %). The EDX analysis suggests that the peptide of gliadin consist of C, N, O with traces of S and P while the source of Na and I detected was possibly due to NaI salt used in the preparation. This implies that the relative ratio of C:O:N:S (weight percentage) in prolamin followed the ascending order, being higher in hordein (113:156:43:1), followed by secalin (110:76:21:1) and gliadin (77:48:24:1). It is noted that O is two times the amount of N in gliadin while 3.6 times in secalin and hordein.

Introduction was used to mill the conditioned grains to obtained flour having extraction rate of 72%, 68% and 60% for wheat, rye, and barley respectively. It was stored at -20 o C and thaws before analysis (25 o C for 2 h). All the chemicals used were of analytical grade.

Methods
Prolamin extraction. Prolamin was extracted from wheat, rye and barley by following 11 . Briefly, 1g of flour was taken in 10 mL extraction solution (0.3 M NaI solution containing 7.5% 1-propanol), shaken for 20 min and centrifuged (5810R, Eppendorf, Hamburg, Germany) for 10 min at 4500xg to collect the supernatant. Extraction process was repeated with the residues. The supernatants of both the fractions were pooled and transferred to round-bottom glass flask. Four times volume of 0.1 M ammonium acetate in methanol was added to the pooled supernatant, shaken thoroughly and kept at -20°C for 48 h to precipitate the prolamins. The solution was centrifuged at 3600xg (5810R, Eppendorf, Hamburg, Germany) to obtain the prolamin fraction as pallet. The precipitates were dissolved in 0.1 M acetic acid and also washed from the sides of the glass tube with 0.1 M acetic acid and freeze dried (HetoPowerDry, Allerod, Denmark). Samples were stored in airtight plastic vials at -20°C for further use.
Protein Determination. Protein content was measured in triplicate by the Kjeldahl method (N × 5.70) following American Association of Cereal Chemists (46-12.01) 14 .
Color of prolamin. The color of prolamin was measured using Hunter Colour lab (Hunter Associates Laboratory Inc., Reston, USA) by following 15 . The instrument was calibrated with black and white standard tiles and then samples (extracted prolamin) were measured using glass cuvette. CIE units consist of L* (lightness) ranges from 0 to 100, a* (+red, -green color) and b* (+yellow, -blue color) were recorded.
The hue angle (H o ) is a color appearance parameter which refers as quantitative attribute of color lies between an angle of 0° -360° with 0° representing red, 90° for yellow, 180° for green and 270° for blue. While, Chroma refers to intensity of color purity or color strength. It is expressed as either grey or the pure hue (red/yellow/green/ blue etc).
Hue and Chroma were calculated using the following Equations: Dynamic Light Scattering (DLS) Measurements. The hydrodynamic diameter (D h ) and zeta potential (ζ-potential) values of proteins were monitored on Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) equipped with a He-Ne laser (632. 8 nm, 4 mW), at back scattering angle of 173° to the incident beam and operated at 25 o C. The instrument monitors, time-dependent fluctuation in the light scattered by molecules present in solution to determine the diffusion rate due to the Brownian motion at a fixed scattering angle. The extracted prolamin(s) (1 mg ml -1 ) were prepared in acetonitrile: water: formic acid (50:50:0.1) solution. To avoid the formation of protein aggregates, the samples were vortexed gently and then filtered through 0.22 µm Millipore filters prior to measurements. An average of three measurements for each sample was considered as an experimental data.
SDS-PAGE of Prolamin. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was used to analyse the variability of extracted prolamin in terms of genetic diversity by adopting the method of 15 . 10mg of prolamin was thoroughly mixed with 1ml of 2x Laemmli sample buffer solution (pH 6.8 containing 62.5mM Tris-HCl, 25% glycerol, 5% ß-mercaptoethanol, 2% SDS, 0.01% bromophenol blue) in 1.5 ml Eppendorf tubes. The suspension was vortexes followed by horizontal shaking in orbital shaker operated (45 0 C) for 1 h at 151 rpm. Then after subjected to heating at 100 0 C for 5 mins followed by centrifugation (RC 4815S, Eltek, Mumbai, India) at 11000xg for 20 mins.
SDS-PAGE of prolamin was performed by loading 10μl of prepared sample supernatant in each well of 4% stacking gel and loaded proteins were resolved in 12% resolving gel at a constant current of 25 mA (Mini-Protean Tetra Cell, Bio-Rad Laboratories, Hercules, USA). When the tracking dye reached at the bottom of gel then gel was removed and stained overnight with straining solution which had a composition of 0.1% Coomassie Brilliant Blue-R250 in 40% methanol along with 10 % acetic acid. To remove stain, destaining solution was used which was prepared by dissolving 20% methanol and 10% acetic acid. The broad range molecular marker (GeNei, Bangalore, India) contains nine ladder Molecular Weight (MW) of myosin (205 kDa), phosphorylase B (97.4 kDa), bovine serum albumin (66.0 kDa), ovalbumin (44.0 kDa), carbonic anhydrase (29.00 kDa), soyabean trypsin inhibitor (20.10 kDa), lysozyme (14.30 kDa), aprotinin (6.50 kDa) and insulin (3.50 kDa) was used for estimation of MW of sample polypeptide. The quantification of destained gel was analysed by using Bio-Rad EZ imager (Bio-Rad Laboratories, Hercules, USA).
