Critical Reviews in Food Science and Nutrition

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Aug 2, 2013 - This article may be used for research, teaching, and private study ... residues of gliadins mainly form intramolecular disulfide bonds, ..... covalent ionic bonds and hydrophobic interactions. ..... Wrigley, C.W., Bekes, F., and Bushuk, W. Eds., American Association of Cereal ..... Chemistry of gluten proteins.

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Biochemical and Functional Properties of Wheat Gliadins: A Review a

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Sheweta Barak , Deepak Mudgil & B. S. Khatkar

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Department of Food Technology , Guru Jambheshwar University of Science and Technology , Hisar , 125001 Accepted author version posted online: 02 Aug 2013.Published online: 02 Aug 2013.

To cite this article: Sheweta Barak , Deepak Mudgil & B. S. Khatkar (2013): Biochemical and Functional Properties of Wheat Gliadins: A Review, Critical Reviews in Food Science and Nutrition, DOI: 10.1080/10408398.2012.654863 To link to this article: http://dx.doi.org/10.1080/10408398.2012.654863

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ACCEPTED MANUSCRIPT Biochemical and Functional Properties of Wheat Gliadins: A Review Sheweta Barak, Deepak Mudgil and B.S. Khatkar* Department of Food Technology, Guru Jambheshwar University of Science and Technology, Hisar-125001 *Corresponding author Email: [email protected]

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Fax: +91-1662-263313; Tel. No.: +91-1662-263313 Biochemical and Functional Properties of Wheat Gliadins: A Review

Abstract: Gliadins account for 40-50% of the total storage proteins of wheat and are classified into four sub-categories, α-, β-, γ- and ω-gliadins. They have also been classified as ω5-, ω1, 2-, α/β- and γ-gliadins on the basis of their primary structure and molecular weight. Cysteine residues of gliadins mainly form intramolecular disulfide bonds, although α-gliadins with odd numbers of cysteine residues have also been reported. Gliadins are generally regarded to possess globular protein structure, though recent studies report that the α/β-gliadins have compact globular structures and γ- and ω-gliadins have extended rod-like structures. Newer techniques such as Mass Spectrometry with the development of matrix-assisted laser desorption/ionization (MALDI) in combination with time-of-flight mass spectrometry (TOFMS) have been employed to determine the molecular weight of purified ω- gliadins and to carry out the direct analysis of bread and durum wheat gliadins. Few gliadin alleles and components, such as Gli-B1b, Gli-B2c and Gli-A2b in bread wheat cultivars, γ-45 in pasta, γ-gliadins in cookies, lower gliadin content for chapatti and alteration in Gli 2 loci in tortillas have been reported to improve the product

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ACCEPTED MANUSCRIPT quality, respectively. Further studies are needed in order to elucidate the precise role of gliadin subgroups in dough strength and product quality. Keywords: Wheat, gluten, gliadins, structure, rheology, product quality INTRODUCTION Wheat is undoubtedly the supereminent cereal crop in the world due to its widespread

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distribution and extensive utilization for baked food products. Hexaploid wheat (Triticum aestivum) accounts for 95% and tetraploid wheat (Triticum durum) for the remaining 5% of the worldwide production of wheat (Shewry, 2009). Wheat kernels comprise of 13-17% bran, 2-3% germ and 81-84% endosperm. The major components of the endosperm are starch (60-75%), proteins (6-20%), moisture (~ 10%) and lipids (1.5-2%). The unusual properties of the wheat are ascribed to the presence of gluten forming storage proteins of the endosperm which are composed of two fractions- the alcohol soluble gliadins and the alcohol insoluble glutenins (Singh et al., 2011; Wieser, 2007). The rheological and functional properties of wheat gluten are found to be dependent upon the ratio of gliadins to glutenins (Pedersen and Jorgensen, 2007; Khatkar et al., 1995), molecular size distribution, structure of the glutenin polypeptides, high/low Mr glutenin polypeptides ratio, bond strength between gliadins and glutenins and reduction or oxidation activity of glutenins. The cohesiveness and extensibility of the gluten is attributed to the monomeric gliadins while glutenins contribute to the elasticity and strength of gluten (Rodrigues et al., 2005). Thus, the ratio of glutenins to gliadins controls the dough strength and extensibility (Wrigley et al., 2006; Khatkar et al., 2002b). Moreover, the property of the wheat flour to form viscoelastic dough is lost upon the removal of gluten proteins from the flour. The influence of the gluten components on the functionality of dough is complex (Khatkar et al.,

