Effects of α-, β-, γ- and ω-Gliadins on the Dough Mixing Properties of Wheat Flour

Journal of Cereal Science 26 (1997) 271–277

Effects of a-, b-, c- and x-Gliadins on the Dough Mixing Properties of Wheat Flour ´ ´ R. J. Fido∗, F. Bekes†, P. W. Gras† and A. S. Tatham∗ ∗IACR Long Ashton Research Station, University of Bristol, Department of Agricultural Sciences, Long Ashton, Bristol BS18 9AF, U.K., and †CSIRO Division of Plant Industry, Grain Quality Research Laboratory, PO Box 7, North Ryde, NSW 2113, Australia Received 30 September 1996

ABSTRACT Gliadins were extracted from wheat and individual groups (a-, b-, c-, x-1 and x-2) purified. The effects of the individual groups of gliadin on the mixing properties of doughs from low and high protein flours were measured on a 2-g Mixograph and a prototype microextension tester. The addition of all groups of gliadin resulted in a decrease in dough strength. The relative weakening effects were x-1>x-2≈a-≈b->c- in the Mixograph, and c->a-≈b-≈x-2≈x-1 in the Extensograph.  1997 Academic Press Limited

Keywords: gliadin, Mixograph, Extensograph, breadmaking.

INTRODUCTION The dough mixing properties of flours and their suitability for breadmaking are largely determined by the gluten proteins. The high molecular weight (HMW) subunits of glutenin have been intensively studied and allelic variation in number and compositions of HMW subunits has been correlated with breadmaking quality1,2; as have the proportions of high Mr glutenin polymers consisting of both HMW and low molecular weight (LMW) glutenin subunits3,4. However, not only the glutenins and their content affect breadmaking quality; the gliadins, which comprise approximately 50% of the gluten proteins, also are associated with functional properties5–8. Data relating specific gluten proteins to dough strength and breadmaking quality parameters have come from three  : HCl=hydrochloric acid; HPLC= high performance liquid chromatography; HPF=high protein flour; LPF=low protein flour; MPF=medium protein flour; SDS-PAGE=sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Corresponding author: A. S. Tatham. E-mail: arthur.tatham @bbsrc.ac.uk. 0733–5210/97/060271+07 $25.00/0/jc970138

types of study: (1) correlative, where statistically significant links are made between quality parameters and gluten protein type(s) and proportions6,7; (2) fractionation/reconstitution where extracted protein fractions are added to a base flour and the effects determined8–10; and (3) nutritional studies in which differences arise in the proportions of proteins in sulphur-deficient wheats, most significantly x-gliadin, and can be related to quality parameters11,12. Correlations between gliadin patterns and technological properties are not as easy to determine as is the case for the HMW subunits of glutenin. Unlike the HMW subunits, gliadins are encoded by large multigene families and inherited in blocks, thus effects attributed to one gliadin(s) may be due to other gliadin(s) or low molecular weight (LMW) subunits of glutenin. Fractionation and reconstitution is, at present, probably the best method for determining the effects of added proteins. However, the effects of the isolation and incorporation procedures on technological properties have to be considered. Dough strength describes the balance between the elastic and viscous properties of a dough. High dough strengths are characterized by long development times and high resistance to extension  1997 Academic Press Limited

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(high elasticity and low viscosity), whereas weak doughs have shorter development times and low resistance to extension (high viscosity and low elasticity). The balance between elasticity and viscosity is important in breadmaking and, generally, high dough strengths are required. Although it has generally been concluded that gliadins have negative effects on dough properties and breadmaking quality6–10, Weegels et al.13 added purified groups of gliadin to base flours and found that they increased loaf volumes. In the present study, five individual groups of gliadins (a-, b-, c-, fast-x and slow-x) were purified and their effects on addition to high, medium and low protein base flours measured using a 2-g Mixograph and a prototype microextension tester.

EXPERIMENTAL Three untreated commercial bakers flours (9·2% (LPF), 10·6% (MPF) and 11·9% (HPF) protein, N×5·7, respectively) were used as base flours. Total gliadins from wheat flour cv. Chinese Spring were extracted using either dilute HCl (pH 5·3) as described by MacRitchie8 or 70% (v/ v) aqueous ethanol as described by Shewry et al.14. Individual gliadins were separated by ionexchange chromatography on carboxymethyl (CM) cellulose, using 3  urea, 0·01  glycineacetate buffer pH 4·6 and were eluted with a linear gradient of NaCl15. The gliadin fractions were dialysed against 1% (v/v) acetic acid at 4 °C for 60 h and freeze dried. Gliadin fractions were identified and assayed for purity by acid-PAGE16 and SDS-PAGE17, the gliadins were free from LMW subunits. Mixing tests were conducted with a 2-g Mixograph using LPF and HPF as previously described18. Mixing experiments were performed by the addition of 2, 4 and 6 mg of gliadin to base flour (2 mg is approximately a 4·3% and 3·4% increase in total gliadin for LPF and HPF, respectively). Parameters recorded were time to peak dough resistance (mixing time, MT), peak dough resistance (PR) and resistance breakdown (RBD). Experiments were performed in duplicate. Mixing was carried out in quadruplicate with a 2 g Mixograph in a temperature controlled bowl at 30 °C and the times to peak dough development determined. Doughs for extension testing were mixed to peak dough development, divided into two 1·7 g samples, rolled in a prototype micro-moulder,