Classification of total prolamin was done according to 16 .The relative proportion of prolamin was calculated using band percentage corresponding to the total extractable proteins. SDS-PAGE gels were performed in duplicates.
Amino acid analysis of prolamin. Analysis of prolamin was performed by following the procedure of 15 with slight modification. Briefly, 5 mg of prolamin were hydrolysed in clean dried screw capped glass test tubes which were priorly dipped whole night in 2N HCl to avoid any sort of contamination. Hydrolysis was carried out using 6N HCl containing 0.1 % ß-mercaptoethanol in autoclave at 110 o C ± 2 o C for 16 h in screw tight capped closure test tubes to minimize the loss of digestion solvent during hydrolysis process. The filtrate was evaporated in a small round bottomed rotary flask under vacuum at 40 o C to dryness in a rotary evaporator (Buchi, Fawil, Switzerland). A suitable volume of 0.1 N HCl was added to each dried film of the hydrolyzed sample to dissolve all the soluble materials and then filtered through 0.22 µm filter paper (Millipore, Merck Life Science Private Limited, Mumbai, India). Amino acid analysis was performed using a Nexera Amino acid Analyzer (Shimazdu, Kyoto, Japan) equipped with a pre-column derivatization using three derivatizing reagents such as mercaptopropionic acid, o-phthaladehyde and 9-fluorenylmethyl chloroformate. A C-18 column (Acclaim Thermoscientific 120Å, 5 µm, 4.6 × 250 mm; Thermo Fisher Scientific, Waltham, USA) having pH stability 2-8 was used for chromatographic separation. Analysis was performed using 20 mmol/L phosphate (potassium) buffer (pH 6.5) as solvent A and 45/40/15 acetonitrile/methanol/water as solvent B. The separation was obtained at a flow rate of 1ml/min using a gradient elution that allowed 0 % B at 0.01 st min, followed by linear raise of eluent B to 50 % at 41 st min and then decreased to 0% B at 43 rd min. The temperature of the column oven was set at 40 o C and injection volume to 1 μL. Resolution monitoring of amino acid derivatives were performed through fluorescence detector with excitation and emission set at 330 nm and 450 nm respectively. Labsolutions LC/GC (Shimadzu, Kyoto, Japan) was used as a working station.
Amino Acid Score (AAS). The Recommended Dietary Allowances (RDA) of amino acid for children (Age: 6 months to 3 years) was used as reference protein for the calculation of amino acid score of each cereal.
Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Analyzer (EDX). The morphology of the isolated freeze-dried prolamin powder was studied with Carl Zeiss Supra-55 SEM (Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 10-15 kV. The samples were mounted on stubs using double-sided adhesive tape. Prior to SEM imaging, all samples were sputter-coated with gold for 2 min by Quorum Sputtered Coater (Q150R ES, Laughton, East Sussex, UK). Further, the elemental composition was analysed with Energy dispersive X-ray spectrometer (Oxford Instruments Nano Analysis & Asylum Research, High Wycombe, UK).
Transmission Electron Microscopy (TEM). The structures of different Prolamin were investigated using Transmission Electron Microscope (JEM-2100, JEOL TEM, Tokyo, Japan). The samples (1mg/ml) were prepared in 50% acetonitrile containing 0.1% formic acid solvent and a drop of the diluted protein suspension solutions (15μl) was poured on a polycarbon film supported on a copper grid and dried at ambient temperature. The samples were examined on TEM at an accelerating voltage of 200 kV. The sizes of the protein nano-structures were obtained using iTEM software (Olympus, Münster, Germany). The radius of the diffraction rings in SAED pattern is the inverse of the d-spacing of the associated lattice planes and radii of the rings were determined using iTEM software.

X-ray Diffraction (XRD).
XRD studies, in the 2θ (Bragg's angle) range of 10-60 o were performed using X-ray diffractometer (Shimadzu 7000, Shimadzu Corporation, Kyoto, Japan) in Bragg-Brentano geometry with Cu K α radiation (λ=1.5405 A o ). Some of the prolamins were found to be nano-crystalline in nature i.e. these are composed of crystallites or grains that are the ordered building blocks units repeated periodically and separated by disordered grain boundaries. Debye-Scherrer equation was used to calculate the particle crystallite size on corresponding diffraction pattern based on the dimension of full-width at half-maximum (FWHM) values.