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ACCEPTED MANUSCRIPT 2002a) and requires extensive studies. Gliadins, one of the major gluten storage proteins of wheat, account for 40-50% of the total storage protein (Anderson et al., 1997) and confer viscous character to wheat gluten (Wieser, 2007; Khatkar et al., 2002a). Significant effects of gliadins on the gluten strength (Metakovsky et al., 1997; Weegels et al., 1996) have been reported. Past researches have suggested that gliadins may play an important role in determining the functional

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property of wheat flour. However, toxicity studies have also revealed that gliadins trigger the occurrence of celiac disease in genetically susceptible individuals with HLA-DQ2 or -DQ8 haplotypes.

In this review paper, efforts have been made to explain the biochemical and

functional properties of gliadins and their relationship with the end product quality.

WHEAT KERNEL PROTEINS Based on the classical fractionation process by Osborne (1907) wheat proteins have been separated into 4 groups- albumins (soluble in water), globulins (soluble in dilute salt solution), gliadins (soluble in 70% ethyl alcohol) and glutenins (soluble in dilute acids and bases). The baking performance of a wheat variety does not depend on the composition of the non-gluten forming proteins- the albumins and globulins (MacRitchie, 1984) as their composition does not vary among different wheat varieties. The ability of the wheat flour to be processed into numerous baked products primarily depends on the quality and quantity of the gluten proteins (Weegels et al., 1996). Gluten proteins are also known as prolamins because of the presence of proline and glutamine amino acid residues in their structures. Extensive and intensive research is being carried out on gluten proteins to determine their properties and structure. Based on their solubility in aqueous alcohol solutions, prolamins of wheat have been divided into two classes,

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ACCEPTED MANUSCRIPT the gliadins and glutenins (Francin Allami et al., 2011). Together, gliadins and glutenins represent 80-85% of the total proteins of wheat flour (Veraverbeke and Delcour, 2002) and impart the unique properties- extensibility and elasticity to the wheat dough. The glutenin polypeptides (30-140 kDa) are further fractionated into- high molecular weight glutenin subunits (HMW-GS: 90 to 140 kDa) (Klindworth et al., 2005) and low molecular weight glutenin

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subunits (LMW-GS: 30 to 75 kDa) by sodium dodecyl polyacrylamide gel electrophoresis under reducing conditions (Wellner et al., 2005; Anjum et al. 2007). Disulphide bonds between the cysteine residues significantly stabilize the glutenin polymers (Lefebvre and Mahmoudi 2007), functioning as interchain bonds between HMW glutenin subunits. Though regarded as the minor components, HMW-GS are the prime determinants of elasticity of the gluten which in turn have a profound effect on the loaf volume of bread (Tatham et al., 1985; Cornish et al., 2006). HMWGS are encoded by genes on the long arm of chromosomes 1A, 1B and 1D at the Glu-A3, Glu-B3 and Glu-D3 loci (Table 1). It has been found that the HMW-GS 1Dx5+ 1Dy10 encoded by Glud1d locus improves the bread quality by increasing the strength of dough. On the other hand, the HMW-GS 1Dx2+1Dy12 encoded by Glu-D1a gives poor loaf volume in bread. Gliadins are the monomeric proteins linked by either no disulphide bonds (ω- gliadins) or by intrachain disulphide bonds (α-, β- and γ- gliadins) (Singh and MacRitchie, 2001). Upon hydration, the gliadins behave as a viscous liquid (Singh and Khatkar, 2005; Song and Zheng, 2008) which imparts extensibility to the dough. Amino acid sequence determination has revealed that α- and γ- gliadins are both related to the LMW-GS (Veraverbeke and Delcour, 2002). The LMW-GS has been subdivided into three groups- B, C and D based on their electrophoretic mobility and isoelectric point. D’ovidio and Masci (2004) reported that out of these, C and D

