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Variation in protein composition of wheat flour and its relationship to dough mixing behaviour
Journal of Cereal Science 40 (2004) 31–39 www.elsevier.com/locate/jnlabr/yjcrs
Variation in protein composition of wheat flour and its relationship to dough mixing behaviour R. Kuktaitea,*, H. Larssonb, E. Johanssona a
Department of Crop Science, The Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53 Alnarp, Sweden b Department of Food Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received 19 December 2003; revised 31 March 2004; accepted 5 April 2004
Abstract Changes in extractability, amount and size distribution of polymeric proteins in the gluten of doughs during mixing were investigated. Ultracentrifugation was used as a non-destructive method to separate the gluten from the dough. Doughs prepared from commercial flour mixtures of different gluten strengths and mixed for varying periods, were analysed. Proteins were detected using RP- and SE-HPLC. The percentages of large unextractable polymeric protein (UPP), total UPP and large unextractable monomeric protein (UMP) were higher in the gluten phases of all flours at minimum and optimum mixing, compared to the flours. After overmixing, the percentages of large UPP, total UPP and large UMP in the gluten phases of the dough decreased to lower levels than in the flours. Differences in percentages of large UPP, total UPP and large UMP between gluten phases of different flours and mixing times originated from the genetic composition of flour proteins. The extractability of the glutenins in the flours reflected the quality of the specific flour. The protein extractability, especially of gliadins, was different in the gluten phase compared to in the flour. Ionic interactions seem to be important forces in the dough. q 2004 Elsevier Ltd. All rights reserved. Keywords: Wheat flour; Protein polymers; Protein composition; Dough mixing; Structure; Function
1. Introduction The end-use characteristics of flours produced from bread wheat are strongly determined by the gluten proteins (Wall, 1979; Weegels et al., 1996). Gluten proteins are particularly important for bread-making quality and consist of two major fractions: the monomeric gliadins and the polymeric glutenins (Sapirstein and Fu, 1998; Shewry and Tatham, 1990; Singh and MacRitchie, 2001). Flour proteins interact in the presence of water by forming gluten, which provides the unique viscoelastic properties needed for bread-making (Shewry et al., 2002). The glutenin fraction Abbreviations: HMW-gs, high molecular weight glutenin subunits; Large, UMP, large unextractable monomeric protein in the total monomeric protein; Large, UPP, large unextractable polymeric protein in the total large polymeric protein; LMP, large monomeric proteins; LMW-gs, low molecular weight glutenin subunits; LPP, large polymeric proteins; Min, mixing, minimum mixing time; Opt, mixing, optimum mixing time; PP, polymeric protein; SMP, smaller monomeric proteins; SPP, smaller polymeric proteins; Total, UPP, total unextractable polymeric protein in the total polymeric protein. * Corresponding author. Tel.: þ 46-40-415563; fax: þ 46-40-415519. E-mail address: [email protected] (R. Kuktaite). 0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2004.04.007
comprises a mixture of polymers, HMW-gs and LMW-gs, with a wide range of size distribution, ranging from dimers to polymers with molecular weights up to millions (Wrigley, 1996). The polymers are formed by intermolecular disulphide bond linked HMW-gs and LMW-gs. These polymeric proteins are known to be the most important determinants of breadmaking quality (Dupont and Altenbach, 2003; Field et al., 1983). Due to the large size and structural complexity of the polymeric proteins, they have been studied to a lesser extent than specific HMW-gs and LMW-gs. To understand more precisely the role and function of the gluten polymers for bread making quality, more information is needed. Most of the studies regarding polymeric proteins are related to the impact of individual protein classes, HMW-gs and LMW-gs, or the genes encoding them (Gras et al., 2001). However, glutenin subunits explain only a small proportion of the variation in quality (Weegels et al., 1996). Variation in the composition of polymeric proteins between wheat cultivars has been demonstrated (Gupta et al., 1996; Johansson et al., 2001; Kuktaite et al., 2000; Lindsay and Skerritt, 2000; Singh and MacRitchie, 2001). The relative
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amount of polymeric protein increases with increasing gluten strength (Johansson et al., 2001; Kuktaite et al., 2000). The effects of the glutenin polymer function in dough and for flour end-use quality have also been investigated (Bekes et al., 1994; Uthayakumaran et al., 1999). Changes in the gluten protein network during dough formation have been investigated using rheological methods (Kuktaite et al., 2003; Larsson et al., 2000), electrophoretical methods (Lin et al., 2003; Shewry et al., 2003), microscopical methods (Bache and Donald, 1998; Le´tang et al., 1999) and gel-filtration techniques (Lee et al., 2002). Many protein structural studies, as well as mixing and baking studies, have postulated that disulphide bonds are present in gluten polymer structure and contribute to the process of dough formation through the disulphide-sulphydryl exchange (Lindsay et al., 2000; Tilley et al., 2001). However, a full understanding of the structure of the gluten polymer during dough processing, as well as of the changes in molecular associations, is still far from being reached. The aim of the present study was to investigate the structure –function changes in the protein polymers during dough processing. Similarities and differences in changes in protein structure between flours of different qualities were also evaluated.