Where, D is the mean size of the ordered (crystalline) domains, λ is the X-ray wavelength in nanometre (nm), β is the peak width of the diffraction peak profile at half of the maximum height (FWHM) in radians, θ is the Bragg angle, which can be in degrees or radians, cos θ corresponds to the same number and K is a constant related to crystallite shape, normally it can be taken as 0.89 or 0.9 for Full Width Half Maximum (FWHM) of spherical crystals with cubic unit cells 17 .
Further, inter-planar spacing (d), was calculated from the peak positions using Bragg's equation Where d is inter-planar spacing, n is order of diffraction.
Statistical analysis. The results were expressed as a mean±SD and compared statistically at p≤0.05, using one-way analysis of variance (ANOVA) with Tukey's post hoc test (Version 17, Minitab Inc., State College, USA).

Result And Discussion
The wheat, rye and barley ours were extracted to isolate the prolamins using 11 protocol. The cream-coloured gliadin, hordein and secalin were obtained on freeze drying the protein isolates ( Fig. 1).
Physiochemical characteristics. The protein content (PC) of the prolamin extracted from wheat, rye and barley followed a decreasing order as gliadin (64.94 -70.26 %), secalin (47.50 -60.70 %) and hordein (34.06 -45.17 %) respectively (Table S1). Among the cultivars, the gliadin from HPW-349 contained the highest PC while hordein from BH-959 contained the lowest PC. The PC of prolamins was found to vary signi cantly (p≤0.05). DuPont et al., 11 reported the protein content of extracted gliadin (40.1%) slightly lowered than our ndings might be due to varietal difference and/ or small-scale extraction process of gliadin.
The water activity (a w ) of the freeze dried prolamin was observed to vary from 0.66 to 0.82, with the highest value in secalin from MCTLG-2 and the lowest value in hordein from BH-902. These a w values of prolamin indicates the microbial and physiochemical stability, which consequently in uences the texture, rehydration and structural properties. The values of a w showed a signi cant (p≤0.05) difference among the cultivars. Variation in the protein content of prolamins may be attributed to the variation of amino acid composition, cereal genetic diversity, fertilizer application and agricultural practices.
The color characteristics of the prolamins are explained via CIE color values which have been recorded in terms of L*, a*, b*, Hue angle (H o ) and Chroma (C*) values. L* value indicates the lightness of the extracted prolamin which was found to be in the range 53.13-68.12 (Table S1). In the investigated cereals, the L* value for hordein from BH-902 was found to be signi cantly (p≤0.05) higher while for secalin from MCTLG-1 lies on lower side. The dark color (lower L*value) of the secalin might be due to the presence of pigment and oxidative changes as compared to hordein and gliadin. In case of coordinates a* and b*, the observed positive values for prolamin falls in the range 0.53 -2.06 and 6.90 -13.93 which implies color had red and yellow tinge respectively. For the investigated cultivars, the a* value was found to be the highest for gliadin (1.53 -2.06) and the lowest for hordein (0.53 -1.28). On the other hand, the high b* values for gliadin (9.68 -13.93) re ects more yellow tint in comparison to secalin (6.90 -10.13) and hordein (7.41 -9.61). On the whole, a signi cant (p≤0.05) difference in a* and b* values were observed among the prolamins at inter as well as at intra-cultivars levels.
Hue angle (H o ) of the extracted prolamin was found to lie in the range 78.51 -86.85 o for different cultivars. This angle represents the color variants on the colour wheel, 90 o represent yellow colour, therefore the colour of prolamins is more towards yellowish tinge. In the present study, a small difference was observed in hue angle among the prolamin. However, the H o was found to be the highest for hordein from BH-959 and the lowest for secalin from MCTLG-5. The chroma values (C*) of the investigated cereals were found to vary signi cantly (p≤0.05) from 6.95-14.05, which represents the intensity of particular color. The higher C* value, in case of gliadin (9.84 -14.05) indicates a profound tinge of its intrinsic color as compared to both secalin (6.95 -10.33) and hordein (7.46 -9.63). The analysis of data revealed that the C* values of different cereals prolamin varied signi cantly (p≤0.05) at inter and intra-cultivar levels except for hordein where the difference was non-signi cant (p≥0.05).
The amount of His was found to be higher in gliadin (2.34 -4.02 %), followed by hordein (1.45 -2.35 %) and secalin (1.38 -2.24 %). This implies that gliadin has high propensity towards stabilizing the insoluble proteins through π-π or π-cation interactions with aromatic groups than hordein and secalin 18  Ileu, Val and Leu which are mostly found in hydrophobic core and thus promote the folding process of protein more favourably than gliadin 18 . β-branched residues such as Thr, Val, Ileu are inclined to form β-sheet con guration, while linear chain residues such as Ala, Leu, Lys, Met, Glu have more propensity towards α-helix con gurations 19 . The total essential amino acids (TEAA) were higher in hordein (30.97 -32.97 %) and secalin (27.75 -33.28 %) while lower in gliadin (24.49 -27.07 %). It is noted that limiting EAA Lys in uences more to TEAA content.