mounted into sample carriers and allowed to rest at 30 °C for 45 min. Rested dough samples were mounted on to the prototype tester and pulled at 1·0 cm/s. Recordings of the dough resistance and the sample carrier position were taken at 100 readings/s and recorded on a personal computer, using LabTech Notebook software. Maximum resistance (Rmax) and extension before rupture were calculated using specially written software19. RESULTS AND DISCUSSION Purification and characterisation of gliadin Figure 1 shows the SDS-PAGE (Fig. 1a) and acidPAGE (Fig. 1b) patterns of the gliadins purified from a 70% (v/v) aqueous ethanol extract of flour. The a-, x-1 and x-2 gliadin fractions are essentially free from other gliadin components (Fig. 1b, tracks a, d and e, respectively) while the b-gliadins show some minor contamination with cgliadins (Fig. 1b, track b). The SDS-PAGE patterns indicate that the x-1 gliadins correspond to the slow and x-2 gliadins to the fast x-gliadins (Fig. 1a, tracks e and f, respectively). Similar patterns were observed for the fractions purified from HCl extracted flour (data not shown). Weegels et al.12 reported and reviewed the problems associated with obtaining purified gliadin fractions without significantly altering their functional properties. Factors reported to affect functional properties include the use of alcohols for extraction, protein aggregation occurring either by precipitation or during dialysis and the use of denaturant containing buffers13. In this study, ureabased buffers were used because they were found to be the most effective in the separation of gliadin by ion-exchange chromatography. Essentially pure groups of gliadin were obtained, whereas the gliadin fractions of Weegels et al.13 were contaminated with other groups of gliadins and proteins of Mr 14 000, which may correspond to protease inhibitors. Care was taken where alteration to functionality could occur due to aggregation during dialysis; this was avoided by the addition of 1% (v/v) acetic acid to the dialysis solutions. Although the effect of 3  urea in the ion-exchange step on gliadin functionality was not determined; however, a recent study has indicated that functionality is retained with the use of urea, providing care is taken to avoid aggregation or flocculation during dialysis20.

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Figure 1 (a) SDS-PAGE of purified gliadin fractions. Track a=Mr markers, 1=76 000–78 000, 2=66 200, 3=42 700, 4= 30 000, 5=17 200 and 6=12 300; track b=a-gliadins; track c=b-gliadins; track d=c-gliadins; track e=x-1 (fast x) gliadins and track f=x-2 (slow x) gliadins. (b) Acid-PAGE of purified gliadin fractions. Track a=a-gliadins; track b=b-gliadins; track c=c-gliadins; track d=x-1 (slow x) gliadins and track e=x-2 (fast x) gliadins.

Effect of addition of gliadin fractions on Mixograph parameters The effects of the addition of ethanol-extracted gliadin fractions on mixing time (MT), peak resistance (PR) and resistance breakdown (RBD) using low and high protein flours, are shown in Figures 2 and 3, respectively. These results are expressed on a mg protein basis but replotting the figures on a molar basis (assuming an average Mr of 31 000 for a- and b-gliadins, 35 000 for cgliadins and 40 000 for x-gliadins) did not significantly alter the plots and did not affect the results (data not shown). Similar results were also obtained for the HCl extracted gliadin fractions (data not shown). All gliadins reduced the mixing time requirement of the base flour. This was not a simple protein dilution effect, as different gliadins produced different effects that were not related to Mr.

