African Journal of
Food Science

  • Abbreviation: Afr. J. Food Sci.
  • Language: English
  • ISSN: 1996-0794
  • DOI: 10.5897/AJFS
  • Start Year: 2007
  • Published Articles: 976

Full Length Research Paper

The composition and extractability of thermo - molded wheat gluten bio-plastics

Abrehet Fisseha
  • Abrehet Fisseha
  • School of Nutrition, Food Science and Technology, Hawassa University, Hawassa, Ethiopia.
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  •  Received: 19 April 2016
  •  Accepted: 20 July 2016
  •  Published: 30 September 2016

 ABSTRACT

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.

Key words: Gluten, gliadin, glutenin, extractability, composition, thermo-molding, amino acids.


 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 disulfide-linked 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
 
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).
(2) LMW-GS (B-, C- and D-types); makes up 20% of the total protein, less characterized group than the HMW-GS. The B- and C-type LMW-GS are sulfur-rich prolamins, while the D-type LMW-GS are sulfur-poor prolamins (Veraverbeke and Delcour, 2002).
 
Gliadins represent a heterogeneous mixture of single-chained 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 ω- were 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 γ-gliadins 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 C-terminal 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, 2007, 2005; Guerrieri et al., 1996).
 
Gluten bioplastic formation
 
The need for environmentally friendly, renewable and biodegradable plastics is increasing due to pollution and  long term health impacts of synthetic polymers (Zhang et al., 2010; Sun et al., 2008); possess suitable gas barrier properties and nutritional value (Lagrain et al., 2010; Cao et al., 2007); poor moisture barrier properties due to its hygroscopic nature; binds with water through hydrogen bridges modified by thermal and chemical treatments (Sun et al., 2008; Carvalho et al., 2008; 2006; Brauer et al., 2007; Dicharry et al., 2006; Gallstedt et al, 2004; Domenek et al., 2004; Woerdeman et al., 2004). The effect  of processing on gluten network formation is determined by the SDS extractability which measures the degree of polymerization: the lower the extractability the higher the degree of polymerization (Sun et al. 2008; Gallstedt et al., 2004; Domenek et al.,  2004; Pommet et al., 2003; Micard et al., 2001; Cuq et al., 2000). Besides process conditions, the presence of additives influences extractability (Lagrain et al., 2010; Sun et al., 2007; Dicharry et al., 2006; Gallstedt et al., 2004; Woerdeman et al., 2004).


 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).


 RESULTS AND DISCUSSION

Gluten composition and extractability
 
Thermo – molded 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%) andglutenin extracts (49%) occurred in equivalent proportions in the SDS media (Goesaert et al., 2005; Belitz et al., 2009).   Reverse    phase   –    high    performance   liquid chromatography (RP-HPLC) profile showed that higher gliadin peak of gliadin fractions than gliadin peak of gluten  explains  that  gliadins  are  more extractable than non-gliadin fractions (Abrehet, 2015). Indeed, all the gliadins are soluble in the SDS buffer, but not all glutenins are soluble. Small gliadin peak as observed in the glutenin fractions shows that pure fractions of neither gliadin nor glutenin was obtained with ethanol extraction (Abrehet, 2015). The three gliadin types exist in a variable proportion; α-gliadin (21%), γ-gliadin (17%) and ω-gliadin (6%)  (Figure   1). The  glutenin  subunits;  B/C-LMW-GS (36%), HMW-GS (11%) and D-LMW-GS (3%) were obtained from gluten protein (Delcour and Hoseney, 2010; Wieser, 2007; Veraverbeke and Delcour, 2002) (Figure 2). There was a reduction in the  extractability of α-gliadin, γ-gliadin and ω-gliadin, HMW-GS and D-LMW-GS with increased molding condition thus explaining the occurrence of polymerization reaction due to the SH- disulfide interchange reactions (Sun et al. 2008; Kieffer et al., 2007; Wieser, 2007; Lagrain et al., 2005; Singh and MacRitchie, 2004; Gallstedt et al., 2004; Domenek et al., 2004; Pommet et al., 2003;) while there was an increase in the extractability of B/C-LMW-GS with increased molding conditions thus explaining the oxidation and protein degradation (Veraverbeke and Delcour, 2002).
 
 
 
 
 
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 thermo-molding 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 thermo-molding 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 ω- gliadin (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 α- and γ-gliadins.
 
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 sulfhydryl-disulfide 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 co-product 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. 


 CONFLICT OF INTERESTS

The author has not declared any conflict of interests.


 ACKNOWLEDGEMENTS

The Laboratory of Food Chemistry and Biochemistry, KU Leuven and Hawassa University, School of Nutrition, Food Science and Technology are duly acknowledged.



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