The composition and extractability of thermo-molded wheat gluten bioplastics

The study was aimed to evaluate the effect of thermo-molding on gluten and gluten fractions composition and its extractability with 1.0 ml 0.05 M sodium phosphate buffer (pH 6.8) with 2.0% sodium dodecyl sulfate (SDS) at 130 and 150°C for 5 and 25 min. The gluten, gliadin and glutenin composition as affected by thermo-molding was analysed with reverse phase high performance liquid chromatography (RP-HPLC) and amino acid was analysed with acid hydrolysis using high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD). The extractability and composition were significantly affected. Gluten extracts were found in equivalent proportions; gliadin (51%) and glutenins (49%). Gluten, gliadin and glutenin subunits (GS) extracts were significantly affected and a reduction with increased molding condition explains the occurrence of polymerization reactions due to SH-disulfide interchange reactions. The glutenin fraction contains 15% gliadin while the gliadin fraction contains 2.5% glutenin after extraction with 60% ethanol. The extractability of the gliadin types and GS decreased with increasing molding time and temperature.


INTRODUCTION
Wheat is among the most important staple food crops in the world with different cultivars classified according to physical hardness, agronomic properties, color of the kernel and end-use characteristics.Wheat proteins are separated by the Osborne fractionation via successive extractions with solvents (Delcour and Hoseney, 2010;Veraverbeke and Delcour, 2002).Gliadins and glutenins are functional gluten proteins in wheat found in more or less equal amounts (Goesaert et al., 2005;Belitz et al., 2009).In contrast to non-gluten proteins, gluten proteins are poorly soluble in water or dilute salt solutions due to low amino acids content with ionizable side chains and high contents of non-polar amino acids.Cysteine, a minor amino acid in glutens is extremely important for its structure and functionality in disulfide bonds within a protein or between different proteins (Wieser, 2007).Glutenins,a heterogeneous mixture of disulfidelinked polymers (80,000 -Millions in MW) of GS is insoluble in alcohol.Glutenins are categorized into; (1) HMW-GS (x-and y-types); a minor component (10%) of the total weight with three structural domains of N-and E-mail: abifish3@gmail.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License C-terminal and more than four cysteine residue of repetitive sequence of proline, glutamine and glycine (Delcour and Hoseney, 2010;Wieser, 2007;Veraverbeke and Delcour, 2002).
Gliadins represent a heterogeneous mixture of singlechained proteins (30,000-80,000 in MW) soluble in alcoholic media.Based on their mobility at low pH in gel electrophoresis, three gliadin groups that is, α-, γ-and ωwere identified (Wieser, 2007).The ω-Gliadins have a MW (39,000 to 55,000) composed of high level of glutamine, proline and phenylalanine, low level of methionine but lacked cysteine residues.The α-and γgliadins overlaps inMW (28,000 to 35,000) with lesser proline, glutamine than the ω-gliadins and increased level of cysteine residues in the C-terminal domain.The Cterminal domain contains non-repetitive sequences of glutamine and proline than the N-terminal domain (Wieser, 2007;Veraverbeke and Delcour, 2002).In general the α-and γ-gliadins exist in higher and equivalent proportion than ω-gliadins (Wieser, 2007).The N-terminal domain constitutes 40 to 50% of the protein with repetitive sequences of glutamine, proline, phenylalanine and tyrosine.
Proteins are cross-linked by covalent bonds between polypeptide chains within a single protein or between different proteins important for structural and functional properties.The protein cross links are either naturally occuring or formed during processing (Gerrard, 2002).Disulfide bonds between cysteine residues are the dominant crosslinks formed by oxidative coupling of two cysteine residues adjacent a protein matrix in the presence of an oxidant which accepts hydrogen atoms from the thiol groups (Veraverbeke and Delcour, 2002;Gerrard, 2002;Shewry and Tatham, 1997).An intramolecular disulfide bond can be converted into an intermolecular disulfide bond by a sulfhydryl (SH)disulfide interchange reaction.Other cross-links include isopeptide bonds, dehydroprotein, Maillard reaction and tyrosine derived during thermal processing; chemical treatment in the formation of covalent bonds from the cysteine and lysine residue (Lagrain et al., 2010;Rombouts et al., 2009;Hanft and Koehler, 2005;Gerrard, 2002;Tilley et al., 2001;Singh, 1991).
The MW of gluten aggregates and protein extractability is dependent on the heating condition, hydrostatic pressure and moisture content attributed to oxidation of SH groups and SH-disulfide interchange reactions which forms intermolecular bonds (Kieffer et al., 2007;Lagrain et al., 2005;Singh and MacRitchie, 2004;Weegels et al., 1994).The formation or cleavage of disulfide bonds is stimulated by the application of redox agents (potassium bromate, potassium iodate and reducing agent dithiothreitol [DTT]) on the gluten proteins.
During hydrothermal treatment, protein extractability was higher in the presence of oxidants thus suggesting a decreased cross-linking which hinders gluten polymerization.In the presence of reducing agents (DTT), protein extractability was lower suggesting increased crosslinking which promotes gluten polymerization due to increased levels of free SH groups and less flexibility of glutenin chains (Lagrain et al., 2008(Lagrain et al., , 2007(Lagrain et al., , 2005;;Guerrieri et al., 1996).