Present results are in close proximity with the work conducted by 12 , who studied the amino acid content of prolamin from wheat, rye and barley except for slightly lower value of phenylalanine (0.92 -1.65 %), lysine (0.25 -1.36 %) while higher value of leucine (3.89 -5.27 %). Wang & Chen 20 reported similar observations on TEAA of gliadin and hordein except for slightly lower content of Lys and Ileu compared to our results. Interestingly, Trp gets destroyed by acid hydrolysis by traditional oven method 15 , although our results showed its presence which may be due to presence of thiol group (β-mercaptoethanol) and relatively mild conditions than traditional oven method 21 . The moist heat, under controlled pressure and relatively short duration could have prevented Trp destruction compared to dry harsh oven method.
Cys was found to be higher in secalin (1.65 -2.38 %), followed by hordein (1.75 -1.97 %) and gliadin (0.56 -0.94 %), this implies that intramolecular disulphide interaction might follow the respective order. Gly was found to be the highest in hordein (5.41 -6.34 %), followed by secalin (2.45 -4.54 %) and gliadin (2.45 -3.50 %), whereas Ala was found to be the highest in gliadin (6.68 -7.59 %), followed by hordein (4.23 -4.78 %) and secalin (3.48 -3.97 %). This implies that gliadin structure is more ordered and stabilized to compact con guration while hordein structure was speculated to be less ordered structure to random coiled con guration. The ordered structure of protein was strengthened by Ala and Glu (α-helix former) while weaken by Pro and Gly (α-helix breaker or β-sheet breaker) 22 . The Tyr residue was found to be higher in hordein (3.39 -5.19 %) followed by gliadin (2.71 -3.56 %) and secalin (1.33 -2.97 %), whereas Arg content was found to be the lowest in gliadin (2.95 -3.62 %) while comparable in secalin (5.84 -7.74 %) and hordein (6.36 -7.12 %). This means that in Tyr-Tyr covalent interactions, the secalin was comparatively less active than hordein and gliadin. Hou et al., 18 suggested that the insolubilizing interactions has been facilitated by Asp, Glu, Arg and aromatic residues (Phe, Tyr, Trp, His). The role of Tyr-Tyr bond in stabilizing the toxic repetitive moiety of 33-mer gliadin was reported by 23 12 reported more serine and threonine in hordein than gliadin and secalin which are similar to our nding, while ala was high in gliadin and secalin than hordein. However, our ndings show higher content of ala in gliadin and relatively comparable in secalin and hordein. Gellrich et al., 25 analysed secalin for amino acid and reported similar results except lower value for Arg, Lys and higher content of pro (20.4%) in comparison to current ndings. The variation in the amino acid pro le in extracted prolamin with the previous ndings might be due to in uence of extraction procedure and to some extent varietal differences.
The amino acid score (AAS) of prolamin (Table S2), indicates the superiority of protein corresponding to reference protein 26 . The AAS determined was found to be higher in hordein (92.02 -97.97 %) followed by secalin (82.48 -98.91 %) while lower in gliadin (72.77 -80.46 %). It implies that the protein pro le of secalin and hordein was relatively superior to gliadin. Similarly many authors reveals the higher biological value and superiority of rye and barley protein 27,28 . The AAS of the prolamin of rye cultivar MCTLG-3 (98.91 %) was the highest whereas gliadin of wheat cultivar HPW-249 (72.77 %) received the lowest score. Lysine, methionine and leucine contents of the extracted prolamin were found below the reference protein 26 . Similar results have been reported by 24 for spring barley cultivar which contained underneath levels of lysine and methionine in comparison to reference protein.
The International ImMunoGeneTics (IMGT) system of information categories the amino acids into three 'hydropathy' groups (Table 1) namely hydrophilic (Lys, Arg, Glu and Asp), hydrophobic (Phe, Trp, Ileu, Leu, Met, Cys, Ala, and Val) and neutral (Tyr, Pro, Gly, Thr, Ser and His) 29 . Out of total amino acids, the hydrophilic amino acids group constituted about 47.35 -52.10 % in secalin, 45.31 -50.02 % in gliadin, and 43.22 -46.34 % in hordein. This implies that secalin contains more amount of soluble protein while hordein contains the least. The highest hydrophilic amino acids content was registered by secalin from MCTLG-5 and the lowest by hordein from BH-393 cultivar. The hydrophilic amino acids of prolamin were further sub categorized into basic and acidic amino acids, which accounts for 6.16 -15.08 % and 30.23 -43.65 % respectively. The acidic AA residues were high in gliadin (39.15 -43.65 %) and low in hordein (30.23 -31.74 %) whereas basic AA residues were high in hordein (11.48 -15.08 %) and low in gliadin (6. 16 -8.42 %). This implies that gliadin has relatively higher solubility than secalin and hordein, due to higher content of acidic AA residues. This fact is con rmed by the ndings of 18 , that Asp/Glu acid residues promotes the protein solubility through the formation of salt bridges by electrostatic interaction of Lys-Asp, Lys-Glu, Arg-Glu and Arg-Asp. On the other hand, acidic AA favours protein insolubility and aggregation when participate in anion-π interaction with aromatic residues. Acidic and basic amino acid fractions of extracted prolamin have shown a signi cant (p≤0.05) difference among intra and inter cultivars levels.