2. Experimental 2.1. Wheat flour Four different types of commercial wheat flours: biscuit (biscuit flour, Ritmo cultivar, containing HMW-gs ‘2 þ 12’), standard (French bread flour, a mixture of Tarso and Vinjett cultivars, both containing HMW-gs ‘5 þ 10’), strong (a flour used for improving weak flour mixtures, imported from Canada, HMW-gs ‘5 þ 10’), durum (pasta flour, imported from Kazakhstan) and one rye flour, supplied by Nord Mills AB (Malmo¨, Sweden) were used in the present study. All five flour samples were used for comparisons of protein polymer structure in the flour. Biscuit, standard and strong flours were used for investigations of protein polymer (gluten phase) structure during dough formation. According to the suppliers, the protein contents were: biscuit 11.15%, standard 13.9%, strong 16.8%, durum 15.0% and rye 7.4%. 2.2. Dough mixing Doughs were prepared from flour and water to investigate the influence of mixing time on the protein structure. Flour (10 g) was mixed with distilled water (water contents differed depending on protein contents and flour moisture contents, 6.04– 6.92 g). Mixing was performed at 25 8C, ¨ ved, using a mixograph (Reomixer, Bohlin Reologi, O Sweden) (Kuktaite et al., 2003). Three mixing times were
chosen for each flour: Min mixing time (half of the highest mixing resistance in the mixograph), Opt (the highest mixing resistance in the mixograph) and Overmixing time (20 min) according to Larsson and Eliasson (1996). At least three doughs were mixed under each set of mixing times and used for ultracentrifugation to separate the gluten phase for HPLC analysis. 2.3. Ultracentrifugation After 30 min resting, the mixed dough was centrifuged for 1 h at 100,000g in an ultracentrifuge (LE 80K OPTIMA, Beckman, USA) (Larsson and Eliasson, 1996). After centrifugation the dough was separated into five phases: liquid, gel, gluten, starch and bottom phase (Kuktaite et al., 2000, 2003). The gluten phase was carefully separated from the rest of the phases, freeze-dried and used for the protein analysis. 2.4. SE-HPLC Proteins from the flour samples and gluten phases were fractionated through size-exclusion high performance liquid chromatography (SE-HPLC) using a BiosepSEC-S-4000 Peek Phenomenex column (Kuktaite et al., 2000, 2003) (Fig. 1). Proteins from the flour samples and gluten phases (11 mg) were extracted with a two-step extraction procedure (Gupta et al., 1993). The first step in this method extracts the SDS-extractable proteins (proteins soluble in dilute sodium dodecyl sulphate (SDS)), whilst the second extract contains the SDSunextractable proteins (proteins soluble only after sonication). The extracted proteins were separated on SEHPLC according to Johansson et al. (2001). At least two replicates of each flour sample were used for the investigation of protein composition. The chromatograms were divided into four sections with decreasing molecular size (Fig. 1): large polymeric proteins (LPP, peak 1), smaller polymeric proteins (SPP, peak 2), large monomeric proteins (LMP, peak 3) and smaller monomeric proteins (SMP, peak 4) (Johansson et al., 2001; Kuktaite et al., 2000, 2003). Molecular weight range and specific protein composition of the proteins within the SE-HPLC chromatograms are described in Larroque et al. (1996). The percentage of large unextractable polymeric protein in the total large polymeric protein (large UPP) was calculated as [peak 1 area (unextractable)/peak 1 area (total)] £ 100. Peak 1 (total) refers to the total of peak 1 (extractable) and peak 1 (unextractable). In the same way, the percentage of total unextractable polymeric protein in the total polymeric protein (total UPP) was calculated as [peak 1 þ 2 area (unextractable)/peak 1 þ 2 area (total)] £ 100. Peak 1 þ 2 (total) refers to the total of peak 1 þ 2 (extractable) and peak1 þ 2 (unextractable) (Johansson et al., 2001; Kuktaite et al., 2000, 2003). The percentage
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Fig. 1. Example of SE-HPLC chromatogram of strong flour at optimum mixing time (a) SDS-extractable and (b) SDS-unextractable proteins. The chromatograms are divided into four parts containing large polymeric proteins (LPP), smaller polymeric proteins (SPP), large monomeric proteins (LMP), and smaller monomeric proteins (SMP).