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ACCEPTED MANUSCRIPT groups are mainly composed of α-, β- , γ- and ω- gliadins mutated in the cysteine residues. LMW-GS can act as either chain terminators or chain extenders, depending on their ability to form disulfide bonds. Typical LMW-GS can act as chain extenders by forming two interchain disulfide bonds while gliadin like LMW-GS are expected to act as chain terminators of glutenin polymers by forming one interchain disulfide bond (Muccilli et al., 2010).

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The composition of the gliadins in wheat differs from variety to variety. As a result of this extensive polymorphism, gliadins are used for the identification of the cultivar in hexaploid and tetraploid wheat. Differences in the gliadin to glutenin ratio among wheat cultivars are considered an important source of inter-cultivar variation in physical properties and end product quality. An inverse relationship exists between the gliadin/glutenin ratio and the elasticity of gluten. Doughs that are too elastic and inextensible give poorer bread making performance than doughs that have an appropriate balance of extensibility and elasticity (Khatkar et al., 2002a). Thus, the knowledge of composition of gliadins and glutenins, their properties and rheological behaviour has become increasingly important in the baking industry.

CLASSIFICATION OF GLIADINS Gliadins have been described as heterogeneous mixtures of single chained polypeptides soluble in 70% aqueous alcohol. They account for about half the gluten proteins and have been divided into 4 groups- α- (fastest mobility), β-, γ-, and ω-gliadins (slowest mobility) based on their electrophoretic mobility in A-PAGE at low pH (Banc et al., 2009; Wieser, 2007). According to the analysis of primary structure and molecular weights (MWs), a new classification is given as ω5-, ω1, 2-, α/β- and γ-gliadins (Wieser, 2007). Because of their structural homology as revealed

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ACCEPTED MANUSCRIPT by the amino acid sequencing, α- and β- gliadins have been grouped under one heading- the αtype gliadins (Zilic et al., 2011). The molecular weight range of gliadins is ≈30,000 to 75,000 Da (Fido et al., 1997). The monomeric gliadins confer their characteristic property- viscosity through non- covalent interactions such as hydrogen bonding, vander Waal’s forces, electrostatic and hydrophobic

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interactions. Furthermore, gliadins can also interact with the glutenin polymers via noncovalent hydrophobic interactions and the glutamine residues via hydrogen bonds (Wellner et al., 2003). The genes coding the gliadin proteins (Table 1) are located on the short arms of group 1 and 6 chromosomes (Wrigley and Shepherd, 1973; Brown and Flavell, 1981). They are tightly linked genes located at three homologous loci of the group 1 chromosome- Gli-A1, Gli-B1, and Gli-D1 and group 6 chromosomes- Gli-A2, Gli-B2, and Gli-D2 loci. Most γ- and ω - gliadins are encoded by Gli-1 genes and all the α-/β- and some of the γ-gliadins are encoded by the Gli-2 genes (Ferranti et al., 2007). There is a tight linkage between the ω- and γ- gliadins encoded at the Gli 1 locus and LMW glutenin subunits. Wieser and Kieffer (2001) pointed out that α/β- and γ-gliadins are major components, accounting for 28-33% and 23-31%, respectively, but the ωgliadins occur in much lower proportions, only 4-7% and 3-6% for the ω1,2- and ω5-gliadins, respectively. Qi and colleagues (2011) recently reported ten novel α- gliadin genes isolated from Triticum aestivum L. Close associations have been found between the gliadin blocks and Zeleny sedimentation value which is regarded as an important criterion for predicting the bread making performance of the wheat variety (Sozinov and Poperelya, 1982). Branlard and Dardevet (1985) reported quality differences between gliadins located on chromosomes 1A, 1B and 1D and on chromosomes 6A,