The hydrophobic AA constituted 26.89 -30.86 % and comprised of aliphatic, S-containing and some aromatic amino acids which accounted for 16.30 -18.90 %, 1.52 -3.39 % and 7.35 -10.04 % respectively. The S-containing amino acids found to be higher in secalin (2.97 -3.39 %) and hordein (2.79 -3.30 %) while lower in gliadin (1.52 -2.18 %). This implies that, due to greater potential for covalent interaction, secalin achieves more symmetry in its protein structure. The S-containing amino acids content of secalin from MCTLG-3 and gliadin from HPW-155 were the highest and the lowest respectively.
The total neutral amino acids of prolamin varied from 19.9 1-28.55 % and constituted of non-polar, polar and aromatic amino acids which accounted for 8.70 -15.73 %, 6.46 -10.44 % and 1.33 -5.19 % respectively. The decreasing trend in polar AA found to be higher in hordein (8.60 -10.44 %) followed by secalin (8.95 -9.88 %) and gliadin (6.63 -7.64 %), while in non-polar group the decreasing trend follows as higher in gliadin (12.08 -15.73 %), followed by hordein (11.32 -14.01 %) and secalin (8.70 -12.28 %). This implies that the higher content of polar AA residues in hordein and secalin have great propensity towards solubility of proteins than gliadin, while gliadin was noticed to have higher relative proportion of non-polar AA which actively take part in protein stability and reduces the solubility of protein. Hordein from BH-393 and gliadin from HPW-147 accounted for the highest and the lowest content of polar amino acids respectively. However, the neutral aromatic group of amino acid was found to be highest in BH-959 and lowest in MCTLG-5secalin.This implies that Tyr-Tyr covalent interactions speculated to be more in hordein which attributed to greater aggregation tendency than gliadin and secalin 23 .
The polar, neutral aromatic and s-containing group of amino acids observed to varied signi cantly (p≤0.05) whereas aliphatic, non-polar, hydrophobic aromatic, total hydrophobic, total hydrophilic and total neutral amino acids varied non-signi cantly (p≥0.05) among different extracted prolamin at inter and intra cultivar level. The variation in amino acid composition is considerably affected by irrigation practices, fertilizer application, agro-climatic conditions, and genotype.
Polypeptide characteristics. The gliadin, secalin and hordein were analysed for their electrophoretic pattern on SDS-PAGE (Fig. 3). The electrograms were categorized broadly into storage protein and metabolically active protein. Schalk et al., 16 sub-categorized the prolamins as HMW-GS, ω-gliadin, LMW-GS, α/β-gliadin and γ-gliadin in wheat cultivars; D-hordein, C-hordein and γ/B-hordein in barley cultivars and HMW-secalin, 75K secalin, ω-secalin and 40K secalin in rye cultivars. Additionally, Lexhaller et al., 30 broadly grouped the gliadin, secalin and hordein into high molecular weight (HMW), medium molecular weight (MMW) and low molecular weight (LMW) peptides groups on the basis of molecular weight, amino acid characteristics (sequence and composition) and their participation in band formation.  (Table S3) showed the relative proportion of HMW group accounted to 1.43 -8.34 % and varied non-signi cantly (p≥0.05) among gliadin and secalin at inter and intra cultivar levels, whereas in hordein a signi cant difference (p≤0.05) was observed in inter and intra cultivar levels. Anjum et al., 31 reported similar ndings that wheat contained 3-5 HMW-GS subunits, while 32 revealed the presence of 1-5 HMW secalin subunits with the relative proportion of HMW group between 6.67 -10.86 % which supports our ndings. The HMW group of prolamin is characterized by large amount of glutamine, glycine and proline amino acid 33 . The elasticity and exibility of polypeptide chain is primarily governed by HMW group due to presence of cystine (involving disulphide linked aggregates) and glycine confers the greater conformational space in reference to its bond angle. HMWgroup is accountable for unique dough elastic property which is favourable for superior baking performance 34 (Table S3), ranged between 6.32 -28.77 % of total extracted prolamin. The ω-gliadin (20.74 -28.73 %); C-hordein (13.43 -23.40 % (except in BH-959 with 28.77 %) and ω-secalin (6.32 -8.68 %) had MMW group of prolamins in decreasing order. The highest relative proportion of MMW group among different cereal extracted prolamin was shown in hordein from BH-959 (28.77%) while the lowest was observed in secalin from MCTLG-5 (6.32%). A signi cant (p≤0.05) difference was observed among different extracted prolamins. MMW group predominantly contains glutamic acid, proline and phenylalanine, which is relatively richer in polar and acidic amino acids while lower in basic amino acids. Therefore, MMW group had less surface hydrophobicity than LMW group 36,37 . The distinctive feature of MMW group of protein is the absence of cysteine amino acid residues which are usually involved in disulphide bonding among proteins in three dimensional gluten network 38 . MMW group mainly occupies the central domain of prolamin which mainly consists of repetitive motifs of glutamine, prolamin and phenylalanine, which tends to form extended non-compact structures due to lack of cysteine and acquires random coiled β-turn con guration 36,39 .