of large unextractable monomeric proteins in the total large monomeric protein (large UMP) was calculated as [peak 3 area (unextractable)/peak 3 area (total)] £ 100. Peak 3 (total) refers to the total of peak 3 (extractable) and peak 3 (unextractable) (Johansson et al., 2003). SE-HPLC analysis was carried out in triplicate. 2.5. RP-HPLC 2.5.1. Protein extraction Wheat proteins were extracted from the freeze-dried gluten phases and flour samples before RP-HPLC analysis. The extraction was performed in nine steps. The extraction buffers during the different steps were as follows: 0.6 ml H2O, 0.6 ml 0.5 M NaCl, 0.3 ml 0.5 M NaCl, 0.6 ml 70% ethanol, 0.6 ml 50% 1-propanol, 0.3 ml 50% 1-propanol þ 1% 1,4-dithio-DL-threitol (DTT), (7) 0.6 ml 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid,
(1) (2) (3) (4) (5) (6)
(8) 0.6 ml 0.5% sodium dodecyl sulphate (SDS) þ 1% DTT, (9) 0.6 ml 6 M urea þ 0.5% SDS þ 1% DTT. The extracting materials were 100 mg of dough and ultracentrifuged gluten phase. After each extraction, the supernatant was collected and a new buffer was added to the pellet. The sample was suspended in the buffer, stirred for 30 min at 2000 rpm and centrifuged for 30 min at 10,000 g to obtain the supernatant. Extractions (2) and (3) were performed at 4 8C, (6) and (7) at 60 8C and for extractions (8) and (9) the samples were heated to 100 8C for 5 min. The other extractions were made at room temperature. The samples were rinsed with H2O between extractions (3) and (4) in order to remove the salt.
2.5.2. Protein separation by RP-HPLC After the protein extractions (9 steps) from the gluten phases and flour samples, protein fractionation was carried out using reversed phase chromatography (RP-HPLC) (Kuktaite et al., 2000). Three replicates of each gluten phase were analysed and at least two replicates of the flour samples were used for the investigation of protein composition.
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3. Results 3.1. Percentages of large UPP and total UPP in flours The flours studied in this work exhibited a variation in percentage of large UPP and total UPP (Fig. 2a). Statistically significant higher percentages of large UPP
Fig. 2. Percentages of (a) large UPP and total UPP in different flour types, (b) large UPP in gluten phase of different flours after different mixing times, and (c) total UPP of different flours after different mixing times. Bars indicate standard deviations.
and total UPP were found for the strong flour compared to the rest of the flours. Rye flour had, significantly, the lowest percentages of large UPP and total UPP (Fig. 2a). 3.2. Protein interactions during dough mixing Compared to flour, the percentages of large UPP and total UPP in gluten phases increased at Min mixing for the three flours investigated (Fig. 2a –c). The influences of mixing on percentage of large UPP and total UPP in gluten phases were similar for all flours investigated. Percentages of large UPP and total UPP in gluten phases were not influenced from Min to Opt, but were decreased after overmixing (Fig. 2b and c). The percentage of large UPP and total UPP was lower for biscuit flour compared to standard and strong flour at Min and Opt mixing. The decrease from Opt to overmixing in percentage of large UPP and total UPP was lower for biscuit flour compared to standard and strong flour. Significantly higher relative amounts of SDS-extractable proteins were found for the biscuit flour compared to the standard and strong flours during Min and Opt mixing (Fig. 3). From opt to overmixing, the percentages of SDSextractable proteins increased (Fig. 3) and the percentages of SDS-unextractable proteins decreased for all flours. The changes were smaller for the biscuit flour compared to the strong and standard flour. The composition of SDS-unextractable large monomeric proteins (large UMP) did not differ significantly between the flours studied (Fig. 4a). A higher percentage of large UMP was found in the gluten phases compared to the flours (Fig. 4). No significant changes in percentage of large UMP were found between Min and Opt mixing for any of the flours, but there was a decrease in percentage of large UMP
Fig. 3. Percentage of SDS-extractable protein (calculated from areas of SEHPLC chromatograms) in gluten phases of different flours mixed at different mixing times. Bars indicate standard deviations.
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For the gluten phases from all the flours and mixing times, a relatively lower amount of gluten proteins extracted in step 4 and 5 were found compared to the flours (Fig. 6b– d). Relatively higher amounts of glutenin proteins extracted with the buffers 6– 9 were found for all gluten phases, independent of flour type and mixing time, compared to flours (Fig. 6). No significant differences in extractability of proteins were detected between gluten phases of different flours or mixing times.
4. Discussion
Fig. 4. Percentage of large UMP in different (a) flours and (b) gluten phases. Bars indicate standard deviations.
at Overmixing. The largest changes in percentage of large UMP were found for strong flour (Fig. 4b). 3.3. Extractability of protein from flours Differences in extractability of gluten proteins were found between the different flours (Fig. 5a). The differences were mainly seen in extraction steps 6 – 8, in which the glutenin subunits were extracted. For biscuit flour, the highest amount of the glutenin subunits was extracted in extraction step 6 (Fig. 5a). In extraction step 7, the highest extracted amounts of glutenin subunits were found for standard flour compared to the other flours. A significantly higher amount of extracted glutenins was found for strong flour compared to the other flours during extraction step 8 (Fig. 5a). 3.4. Extractability of protein from dough Proteins extractable with all the types of extraction buffers used were found in the gluten phases (results not shown). A comparison of the amount of gluten proteins extracted in extraction steps 4– 9 showed large differences between the flour and the gluten phases (Fig. 6).