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ACCEPTED MANUSCRIPT 6B and 6D. However, the correlation between the wheat quality parameters and gliadins located on chromosome group 1 has been attributed to the LMW subunits of the glutenins as the genes encoding both these proteins have been found to be tightly linked. Furthermore, deletions in the gliadin locus (Gli 1) increase the dough strength and percentage of polymeric proteins. Due to high structure heterogeneity of gliadins, powerful separation techniques are needed for

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isolation and characterization of gliadins (Piergiovanni and Volpe, 2003). At present, various modes of electrophoresis and chromatography are widely used methods for gliadin separation and wheat varietal identification. Great numbers of biochemical, genetical and technological investigations of gliadin proteins have been conducted by electrophoresis (Seva et al., 2005; Ojaghi and Akhundova, 2010; Brezhneva et al., 2010). Electrophoregrams of gliadin proteins provide information about identity of cultivars, protein polymorphisms, technological quality of grain, flour and dough of wheat (Rashed et al., 2007; Wu et al., 2007). Gliadin markers are easier and more powerful tools for wheat genotype identification than DNA molecular markers, which normally exhibits lower level of intervarietal polymorphism. pH 3.1 is extensively used in all polyacrylamide gel electrophoresis (PAGE) and starch gel electrophoresis methods. Various gliadin components have also been separated by ion-exchange and gel-filtration column chromatography. Bietz (1983) first reported the use of High Performance Liquid Chromatography (HPLC) to characterize wheat proteins. Another powerful technique, Capillary Zone Electrophoresis (CZE) has shown much potential for protein analysis and varietal identification (Bean and Lookhart, 2000; Siriamornpun et al., 2001). Capillary electrophoresis is increasingly being recognized as an important separation technique because of its speed, efficiency, reproducibility, ultra-small samples volume and low consumption of solvents. It

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ACCEPTED MANUSCRIPT overcomes some of the disadvantages of gel electrophoretic methods. CZE gives better resolution and shorter analysis times than either A-PAGE or HPLC (Rodriguez-Nogales et al., 2006). Recently, mass spectrometry (MS), with the development of ‘soft’ desorption/ionisation methods such as electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), has become important as an alternative and powerful technique in the genomic and

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proteomic fields. Particularly, MALDI in combination with time-of-flight mass spectrometry (TOFMS) has been used to determine the molecular masses of purified wheat α-gliadins. Furthermore, MALDI-TOF-MS has also been used to study the alteration of gliadins during the baking process (Sorensen et al., 2002). This methodological approach has been extended to the direct analysis of bread and durum wheat gliadins (Camafeita et al., 1998). Compared with conventional methods for gluten protein separation (gel electrophoresis and reversed-phase HPLC), MALDI-TOF is much more accurate and much faster, requiring less than 1 pmol of sample and only a few minutes per sample to perform the measurement (Cunsolo et al., 2003). Alpha and Gamma Gliadins The average molecular mass of α- and γ- gliadins have been reported to be 31,000 and 35,000 Da, respectively. The composition of amino acids of α- gliadins is quite similar to that of γgliadins. Both are relatively rich in sulphur containing amino acids such as methionine and cysteine but contain few proline, glutamine and phenylalanine residues. Thus, they have also been classified as the S- rich prolamins by Shewry et al. (1986). Presence of even number of sulphur rich cysteine residues in α- and γ- gliadins leads to the formation of intrachain disulphide bonds responsible for their folded structure which further determines the nature of non covalent bonding. These non-covalent protein-protein interactions (mainly hydrogen bonds and

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ACCEPTED MANUSCRIPT hydrophobic interactions) are mainly responsible for the viscosity of gliadins and extensibility of gluten (Shewry and Tatham, 2000). Omega Gliadins These belong to the medium-molecular-weight (MMW) group of gluten protein with their molecular weight ranging from 44,000-80,000 Da. The ω- gliadin differs from the other gliadin