According to 30 , the LMW group covers the monomeric peptides (identi ed as α/β-gliadins, γ-gliadins in wheat; γ-40k-secalins in rye and γ-hordeins in barley) and polymeric peptide (recognized as LMW-GS in wheat, γ-75k-secalins in rye and B-hordeins in barley). The LMW peptides ranged from 28-35 kDa except in γ-75k-secalins (MW~50 kDa). This group of prolamin is sulphur rich due to presence of cysteine in hydrophobic part of C-terminal domain, while N-terminal domain have glutamine, proline and aromatic (especially phenylalanine, tyrosine) amino acid residues 33 . It leads to a compact globular structure due to formation of intra-molecular covalent disulphide bridge between two cysteine molecules by oxidation of its thiol group. Therefore, threedimensional structure of protein usually contains helical and beta sheet con guration by active participation of hydrophobic interactions which provides characteristic viscous property and extensibility to dough and improve bread loaf volume 36 Table S3. Petrovičová et al., 32 reported slightly higher values for LMW group of secalin than present study. The highest relative proportion of LMW group was revealed in secalin from MCTLG-3 (45.86 %) and the lowest in hordein from BH-946 (12.18 %). The LMW group of rye secalin comprises of γ-75k-secalins and γ-40k-secalins with relative proportion of 26.53 -31.96 % and 13.39 -14.82 % respectively. The γ-75k-secalins were obtained in high concentration in rye cultivar MCTLG-3 and in low concentration in rye cultivar MCTLG-5 whereas the highest concentration of γ-40k-secalins was attained in rye cultivar MCTLG-2 and the lowest in MCTLG-1.
Schalk et al., 16 reported 80-120 kDa for HMW group, 43-68 kDa for MMW group and 32-45 kDa for LMW group of gliadins, whereas 100 kDa for Dhordein, 45-65 kDa for C-hordein and 32-40 kDa for B-hordein. Socha et al., 40 showed the MW of 30-45 kDa for α/β, γ-gliadins in wheat while, 37 kDa for 40k-γ-secalins and 66.2 kDa for 75k-γ-secalins in rye ours respectively. Field et al., 12 also reported the MW of 40k-γ-secalins and 75k-γ-secalins as 33 kDa and 54 kDa respectively, while 41 also reported the MW of secalin as 35 kDa and 66 kDa, which con rms our ndings. The slight variation in MW among present study with that of previously studied MW might be in uenced by genetic makeup, agro-climatic differences and protocol employed for extraction of prolamin.
The extracted prolamin via DuPont extracted protocol were found to be contaminated with metabolic active protein having molecular weight ranges between 9.30-24.19 kDa, 6.50-23.93 kDa and 7.21-25.38 kDa for gliadin, secalin and hordein respectively. Densitometric evaluation (Table S3) reveals the relative proportion of ALB+GLO group were varied between 38.13 -69.10 % wherein a more diversity has been observed in hordein (38.13 -69.10 %), followed by secalin (43.51 -51.93 %) and least in gliadin (45.00 -53.59 %). A signi cant difference was observed in ALB+GLO group including intracultivar differences. Petrovičová et al., 32 reported that the relative share of ALB+GLO was estimated to be 20.97 -31.02 % of total protein from rye cultivars which is slightly less than our ndings. The variation in the relative proportion in ALB+GLO between present studies and reported values in the literature might be due to different extraction procedure employed.
Genetic proximity of various accessions in the present study was further evaluated through Jaccard Similarity Matrix by means of Unweighted Pair Group Method with Arithmetic Averages (UPGMA) to construct a dendrogram (Fig. S2). Three distinct groups could be identi ed from the dendrogram of gliadin, secalin and hordein, moreover, gliadin and hordein were closer to each other than secalin. In case of wheat gliadin, group-I had two accessions where HPW-42 was found to be dissimilar from rest of the other extracted gliadins with similarity coe cient of 0.58; Group-II had two accessions of closely This variation among cereals might be due to dissimilarity in genotypic diversity, inherent surface amino acid pro le, interaction with extraction solvents as well as protocols working conditions. Particle Characteristics. The prolamins exist as three-dimension structured particle. This microstructure was analysed using sophisticated instruments to elucidate its properties.