In the current investigation, the different flours of various gluten strengths exposed differences in percentage of large UPP and total UPP, and in extractability of glutenin subunits. The different flours did not show any statistical differences in percentage of large UMP. Several authors have reported the positive relationship between percentage of large UPP and total UPP and gluten strength (Johansson et al., 2001; Singh and MacRitchie, 2001). In this study, as well as in previous studies (Johansson et al., 2001), no relationship between percentage of monomeric proteins (e.g. large UMP) and gluten strength has been found. Quality varies considerably as a result of environmental conditions and genetic composition of wheat cultivars (Dupont and Altenbach, 2003; Johansson and Svensson, 1998, 1999). The composition of proteins and protein subunits is genetically determined (Payne, 1987). The protein composition of the flours used in our study varied, with biscuit wheat containing HMW glutenin subunits 2 þ 12, while standard and strong flours contained subunits 8 þ 10. One of the cultivars, Tarso, included in the standard flour, also contained a 1BL/1RS rye translocation (Johansson et al., 2002). Thus, the gluten strength of the flours reflected well the protein composition of the cultivars, with HMW gluten subunits 5 þ 10 implying strong gluten properties, and 2 þ 12 and the 1BL/1RS rye translocation resulting in weak gluten properties (Lee et al., 1995; Payne et al., 1987). Variations in flour strength are governed by a shifting of the polymeric protein (PP) fraction from SDSsoluble to SDS-insoluble large polymeric proteins (LPP), leading to changed percentages of large UPP and total UPP (Johansson et al., 2001). This shifting was seen in the wheat flours in our study, and is most likely related to the protein composition. The influence of the protein composition was also evident in the results from the extractability investigations performed in the present study. While it was possible to extract the largest part of the glutenin subunits with propanol and DTT in the biscuit flour, acetic acid was also needed for the standard flour. Reducing pH with acetic acid influenced the charged amino acids in the proteins, causing reductions of disulphide bonds of the polymeric proteins (Horton et al., 1993). However, the selective cleavage of inter-molecular bonds over intra-molecular bonds at low reducing agent conditions (i.e. DTT concentration) results
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Fig. 5. Relative amounts of extractable proteins (calculated from areas of RP-HPLC chromatograms) in (a) different flours and gluten phases after (b) min mixing, (c) opt mixing and (d) overmixing. Extraction buffers used during the different extraction steps were: 4 ¼ 70% ethanol, 5 ¼ 50% 1-propanol, 6 ¼ 50% 1-propanol þ 1% 1,4-dithio-DL-threitol (DTT), 7 ¼ 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid, 8 ¼ 0.5% sodium dodecyl sulphate (SDS) þ 1% DTT, 9 ¼ 6 M urea þ 0.5% SDS þ 1% DTT. Bars indicate standard deviations.
in subunits being released from the gluten polymer before they are fully reduced (Lindsay and Skerritt, 1998), as for the flours at the extraction step 6 (50% 1-propanol þ 1% DTT). For the strong flour, SDS was needed to reduce the amount of hydrophobic interactions within the protein polymers, leading to conformational rearrangements (unfolding) of the protein structure. It is known that the composition of the polymeric protein results in differences in reduction of individual subunits from the polymer, particularly between flours of different HMW-gs composition (Lindsay and Skerritt, 1998). This means that the different extractability of the strong flour proteins, compared to the other flours, might be related to a higher number of disulphide bonds between protein subunits, strength of bonds, and/or branching patterns of the subunits. Thus, the disulphide bonds between particular polypeptides of strong flour were not more susceptible for the DTT reduction until after SDS treatment. Protein composition and gluten strength of the cultivars thereby reflect well the polymeric structure of protein in the flour, with more hidden and harder to reach disulphide bonds in the cultivars with higher gluten strength. Dough mixing results in changes in gluten structure (Lindsay et al., 2000; Skerritt et al., 1999a,b; Weegels et al., 1997). As the structure of gluten changes, functionally
induced alterations in the protein composition take place (Bekes et al., 2001), and cross-links between tyrosine residues of HMW-gs (Tilley et al., 2001) and disulphide bonds between cystein residues (Shewry and Tatham, 1997) are formed. In the present investigation, the observed increase in the percentage of large UPP, total UPP and large UMP when mixing began, indicated such an effect on gluten structure. However, contrary to earlier findings showing that the amount of HMW glutenins increased with progressive stages of dough mixing (Lee et al., 2002), the percentages of large UPP, total UPP and large UMP did not increase significantly from Min to Opt mixing time. After Overmixing, percentages of large UPP, total UPP and large UMP decreased below the level in the respective wheat flours. This is in accordance with earlier findings (Skerritt et al., 1999a,b) of overmixing leading to a breakdown of disulphide bonds and a disruption of the protein matrix. In earlier investigations, it was indicated that the size and the amount of HMW glutenins are related to the strength of the dough and the amount of protein matrix present in the dough (Lee et al., 2002). A higher percentage of large UPP, total UPP and large UMP were also found for the standard and strong flour after Min and Opt mixing in the present investigation. However, the differences in percentage of large UPP and total UPP were recorded in the flours already
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Fig. 6. SDS-PAGE of gliadins and glutenins extracted in steps with different extraction buffers of gluten phase (a) proteins extracted with extraction buffer 7 ¼ 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid; (b) proteins extracted with extraction buffer 8 ¼ 0.5% SDS þ 1% DTT; (c) proteins extracted with extraction buffer 9 ¼ 6 M urea þ 0.5% SDS þ 1% DTT) and flour (d) proteins extracted with extraction buffer 1 ¼ H2O; (e) proteins extracted with extraction buffer 2 ¼ NaCl; (f) proteins extracted with extraction buffer 4 ¼ 70% ethanol; (g) proteins extracted with extraction buffer 5 ¼ 50% 1-propanol; (h) proteins extracted with extraction buffer 6 ¼ 50% 1-propanol þ 1% DTT; (i) proteins extracted with extraction buffer 7 ¼ 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid; (j) proteins extracted with extraction buffer 8 ¼ 0.5% SDS þ 1% DTT; (k) proteins extracted with extraction buffer 9 ¼ 6 M urea þ 0.5% SDS þ 1% DTT). The parenthesis in slots a, b, c, and g mark some omega gliadins. The parenthesis in slot k mark HMW albumins (Gupta et al., 1991).