MATERIALS AND METHODS
Wheat gluten was fractionated into gliadin and glutenin with 60% ethanol and protein composition was analysed with RP-HPLC in the presence of redox agents (SDS) and thermo-molded at 130 and 150°C for 5 and 25 min in a Pinette Press Zenith 2 (Pinette Emidecau Industries, Chalon sur Saône, France) at 5 bars after conditioning for 2 days at 50% relative humidity and 20°C (the moisture was adjusted to 7%).Hydrophilic molecules elute first, followed by hydrophobic molecules which interact stronger with the column (Figure 1).Amino acid analysis was performed after acid hydrolysis of high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD) as described by Rombouts et al. (2009).

Gluten composition and extractability
Thermomolded gluten and gluten fractions composition and extractability with 1.0 ml 0.05 M sodium phosphate buffer (pH 6.8) and 2.0% sodium dodecyl sulfate (SDS) are presented in Tables 1 to 4. The gliadin (51%) and The standard deviation is given in brackets.
Table 3.The extractability and composition of gliadin protein before and after thermo-molding at 130 and 150 °C for 5 and 25 min analysed by reverse phase-high performance liquid chromatography (RP-HPLC) expressed in percentage.The standard deviation is given in brackets.

Glutenin composition and extractability
Upon extraction with 60% ethanol, the glutenin rich fractions contained 15% gliadins.The proportion of gliadin in the glutenin fractions indicates that the alcohol strength for the fractionation of gluten was not enough to separate the pure fractions of the gliadin and glutenins (Abrehet, 2015).The extractability of the GS decreases with increased molding condition which explains the occurrence of cross links (Abrehet, 2015).The distribution of ω-α-and γ-gliadins in the 15% gliadin of the glutenin enriched fractions was 2, 7 and 5% (Wieser, 2007), respectively, which further decreased with thermomolding at 130 and 150°C in 5 and 25 min.GS extractability in the glutenin fraction was found to be 4, 23 and 57% for the D-LMW-GS, HMW-GS and B/C-LMW-GS, respectively (Delcour and Hoseney, 2010;Wieser, 2007;Veraverbeke and Delcour, 2002).With thermomolding at 130 and 150°C in 5 and 25 min was a reduction and distribution in D-LMW-GS and HMW-GS as protein polymerization occurred due to the repetitive sequence of cysteine residue (Kieffer et al., 2007;Lagrain et al., 2005;Singh and MacRitchie, 2004;Weegels et al., 1994).There was increased extractabililty of B/C-LMW-GS with thermo-molding as the repetitive amino acid chains were broken down due to the thermal effects.

Gliadin composition and extractability
The gliadin fractions composed of 2.5% glutenin with higher proportion of B/C-LMW-GS while the D-LMW-GS and HMW-GS excites at lower concentration.The GS (B/C-LMW-GS, D-LMW-GS and HMW-GS) extractability was significantly affected with increased thermo-molding due to its hydrophobicity property similar to ω-gliadins in the gliadin enriched fractions.97% of the gliadin fraction contained α-gliadin (46%) and γ-gliadin (37%) susceptible to covalent incorporation in the glutenins network formation as they are rich sources of cysteine residues than ωgliadin (14.5%).Extractability was significantly reduced by more than 50% after molding at 130°C for 5 min due to the incorporation of gliadins in network formation and occurrence of non-disulfide bonds (Wieser, 2007;Veraverbeke and Delcour, 2002).The increased molding condition diminishes the extraction even though ω-gliadin exists in lesser amount and is unextractable with the application of thermo-molding than αand γgliadins.

Amino acid composition
The three amino acids (cysteine, lysine and lanthionine (LAN)) were significantly affected by thermo-molding at 130 and 150°C for 5 and 25 min (Table 4).The reduction in cysteine and lysine amino acids with increased molding condition was due to sulfhydryl-disulphide interchange, oxidation and β-elimination reactions.LAN was undetectable after thermomolding at 130°C for 5 and 25 min for gluten and gliadin fraction and was detectable during thermomolding at 130°C for 25 min due to the occurence of repetitive sequence of cysteine due to oprotein agrigation.LAN formation increased with increased molding condition due to the sulfhydryldisulfide interchange reaction and β-elimination reaction occurence.While cysteine and lysine amino acids were observed to be relatively similar with a slight reduction.
The reduction in lysine with increased molding condition was due to oxidation and Maillard reactions (darkening of thermo molded plastic plates).Cysteine was converted to dehydroalanine (DHA) and atomic sulphur.Dehydroalanine was convereted in to lysinoalanine (LAL) (not detected) and lanthionine (LAN).The level of LAN formation is an indication of improved network formation.

Conclusion
Gluten proteins, the storage proteins of wheat and coproduct of starch industry display interesting features for non-food applications.Understanding the composition and processing of gluten on network formation is important for biodegradable, renewable organic material formation.The fractionation of gluten with 60% ethanol yielded a glutenin-(49%) and a gliadin-rich (51%) fractions.Among the different amino acids; cystine, lysine and LAN were significantly affected by thermo-molding; the level of cysteine and lysine amino acids decreased with increased modling condition due to protein polymerization reactions.While the LAN increased with molding at 150°C.The reduction of extrability with increased thermo-molding condition improves protein polymerization and mechanical properties.

Table 1 .
The extractability and composition of gluten protein before and after thermo -molding at 130 and 150°C for 5 and 25 min analysed by reverse phase -high performance liquid chromatography (RP-HPLC) (expressed in percentage).

Table 2 .
The Extractability and composition of glutenin protein before and after thermo-molding at 130 and 150 °C for 5 and 25 min analysed by reverse phase-high performance liquid chromatography (RP-HPLC) (expressed in percentage).

Table 4 .
The lysine, LAN and cystine contents of gluten, gliadin and glutenin proteins (mole/10 5 g of protein) thermo-molded at 130 and 150 °C for 5 and 25 min.