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subgroups in its amino acid composition. Shewry et al. (1986) have classified ω- gliadins as the S-poor prolamins as it lacks the sulphur containing amino acid (cysteine or methionine) while contains low amounts of amino acids of basic nature. Thus, it does not form disulphide bonds and interacts in dough through the hydrogen bonds (Shewry and Tatham, 2000). Omega gliadins are reported to be more polar than α-, β- and γ- gliadins (Banc et al., 2009). Charbonnier (1974) reported that nearly 80% of the amino acids comprising the ω- gliadins are Glx (45–56%), Pro (20–30%), and Phe (9–10%). Alanine, threonine or seronine formed the N-terminal region. The reported value of amide content revealed that Asx and Glx had a degree of amidation of 99.6%. STRUCTURE AND AMINO ACID COMPOSITION OF GLIADINS The overall structure of gliadins consists of a central domain (CD) containing repetitive amino acid (AA) sequences rich in proline (Pro) and glutamine (Gln), and two terminal non-repetitive domains which are hydrophobic and contains most of the ionizable amino acids (histidine, arginine and lysine), although the latter are present only in low levels (Gianibelli et al., 2001). The knowledge of the complete gliadin amino acid sequences come from the analysis of cDNA and genomic DNA sequences. Glutamic and aspartic acids exist as amides. The sequence of gliadin proteins is of prime importance as they are the major determinants of the toxicity and functionality in dough (Bietz et al., 1977).

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ACCEPTED MANUSCRIPT Different types of gliadins have different secondary structure associated with the molecules. Tatham and Shewry (1985) studied the secondary structures of gliadins using circular dichroism spectroscopy and found that ω-gliadins were rich in randomly coiled β-turns without detectable α-helix or β-sheet, but α/β- and γ-gliadins contained 30-35% α-helix and 10-20% β-sheet conformations. They also reported that ω-gliadins were mainly stabilized by strong hydrophobic

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interactions and α/β- and γ-gliadins were stabilized by covalent disulphide bonds and noncovalent hydrogen bonds in their α-helices and β-sheets. However there has been no consensus on the structure of gliadins in solutions. Friedli (1996) proposed a doughnut-like structure for gliadin molecules in which there was a large central hole. However, gliadins are still widely considered to adopt a globular protein structure in 70% aqueous ethanol (Foulk and Bunn, 2001). But recent researches reveal that α/β-gliadins have compact globular structures and γ- and ωgliadins have extended and rod-like structures (Paananen et al., 2006; Ang et al., 2010). Alpha and Beta Gliadins These two types of gliadins proteins have similar primary structures consisting of around 250 and 300 amino acid residues. The sequences are composed of the N-terminal domain with five residues, repetitive central domain consisting of about 113-134 amino acid residues particularly rich in proline and glutamine sequences ((heptapeptide: Pro-Gln-Pro-Gln-Pro-Phe-Pro and pentapeptide: Pro-Gln-Gln-Pro-Tyr) (Ferranti et al., 2007) and the C-terminal domain of 144-166 residues (non-repetitive domain). The protein structure is stabilized by the disulphide bonds formed between the cysteine residues (Shewry and Tatham, 1997). About 90% of the amino acid residues in these gliadins are present as the glutamic and aspartic acid residues in amide form (Ewart, 1983). The repetitive domain is composed of repeat units of PQPQPFP and PQQPY

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ACCEPTED MANUSCRIPT (Shewry and Tatham, 1990). Alpha gliadins contain six cysteine residues which form three intrachain crosslinks (Altenbach et al., 2010) and thus there are no free cysteines preventing gliadins from participating in the polymeric structure of glutenin. However, α–gliadins with odd numbers of cysteine residues have also been reported (Anderson et al., 1997). Such gliadins can form one intermolecular S-S bond and act as chain terminators of glutenin and probably decrease