before mixing started, indicating that the differences in protein matrix are already determined genetically or environmentally (Johansson et al., 2003) in the wheat grain. Earlier findings (Lee et al., 2002) have shown that the amount of gliadins decreased with progressive stages of dough development. In the present investigation large UMP increased when dough mixing started compared to in the flour, similarly to large UPP and total UPP. This indicates that the amount of gliadins unextractable with SDS increases during min and opt mixing. This might be related to the entanglements and specific non-covalent interactions in the gluten network during dough mixing. Large differences in glutenin macropolymer (GMP) extractability with acetic acid have been observed between flours during mixing. Weaker flours have higher rates of extractability compared to strong flours (Weegels et al., 1996) and the extractability of proteins from the GMP of doughs changes after different dough resting times (Weegels et al., 1997). Directly after mixing the extractability of HMW-gs from GMP is much lower compared to flour but after resting the amount of extractable HMW-gs from GMP again reaches a similar level to that in flour, although the composition changes (Weegels et al., 1997).
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In the present investigation, the composition of extractable gluten proteins changed dramatically in the gluten phases compared to the flours, although the doughs were allowed to rest before the ultracentrifugation (Kuktaite et al., 2003; Larsson and Eliasson, 1996). In the wheat flours, about 25% of the gluten proteins were extracted in step 4 and 5, i.e. with 70% ethanol and 50% propanol, extracting mainly all the gliadins in wheat flour. These are roughly similar to amounts found in an earlier investigation, covering four bread wheat cultivars grown during three different years (Johansson, unpublished results). In the gluten phases, irrespective of flour and mixing time used, only about 5% of the gluten proteins were extracted at step 4 and 5. However between 35– 40% of the gluten proteins were extracted at extraction step 7, i.e. with 50% propanol, 1% DTT and 1% acetic acid. The influence of the acetic acid, lowering the pH of the readily extractable proteins, indicates strong ionic interactions within the polymeric proteins in the gluten phase compared to the flour. These ionic interactions seem to involve both the glutenins and gliadins, mainly the omega gliadins, since the gliadins are not extracted at extraction steps 4 and 5. In addition, a comparison of the protein composition, revealed by SDSPAGE, at extraction steps 7, 8, 9 of flour and gluten phases, shows the differences in gliadin composition between the flour and the gluten phases (Fig. 6).
5. Conclusions The amount and size distribution of polymeric protein in the flour sample, genetically and environmentally determined, form the basis for the build-up (cross-linking) of protein polymers during dough formation. The pattern of cross-links and breakdown of the protein polymer during dough formation is similarly independent of flour type, although is not as pronounced in weak flours as in stronger flours. Glutenins, and particularly omega-gliadins, seem to take part in polymer formation during dough processing. The omega-gliadins interact with the glutenins during dough processing through non-covalent interactions. During dough formation the protein polymer seems to have hydrophilic residues orientated on the outside, so they can interact with the aqueous environment, and the hydrophobic part inside. This explains the differences in protein extractability between flour and gluten, i.e. it is difficult to extract gliadins inside the protein polymer due to non-covalent interactions.
Acknowledgements The study was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, the Royal Physiographical Society
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and the Swedish University of Agricultural Sciences. We thank Maria-Luisa Prieto Linde for technical assistance.