Dynamic Light Scattering (DLS). The prolamins were analysed for particle size distribution (PSD) in terms of polydispersity index (PDI) and hydrodynamic diameter (D h ) in solution as presented in Table 2, Fig. S3. PDI values for prolamins fall in the range 0.49 Wang, Y. & Chen 0.87. Lower PDI value i.e. less than 0.5 indicates more uniform PSD and existence of monodisperse system 42 . In the present study, PDI of the secalin (0.66 -0.87), hordein (0.58 -0.83) and gliadin (0.49 -0.64) were higher indicating disparity in particle size.
The gliadin and hordein mainly existed in monomeric form with hydrodynamic diameter (D h ) in the ranges between 1.23 -1.83 nm and 6.67 -7.62 nm respectively. The hordein from BH-946 (7.62 nm) had higher and gliadin from HPW-42 (1.23nm) had lower hydrodynamic diameter. Gliadin showed monomeric units nonetheless hordein shows the existence of some higher ordered aggregates. This fact is also supported by amino acid composition that the higher proportion of basic AA and S-containing AA residues in hordein shows its tendency in formation of aggregates through strong electrostatic and hydrostatic interaction 9 .
The secalin showed a bimodal PSD with two populations in the solution. The particle size of small and large sized population varied from 7.73 -18.44 nm and 26.48 -68.33 nm, whereas, their proportion varied from 99.60 -99.90 % and 0.10 -0.40 % respectively. It indicated that the small sized particles were abundant in number while large sized particle were scanty. The large sized particles were observed in secalin from MCTLG-3 (18.44 nm; 68.33 nm) and the small sized from MCTLG-4 of 7.73 nm; 26.48 nm in peak 1 and 2 respectively. The PDI and D h of the investigated prolamin have been found to vary signi cantly (p≤0.05) among different extracted prolamin samples including intra-cultivar differences except wheat cultivars which shares non-signi cant differences (p≥0.05).
The zeta potential (ZP) pro le of the gliadin, secalin and hordein have shown a positive value in the range of 23.53-27.00 mV, 11.23-16.60 mV and 4.10-7.98 mV respectively. The ZP values were found to be signi cantly (p≤0.05) different at inter as well as intra-cultivar levels. The relatively higher zeta potential values of gliadin indicate the high stability of their monomeric protein subunits. Gliadin and hordein had a monomodal while secalin had a bimodal PSD. The secalin had relatively lower ZP than gliadin which might be due to its bimodal PSD and large particle size of the minor component. On the other hand, the substantially lower ZP values for hordein re ected the greater tendency of protein motifs to aggregates due to lower surface charge.
The constituent protein units predominantly exist in higher ordered form in secalin and hordein compared to gliadin, which was correlated by PDI value.
The low ZP in case of hordein is probably due to its high basic amino acid contents which might be involved in π-interaction and increase aggregation propensities 18 .
Zavareze et al., 43 has reported that zeta-potential of gliadin was about 30mV and had remarkable colloidal stability. However, gliadin nano-particles prepared by controlled aggregation had D h , PDI and ZP of 190 -220.6 nm, 0.067 -0.232 and 14.8 -18.3 mV respectively 44 . Peng et al., 45 has also demonstrated the high zeta potential which indicated greater electrostatic repulsions among the protein molecules and poses higher electrostatic barrier which prevents protein aggregation. The overall zeta-potential of gliadin, secalin and hordein was observed to be in decreasing order. It further indicated the solution stability of these proteins was also in the same order. The higher colloidal stability of gliadin suspension is attributed to the high glutamine content which is engaged in hydrogen bonding with water 46 . The variation in zeta potential between inter and intra cultivar of present and earlier studies might be due to difference of dispersion media, amino acid side chains, pH, ionic strength of solution and temperature during extraction/analysis process 43,46 .
SEM and EDX. Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Analyzer (EDX) were used to investigate the morphology and elemental composition of prolamin respectively as elucidated in Fig. 4 and Table S4. Morphology by SEM illustrated the globular particle arrangement of gliadin while sheet-like and stacked aky structure was observed for secalin and hordein respectively. Similar globular particle arrangement has been reported in gliadin nanoparticles 5 which seems consistent with our ndings. Due to irregular pattern found in SEM images of gliadin, the size of observed morphology could not be quanti ed. and hordein (BH-946). Jia et al. 47 also reported crystalline peaks in XRD analysis of gliadin, however these peaks were at different position.