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the molecular weight of the glutenin in the network. Gamma Gliadins The γ- type gliadins starts with a 20 residue signal peptide, followed by a short twelve residue Nterminal non-repetitive domain, a highly variable repetitive domain of 72-161 residues, a nonrepetitive domain containing most of the cysteine residues and the C-terminal non-repetitive domain containing the final two conserved cysteine (Fig. 1) residues (Cassidy et al., 1998). All the cysteine residues form intramolecular disulfide bonds. About one quarter of γ- gliadins have been reported to contain an uneven number of cysteine residues (Anderson et al., 2001). The free-SH groups left after the formation of intramolecular disulfide bonds form intermolecular disulfide bonds. Thus α-, β- and γ- gliadins have a possibility of a greater interaction with the gluten because of the higher number of cysteine residues as compared to the ω- gliadins. The repetitive domain in γ- gliadins forms the extended structure and is rich in β reverse turns, while the non-repetitive domain is rich in helices (Tatham et al., 1990). Omega Gliadins Proline, glutamine and phenylalanine residues account for 80% of the total amino acids in ωgliadins compared to 50-60% for the other gliadins (Hisa and Anderson, 2001). They may also contain few or no methionine and cysteine (sulphur containing amino acids). Their methionine

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ACCEPTED MANUSCRIPT level may be less than 0.1%, lack cysteine amino acid and are not able to produce S-S-type bonding. Thus, a compact structure (Fig. 2) cannot be observed in ω- gliadins. These gliadins have few charged amino acids such as lysine (Kasarda et al., 1976). Also, they have few basic amino acids and comparatively higher level of phenylalanine than other gliadin subgroups (Kasarda et al., 1983). Their surface hydrophobicity is lower than that of α- and γ- gliadins. They

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are the first peptides to elute out from the RP-HPLC column (Popineau and Pineau, 1987). Moreover, ω- gliadins have also been shown to include modified gliadins having one cysteine residue and therefore can act as chain terminators (Gianibelli et al., 2002). On the basis of the N-terminal sequences, three different types of ω- gliadins have been observed in wheat and in related proteins such as C-hordeins and ω-secalins. These sequences have been named ARQ-, KEL- and SRL-types depending on the first three amino acids of their N-terminal sequences (Tatham and Shewry, 1995). According to Kasarda et al. (1983) and Tatham and Shewry (1995), the KEL-type differs from the ARQ-type due to the absence of the first eight residues in its structure. The third type of ω- gliadin - SRL-type is a characteristic of ω- gliadins encoded by chromosome 1B (Du Pont et al., 2000). ROLE OF GLIADINS IN DOUGH RHEOLOGY Basic rheological instruments provide the fundamental rheological behaviour of a material. Viscoelastic properties of gluten affect the rheological properties of wheat dough. When the dough is developed by mixing, the gluten proteins form continuous three-dimensional viscoelastic network throughout the dough with starch granules behaving as filler. The threedimensional structure of gluten matrix is stabilised by covalent (disulfide), hydrogen and noncovalent ionic bonds and hydrophobic interactions. The balance between gliadins and glutenins

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ACCEPTED MANUSCRIPT is responsible for important rheological properties such as viscosity and elasticity (Gomez et al. 2011; Khatkar et al., 1995). Understanding of the relationship between the gliadins and the dough rheology still remain the major objectives of cereal biochemistry and rheology. In an attempt to deduce the relationship between seed storage proteins and gluten strength, Damidaux et al. (1978) studied different seed storage proteins of durum wheat cultivars. They found that the

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cultivars having γ- 45 gliadin component exhibited a stronger gluten as compared to cultivars with γ-42 gliadin component. The component was named depending on its mobility in A-PAGE (Bushuk and Zillman, 1978). The proteins encoded by gliadin alleles vary in their structure, level of expression and influence on the quality of flour (Payne, 1987). Certain gliadins have been reported to contain long repetitive domains which could have an effect on the flour quality. Kasarda (1983) proposed that gliadins with an odd number of cysteines could form intermolecular disulfide bond and thus participate in the gluten polymer. Significant positive effects of certain gliadin alleles have been reported on gluten strength (Metakovsky et al., 1997). For example, wheats giving flour of the strong type have been found to carry gliadin blocks 1A3 (or 1A4), 1B1, 1D1 (or 1D5), 6A3, 6B1 and 6D1 (or 6D2), which are markers of high gluten quality, frost hardness and drought resistance. Functional studies of single gliadin by reduction and oxidation of flours containing proteins are useful in assessing the effect of a particular protein on functional properties of dough (Clarke et al., 2003). Redaelli et al. (1997) have shown strong positive effects on dough extensibility by Gli-D1 (gliadins)/Glu-D3 (LMW-GS) alleles. Khatkar et al. (1995) reported that alteration in the glutenin to gliadin ratio affect the rheological property of gluten dough. They suggested that glutenin contribute to elasticity and gliadin to the viscous property of hydrated

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ACCEPTED MANUSCRIPT gluten. Uthayakumaran et al. (2001) showed that upon increasing the glutenin content, the rupture viscosity increased, whereas increased level of gliadins lowered the rupture viscosity.