Therefore, the different gliadin groups clearly had different functional activities. No significant differences were found between gliadin fractions purified by HCl or ethanol extraction. In MT experiments x-gliadins showed the largest weakening effect on the flours, followed by a-/b-gliadins, with c-gliadins showing the least effect (Figs 2a and 3a). With the high protein flours, the overall reduction in MT was reduced on the addition of the gliadin fractions, compared with the low protein flours (Figs 2a and 3a). All gliadins reduced the PR of the flour, indicating a weakening effect on the dough (Figs 2b and 3b). The behaviour is more complex than that for MT, with the order of effects being x-1>x-2>c>a/b, at the 6 mg level of addition (Figs 2b and 3b). All gliadin fractions increased the RBD; the x-gliadins having the largest effect, with x-1>x-2>c>a/b gliadins (Figs 2c and 3c). Addition of 2 mg of a gliadin fraction to flour

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would increase the total protein content of the low protein flour by about 1·2% and the high protein flour by about 1%. It would also alter the gliadin: glutenin ratio that is known to affect baking quality. At these levels all of the gliadin fractions had a weakening effect on dough strength. Effect of the addition of gliadin fractions on Extensograph parameters Two parameters, Rmax and extensibility, were measured at 6 mg addition in a 10·6% (MPF) protein flour. Addition of gliadin resulted in a decrease in Rmax (Fig. 4), the effects of the different

gliadin groups being in the order c-≈x-2>a-≈b≈x-1. The effects on extensibility fell into two groups. Addition of a- and x-1 were similar to that of the control, whereas the addition of b-, cand x-2 gliadin fractions resulted in increased extensibility (Fig. 4). The Rmax and extensibility values indicate a weakening effect on addition of gliadins to these flours. Comparison of Mixograph and Extensograph results A dough, even the simple flour and water system used in our studies, is a complex mixture of differ-

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Figure 4 (a) Effect of the addition of groups of gliadins to the Extensograph. 1=control, 2=a-gliadin, 3=b-gliadin, 4=cgliadin, 5=x-1 gliadin and 6=x-2 gliadin. (b) Rmax and Ext (extensibility), AU: arbitrary units.

ent components. Its rheological properties depend on the structures of the components, their spatial arrangements, their interactions and changes that occur in these structures/interactions during the mixing process. The Mixograph and Extensograph measure different properties of doughs. The Mixograph is a recording dough mixer, with the mixing bowl containing moving and fixed pins. The moving pins stretching the dough between fixed pins, the resulting forces being recorded, developing the dough from flour and water to its optimal state. The Extensograph measures extensibility and the resistance to extension of a piece of dough which has been rested, in our study for 45 min, so that the structure of the dough has relaxed after mixing in the Mixograph21. The effects of addition of gliadins to the flours and their subsequent analysis by Extensograph and Mixograph show different orders of effects of the different gliadin fractions. Both show a weakening effect on dough strength, as would be expected on the addition of monomeric proteins and the consequent alteration of the gliadin:glutenin ratio. Comparison between the effects of the different gliadin fractions on the Mixograph parameters (MT, PR and RBD) and Extensograph

parameters (Rmax and extensibility) indicated no strong correlations.

General discussion There have been a number of studies of the effects of specific gliadins on the baking performance and properties of wheat flours, often with mixed and ambiguous results. In the case of x-gliadin, Wrigley et al.5 correlated specific x-gliadin bands with both dough strength and dough weakness; Branlard and Dardevet6 showed that the x-gliadin generally had negative effects on a number of quality parameters and Campbell et al.22 found a correlation between some bands and dough extension. MacRitchie23 carried out reconstitution experiments and found that the addition of gliadins to base flours decreased the mixing requirement and resulted in decreased loaf volumes. MacRitchie et al.24 fractionated a range of flours and analysed the fractions by electrophoresis and densitometry, but could not draw any firm conclusions about the effects of x-gliadins. Branlard and Dardevet 6 found that c-gliadins were positively correlated with dough strength and Dong et al.25 reported an association

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between c-gliadins and mixing tolerance. Lonkhuijsen et al.26 found that c-gliadins had a positive effect on breadmaking, as did Weegels et al.13, who reported that the addition of c-gliadins to flours increased loaf volume. Data for the effects of the structurally similar a- and b-gliadins is more ambiguous, certain bands being correlated with positive and others with negative technological properties5–10,13,24–27. Correlations have been reported between loaf volumes and gliadin surface hydrophobicity, as measured by reversed-phase HPLC, where the order of hydrophobicity is x-
of molecules with which the gliadin can interact. Thus, x-gliadins may disrupt interactions between larger numbers of protein molecules in the gluten matrix than the more compact a-/b-/c-gliadins. Alteration of the gliadin:glutenin ratio by the addition of gliadins may also affect polymer:polymer interactions, by introducing polymer:monomer interactions, weakening the interactions of the polymer networks. This is, however, a simplistic view taking into account only the hydrogen bonding and, to a limited extent, the tertiary structures of gliadins. The use of gliadin domains, produced either by enzymic or chemical cleavage or by protein engineering, should allow a better understanding of the protein interactions in dough systems that contribute to the complex behaviour observed for the different gliadins in the Mixograph and Extensograph results. Acknowledgements IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.

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