The inter-planar spacing, 'd', was calculated from the peak positions using Bragg's equation (  9 reported that α-gliadin and LMW-GS showed a similar secondary structure propensity in the absence of intra-molecular disulphide bonds with a relatively uniform distribution of α-helix and/or β-sheet whereas when intra-molecular disul de bonds are involved there is an increased tendency for β-sheet/strand for the amino acid involved in these speci c bonds. It can be inferred from the previous study that the prolamins under investigation also had α-helix and/or β-strand structures. Transmission Electron Microscopy. Transmission Electron Microscopy was used to study the morphology and microstructure of prolamins. The micrograph was analysis for morphology (Fig. 6), lattice planes (Fig.7) and ring diffraction patterns of selected area (Fig. 8). Morphology of gliadin, secalin and hordein showed a compact spherical structure (Fig 6) which might be due to active participation of α/β-gliadins polypeptides 36 . A rod like structure was also observed in gliadin due to folding of a sheet with an average diameter of 17.94 nm and length of 250 nm. Formation of such structures is attributed to involvement of ω and γ-gliadin polypeptides 36 . Ang et al., 49 also supported the existence of two structures of gliadin as compact globular and rod-shaped. The histogram representing the particle size distribution of compact globular structures in prolamin and rod structure of gliadin (Fig. S5). The average particle size was found to be smallest for gliadin (3.88nm), followed by hordein (4.32 nm) and largest in secalin (5.79nm).
The inter-planar spacing calculated from HR-TEM images are presented in Fig. (7 and 8), shows the SAED patterns of the gliadin, secalin and hordein. Two kinds of patterns were observed for different samples, one consisting of sharp, concentric rings and each ring corresponds to speci c lattice plane, second diffused rings which indicate lesser orderly arrangement of molecules i.e. poor crystallinity. The patterns show the gliadin and secalin possess higher crystallinity as compared to hordein and this observation is consistent with that made from XRD patterns of the samples. The reciprocal of the radius of each ring in the SAED ring pattern is a measure of inter-planar spacing corresponding to speci c lattice planes. Comparison between advance analytical techniques. Particle size of the prolamins observed for gliadin (1.12 -6.06 nm), secalin (3.23 -9.47 nm) and hordein (2.13 -6.83 nm) using TEM whereas for gliadin (1.23 -1.83 nm), secalin (6.45 -11.96 nm) and hordein (6.67 -7.62 nm) using DLS. Particle size of gliadin was on the higher side in TEM but in case of secalin and hordein, it was on the higher side in DLS. However, the particle size range overlapped in all the three prolamins.
The crystalline microstructure of prolamin was shown by both XRD and TEM studies. The d-spacing was estimated from HR-TEM lattice plane pattern, Selected Area Electron Diffraction (SAED) ring diffraction pattern and XRD data. The d-spacing observed by HR-TEM, SAED  respectively. HR-TEM and SAED gave one value of d-spacing whereas XRD has given three values of d-spacing. It indicates that there are three types of crystalline structure in the prolamins as per XRD and one crystalline structure as per TEM analysis. Further the crystalline pattern showed that gliadin and secalin possessed relatively high ordered crystallinity than hordein.
These periodic regular arrangements probably attributed to the subsistence of β-sheet structure among the prolamin and disulphide bonding as a result of intra-molecular interaction which mainly involves cysteine residues in polypeptide chains 50 . Markgren et al., 9 highlighted the role of cysteine amino acid in intra-molecular disulphide bonding, which when located in more hydrophobic motif and stated that the intra-molecular interaction enhances the tendency of formation of more ordered β-sheet con guration. Similarly, 51 also reported the crystalline structure of proteins by XRD studies and attributed it to the βsheet structure. Rasheed et al., 52 have also reported that the modi ed gliadin with nano-crystalline structure consists of high proportion of β-sheet structure along with irreversible linkages such as covalent disulphide bonds.

Conclusion
The gliadin, secalin and hordein were extracted following 11 method and examined using advance techniques. Gliadin has more protein content than secalin and hordein. The amino acid composition reveals that prolamin is abundant in glutamic acid and proline content among the non-essential amino acids while isoleucine and phenylalanine among the essential amino acids. Glutamic acid and proline sequence triggers the autoimmune response resulting in celiac disease. Tryptophan remained intact in the protocol due to mild degradation conditions and presence of mercaptoethanol. The extraction method was able to exclude LMW-GS in all the prolamin which was the major problem in other extraction protocols. SEM images shows globular morphology of gliadin while sheet like or stacked aky morphology of hordein and secalin. Prolamin sizes determined by TEM and DLS were in close proximity. Results indicated that prolamin had both amorphous as well as crystalline structure.

Declarations
Con ict of interests None Tables   Table 1. IMGT (ImMunoGeneTics) Amino Acid Classification (Pommie et al.,2004) of different cultivars of gliadin, secalin and hordein (g amino acid/100 g protein). Mean±SD with different superscripts in column differ significantly (p ≤ 0.05); n = 3 for each treatment.