During formation of the dough, the gliadins act as a ‘plasticiser’ and promote the viscous flow and extensibility, considered as important rheological characteristics of dough. They might also

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interact through hydrophobic interactions and hydrogen bonds. Addition of total gliadins to the dough has shown to decrease the overall strength of dough. Gliadin-supplemented doughs generally have a shorter mixing time, greater resistance breakdown, lower maximum resistance to extension and decreased loaf volume (Uthayakumaran, 1999). Analogous results were observed by MacRitchie (1987) while studying the effect of addition of increased levels of gliadin rich fraction to base flour. He found that the gliadin addition shortened the mixing time and stability of dough. Khatkar and colleagues (2002b) studied the effects of addition of total gliadins on the dynamic rheology of gluten proteins. The addition of total gliadins increased the storage modulus (G') upto 0.5% total gliadin addition (Fig.3), but any further increments decreased it. However, tan δ values increased with the addition of total gliadins. This was primarily due to differential rate of change in the values of storage modulus (G') and loss modulus (G") as a result of total gliadins addition. Pioneering work on the effect of various gliadin subfractions on the mixograph properties was carried out by Khatkar et al. (2002a). They observed that the RBD (resistance breakdown) values increased with addition of gliadin and its subgroups. Within gliadin subgroups RBD values increased in the sequence ω1- < γ- < α- < β- gliadins (Fig. 4). They postulated that the differential interactive behaviour of gliadin with the gluten proteins was responsible for the

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ACCEPTED MANUSCRIPT above stated differential increase of RBD values. They also pointed out that addition of gliadin subgroups (α-, β-,γ- gliadins) increased the PDR (peak dough resistance) of the base flour to a greater extent than gluten and ω1- gliadin addition. Contrary to this, Uthayakumaran et al. (2001) while studying the effect of gliadins and its subgroups on the rheological parameters and functional properties of the dough observed that addition of gliadin subgroups reduced PDR and

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RBD. They further reported that among all the gliadin fractions γ-gliadin caused maximum reduction in the mixing time and resistance to extension. They postulated that the mixing time and maximum resistance to extension decreased as the hydrophobicity of the gliadin fractions increased in the order ω- < α- and β- < γ-gliadins. Similarly, Fido et al. (1997) reported that among all the gliadin fractions, ω- gliadins showed the largest weakening effect on the flours, followed by α/β gliadins, while γ- gliadins demonstrated the least effect on the mixing time. However, Khatkar (1996) found that α- gliadins reduce the mixing time to the maximum. He postulated that size/ charge of the gliadin is mainly responsible for the reduction of mixing time. It appears more reasonable that the smallest gliadins should have maximum weakening effect on the mixing time of the flour due to their action similar to ball-bearing. Thus, it is evident that there are conflicting thoughts with regard to the effect of specific group of gliadins on the mixing time of the flour. It can be concluded that wheat quality can be improved by acquiring the desired knowledge linked to the role of gliadins and its subfractions on the rheological and end product properties. INFLUENCE OF GLIADINS ON PRODUCT QUALITY The studies on the significance of gliadins and its subgroups on the wheat product quality have been carried out in various parts of the globe. But no single conclusion has been drawn even after

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ACCEPTED MANUSCRIPT numerous studies. Some researchers suggest that gliadin affects the loaf volume of bread whereas others regard glutenin as the sole determinant of the bread quality. A few gliadin alleles and components, such as Gli-B1b, Gli-B2c and Gli-A2b, in bread wheat cultivars (Wrigley et al., 1982) and γ-45 in durum wheat (D’Ovidio and Masci, 2004) have been found to contribute

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significantly to gluten strength.