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Larroque, O.R., Gianibelli, M.C., Batey, I.L., MacRitchie, F., 1996. Identification of elution subfractions from the first peak in SE-HPLC chromatograms of wheat storage protein. In: Wrigley, C.W., (Ed.), Proceedings of Sixth International Gluten Workshop, Cereal Chemistry Division, Royal Australian Chemical Institute, North Melbourne, Australia, pp. 288 –293. Larsson, H., Eliasson, A.-C., 1996. Phase separation of wheat flour dough studied by ultracentrifugation and stress relaxation. I. Influence of water content. Cereal Chemistry 73, 18–24. Larsson, H., Eliasson, A.C., Johansson, E., Svensson, G., 2000. Influence of added starch on mixing of dough made with three wheat flours differing in high molecular weight subunit composition: rheological behavior. Cereal Chemistry 77, 633–639. Lee, J.H., Graybosch, R.A., Peterson, C.J., 1995. Quality and biochemical effects of a 1BL/1RS wheat-rye translocation in wheat. Theoretical Applied Genetics 90, 105–112. Lee, L., Ng, P.K.W., Steffe, J.F., 2002. Biochemical studies of proteins in nondeveloped, partially developed, and developed doughs. Cereal Chemistry 79, 654 –661. Le´tang, C., Piau, M., Verdier, C., 1999. Characterization of wheat flour– water doughs. Part I: rheometry and microstructure. Journal of Food Engineering 41, 121–132. Lin, P.F., Chiang, S.H., Chang, C.Y., 2003. Comparison of rheological properties of dough prepared with different wheat flours. Journal of Food and Drug Analysis 11, 220 –225. Lindsay, M.P., Skerritt, J.H., 1998. Examination of the structure of the glutenin macropolymer in wheat flour and doughs by stepwise reduction. Journal of Agriculture Food Chemistry 46, 3447– 3457. Lindsay, M.P., Skerritt, J.H., 2000. Immunocytochemical localisation of gluten proteins uncovers structural organization of glutenin macropolymer. Cereal Chemistry 77, 360–369. Lindsay, M.P., Tamas, L., Appels, R., Skerritt, J.H., 2000. Direct evidence that the number and location of cysteine residues affect glutenin polymer structure. Journal of Cereal Science 31, 321–333. Payne, P.I., 1987. Genetics of wheat storage proteins and the effect of allelic variation on bread-making quality. Annual Review of Plant Physiology 38, 141–153. Payne, P.I., Nightingale, M.A., Krattiger, A.F., Holt, L.M., 1987. The relationship between HMW glutenin subunit composition and the bread-making quality in British-grown wheat varieties. Journal of Food Science and Agriculture 40, 51–65. Sapirstein, H.D., Fu, B.X., 1998. Intercultivar variation in the quantity of monomeric proteins, soluble and insoluble glutenin, and residue protein in wheat flour and relationships to breadmaking quality. Cereal Chemistry 75, 500 –507. Shewry, P.R., Tatham, A.S., 1990. The prolamin storage proteins of cereal seeds: structure and evolution. Journal of Biochemistry 267, 1–12. Shewry, P.R., Tatham, A.S., 1997. Disulphide bonds in wheat gluten proteins. Journal of Cereal Science 25, 207 –227. Shewry, P.R., Halford, N.G., Belton, P.S., Tatham, A.S., 2002. The structure and properties of gluten: an elastic protein from wheat grain. Philosophical Transactions of the Royal Society of London-Series B: Biological Sciences 357, 133 –142. Shewry, P.R., Halford, N.G., Lafiandra, D., 2003. Genetics of wheat gluten proteins. Advances in Genetics 49, 111 –184. Singh, H., MacRitchie, F., 2001. Application of polymer science to properties of gluten. Journal of Cereal Science 33, 231–243. Skerritt, J.H., Hac, L., Bekes, F., 1999a. Depolymerization of the glutenin macroplymer during dough mixing: I. Changes in levels, molecular weight distribution, and overall composition. Cereal Chemistry 76, 395 –401. Skerritt, J.H., Hac, L., Lindsay, M.P., Bekes, F., 1999b. Depolymerization of the glutenin macropolymer during mixing: II. Differences in retention of specific glutenin subunits. Cereal Chemistry 76, 402 –409. Tilley, K.A., Benjamin, R.E., Bagorogoza, K.E., Okot-Kotber, B.M., Prakash, O., Kwen, H., 2001. Tyrosine cross-links: molecular basis
R. Kuktaite et al. / Journal of Cereal Science 40 (2004) 31–39 of gluten structure and function. Journal of Agriculture Food Chemistry 49, 2627–2632. Uthayakumaran, S., Gras, P.W., Stoddard, F.L., Bekes, F., 1999. Effect of varying protein content and glutenin-to-gliadin ratio on the functional properties of wheat dough. Cereal Chemistry 76, 389 –394. Wall, J.S., 1979. The role of wheat proteins in determining baking quality. In: Laidman, D.L., Wyn Jones, R.G. (Eds.), Recent advances in the biochemistry of cereals, Academy, London, pp. 275–311.
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Weegels, P.L., Hamer, R.J., Schofield, J.D., 1996. Functional properties of wheat glutenin. Journal of Cereal Science 23, 1–18. Weegels, P.L., Hamer, R.J., Schofield, J.D., 1997. Depolymerisation and re-polymerisation of wheat glutenin during dough processing. II Changes in composition. Journal of Cereal Science 25, 155–163. Wrigley, C.W., 1996. Giant proteins with flour power. Nature 381, 738– 739.
Variation in protein composition of wheat flour and its relationship to dough mixing behaviour R. Kuktaitea,*, H. Larssonb, E. Johanssona a
Department of Crop Science, The Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53 Alnarp, Sweden b Department of Food Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received 19 December 2003; revised 31 March 2004; accepted 5 April 2004
Abstract Changes in extractability, amount and size distribution of polymeric proteins in the gluten of doughs during mixing were investigated. Ultracentrifugation was used as a non-destructive method to separate the gluten from the dough. Doughs prepared from commercial flour mixtures of different gluten strengths and mixed for varying periods, were analysed. Proteins were detected using RP- and SE-HPLC. The percentages of large unextractable polymeric protein (UPP), total UPP and large unextractable monomeric protein (UMP) were higher in the gluten phases of all flours at minimum and optimum mixing, compared to the flours. After overmixing, the percentages of large UPP, total UPP and large UMP in the gluten phases of the dough decreased to lower levels than in the flours. Differences in percentages of large UPP, total UPP and large UMP between gluten phases of different flours and mixing times originated from the genetic composition of flour proteins. The extractability of the glutenins in the flours reflected the quality of the specific flour. The protein extractability, especially of gliadins, was different in the gluten phase compared to in the flour. Ionic interactions seem to be important forces in the dough. q 2004 Elsevier Ltd. All rights reserved. Keywords: Wheat flour; Protein polymers; Protein composition; Dough mixing; Structure; Function
1. Introduction The end-use characteristics of flours produced from bread wheat are strongly determined by the gluten proteins (Wall, 1979; Weegels et al., 1996). Gluten proteins are particularly important for bread-making quality and consist of two major fractions: the monomeric gliadins and the polymeric glutenins (Sapirstein and Fu, 1998; Shewry and Tatham, 1990; Singh and MacRitchie, 2001). Flour proteins interact in the presence of water by forming gluten, which provides the unique viscoelastic properties needed for bread-making (Shewry et al., 2002). The glutenin fraction Abbreviations: HMW-gs, high molecular weight glutenin subunits; Large, UMP, large unextractable monomeric protein in the total monomeric protein; Large, UPP, large unextractable polymeric protein in the total large polymeric protein; LMP, large monomeric proteins; LMW-gs, low molecular weight glutenin subunits; LPP, large polymeric proteins; Min, mixing, minimum mixing time; Opt, mixing, optimum mixing time; PP, polymeric protein; SMP, smaller monomeric proteins; SPP, smaller polymeric proteins; Total, UPP, total unextractable polymeric protein in the total polymeric protein. * Corresponding author. Tel.: þ 46-40-415563; fax: þ 46-40-415519. E-mail address: [email protected] (R. Kuktaite). 0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2004.04.007
comprises a mixture of polymers, HMW-gs and LMW-gs, with a wide range of size distribution, ranging from dimers to polymers with molecular weights up to millions (Wrigley, 1996). The polymers are formed by intermolecular disulphide bond linked HMW-gs and LMW-gs. These polymeric proteins are known to be the most important determinants of breadmaking quality (Dupont and Altenbach, 2003; Field et al., 1983). Due to the large size and structural complexity of the polymeric proteins, they have been studied to a lesser extent than specific HMW-gs and LMW-gs. To understand more precisely the role and function of the gluten polymers for bread making quality, more information is needed. Most of the studies regarding polymeric proteins are related to the impact of individual protein classes, HMW-gs and LMW-gs, or the genes encoding them (Gras et al., 2001). However, glutenin subunits explain only a small proportion of the variation in quality (Weegels et al., 1996). Variation in the composition of polymeric proteins between wheat cultivars has been demonstrated (Gupta et al., 1996; Johansson et al., 2001; Kuktaite et al., 2000; Lindsay and Skerritt, 2000; Singh and MacRitchie, 2001). The relative
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amount of polymeric protein increases with increasing gluten strength (Johansson et al., 2001; Kuktaite et al., 2000). The effects of the glutenin polymer function in dough and for flour end-use quality have also been investigated (Bekes et al., 1994; Uthayakumaran et al., 1999). Changes in the gluten protein network during dough formation have been investigated using rheological methods (Kuktaite et al., 2003; Larsson et al., 2000), electrophoretical methods (Lin et al., 2003; Shewry et al., 2003), microscopical methods (Bache and Donald, 1998; Le´tang et al., 1999) and gel-filtration techniques (Lee et al., 2002). Many protein structural studies, as well as mixing and baking studies, have postulated that disulphide bonds are present in gluten polymer structure and contribute to the process of dough formation through the disulphide-sulphydryl exchange (Lindsay et al., 2000; Tilley et al., 2001). However, a full understanding of the structure of the gluten polymer during dough processing, as well as of the changes in molecular associations, is still far from being reached. The aim of the present study was to investigate the structure –function changes in the protein polymers during dough processing. Similarities and differences in changes in protein structure between flours of different qualities were also evaluated.