Bread Quality The influence of gliadins and its subgroups on the quality of bread has been debatable for many years. The type and quantity of the gluten proteins are important in determining the bread making properties of the wheat flour (Gomez et al., 2011). Certain gliadin bands have shown significant correlation with hardness of grain and dough strength (Wrigley et al., 1981). Huebner and Beitz (1986) identified a correlation between a specific gliadin fraction and a general breadmaking score and named it ‘anti-baking-quality fraction’ as it had negative effect on the bread quality. Ohm et al. (2010) also observed negative relationship of gliadins on the loaf volume of bread. The effect of genetic variation in the glutenin and gliadin protein alleles of wheat on the mixing characteristics of the dough and quality of bread and noodle were evaluated by Wesley et al. (1999). They concluded that both gliadin and glutenin influence bread and noodle-making properties of wheat flour. They suggested that differences at the Glu-3/Gli-1 loci, coding for LMW subunits and gliadins or any other biochemical components that affect dough rheology, could be a reason for variation in the bread and noodle making performance. Many researchers have observed positive relationship between bread loaf volume and gliadins. Park et al., (2006) measured the protein and protein fractions in wheat flours to investigate their

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ACCEPTED MANUSCRIPT relationship to breadmaking properties. The percentage of gliadins based on flour and protein content of the wheat varieties exhibited a positive correlation with the bread loaf volume (r = 0.73, P < 0.0001 and r = 0.46, P < 0.001, respectively). Composition of gliadins and HMWglutenin subunits were studied by SDS-PAGE and A-PAGE method by Lan et al., (2009) to deduce the relationship of gliadins with the bread making performance of the wheat varieties. A

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close association was observed between the ω- gliadin fraction and bread making quality. Certain gliadin bands correlated positively with baking quality of bread. Thus, they suggested that gliadins could be used as an important parameter when breeding for bread making quality. Different subfractions of gliadins have found to be differently associated with the bread quality. Lonkhuijsen et al. (1992) carried out research on 32 wheat samples having similar composition of HMWGS (null, 7 and 2+12) and different gliadin composition. The bread loaf volumes ranged from 445 to 616 ml per 100g of flour. The gliadin composition of the wheat samples accounted for 82% of the observed variation. The ω-gliadin fraction was negatively associated with the bread quality whereas γ-gliadin was found to be present in the good bread quality wheat line. They concluded that both gliadins and HMW glutenin subunits control the bread making properties of wheat flour. Weegels et al. (1994) also observed similar effects of γ-gliadin on the loaf volume of bread. Khatkar et al., (2002a) reported that addition of total gliadins and its subgroups (α-, β-, γ- and ω- gliadins) to the dough significantly improved its bread making performance. The addition of total gliadins at ≥ 0.5% level increased the loaf volume significantly by about 10.6% while among the different subgroups α- increased the loaf volume of bread by 13.9%, followed by γ- and β- gliadins while ω1-gliadin sub-fraction caused 11% increase. Omega gliadin proved less effective than α-, β- and γ-gliadins in improving the loaf

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ACCEPTED MANUSCRIPT volume of the bread as they lack the sulphur containing amino acid cysteine (Tatham and Shewry, 1995), therefore, ω-gliadins interact with other proteins only non-covalently and thus are probably less effective in influencing viscoelastic properties and bread making quality. Dynamic rheological measurements determined by Khatkar (1996) for different gliadin subgroups revealed that the dynamic moduli, G’ and G’’, had significant (r =0·74 and 0·77)

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positive relationship with loaf volume for gliadin subgroups added (Fig. 5). Contrary to this, Uthayakumaran et al., (2001) reported that addition of gliadins decreased the loaf volume of bread with ω- gliadin reducing the loaf height to the maximum while α- + β- gliadins causing least reduction in loaf height. Furthermore, some investigators have reported a correlation between gliadin surface hydrophobicity and loaf volume; the order of hydrophobicity being ω

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