2. Experimental 2.1. Wheat flour Four different types of commercial wheat flours: biscuit (biscuit flour, Ritmo cultivar, containing HMW-gs ‘2 þ 12’), standard (French bread flour, a mixture of Tarso and Vinjett cultivars, both containing HMW-gs ‘5 þ 10’), strong (a flour used for improving weak flour mixtures, imported from Canada, HMW-gs ‘5 þ 10’), durum (pasta flour, imported from Kazakhstan) and one rye flour, supplied by Nord Mills AB (Malmo¨, Sweden) were used in the present study. All five flour samples were used for comparisons of protein polymer structure in the flour. Biscuit, standard and strong flours were used for investigations of protein polymer (gluten phase) structure during dough formation. According to the suppliers, the protein contents were: biscuit 11.15%, standard 13.9%, strong 16.8%, durum 15.0% and rye 7.4%. 2.2. Dough mixing Doughs were prepared from flour and water to investigate the influence of mixing time on the protein structure. Flour (10 g) was mixed with distilled water (water contents differed depending on protein contents and flour moisture contents, 6.04– 6.92 g). Mixing was performed at 25 8C, ¨ ved, using a mixograph (Reomixer, Bohlin Reologi, O Sweden) (Kuktaite et al., 2003). Three mixing times were
chosen for each flour: Min mixing time (half of the highest mixing resistance in the mixograph), Opt (the highest mixing resistance in the mixograph) and Overmixing time (20 min) according to Larsson and Eliasson (1996). At least three doughs were mixed under each set of mixing times and used for ultracentrifugation to separate the gluten phase for HPLC analysis. 2.3. Ultracentrifugation After 30 min resting, the mixed dough was centrifuged for 1 h at 100,000g in an ultracentrifuge (LE 80K OPTIMA, Beckman, USA) (Larsson and Eliasson, 1996). After centrifugation the dough was separated into five phases: liquid, gel, gluten, starch and bottom phase (Kuktaite et al., 2000, 2003). The gluten phase was carefully separated from the rest of the phases, freeze-dried and used for the protein analysis. 2.4. SE-HPLC Proteins from the flour samples and gluten phases were fractionated through size-exclusion high performance liquid chromatography (SE-HPLC) using a BiosepSEC-S-4000 Peek Phenomenex column (Kuktaite et al., 2000, 2003) (Fig. 1). Proteins from the flour samples and gluten phases (11 mg) were extracted with a two-step extraction procedure (Gupta et al., 1993). The first step in this method extracts the SDS-extractable proteins (proteins soluble in dilute sodium dodecyl sulphate (SDS)), whilst the second extract contains the SDSunextractable proteins (proteins soluble only after sonication). The extracted proteins were separated on SEHPLC according to Johansson et al. (2001). At least two replicates of each flour sample were used for the investigation of protein composition. The chromatograms were divided into four sections with decreasing molecular size (Fig. 1): large polymeric proteins (LPP, peak 1), smaller polymeric proteins (SPP, peak 2), large monomeric proteins (LMP, peak 3) and smaller monomeric proteins (SMP, peak 4) (Johansson et al., 2001; Kuktaite et al., 2000, 2003). Molecular weight range and specific protein composition of the proteins within the SE-HPLC chromatograms are described in Larroque et al. (1996). The percentage of large unextractable polymeric protein in the total large polymeric protein (large UPP) was calculated as [peak 1 area (unextractable)/peak 1 area (total)] £ 100. Peak 1 (total) refers to the total of peak 1 (extractable) and peak 1 (unextractable). In the same way, the percentage of total unextractable polymeric protein in the total polymeric protein (total UPP) was calculated as [peak 1 þ 2 area (unextractable)/peak 1 þ 2 area (total)] £ 100. Peak 1 þ 2 (total) refers to the total of peak 1 þ 2 (extractable) and peak1 þ 2 (unextractable) (Johansson et al., 2001; Kuktaite et al., 2000, 2003). The percentage
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Fig. 1. Example of SE-HPLC chromatogram of strong flour at optimum mixing time (a) SDS-extractable and (b) SDS-unextractable proteins. The chromatograms are divided into four parts containing large polymeric proteins (LPP), smaller polymeric proteins (SPP), large monomeric proteins (LMP), and smaller monomeric proteins (SMP).
of large unextractable monomeric proteins in the total large monomeric protein (large UMP) was calculated as [peak 3 area (unextractable)/peak 3 area (total)] £ 100. Peak 3 (total) refers to the total of peak 3 (extractable) and peak 3 (unextractable) (Johansson et al., 2003). SE-HPLC analysis was carried out in triplicate. 2.5. RP-HPLC 2.5.1. Protein extraction Wheat proteins were extracted from the freeze-dried gluten phases and flour samples before RP-HPLC analysis. The extraction was performed in nine steps. The extraction buffers during the different steps were as follows: 0.6 ml H2O, 0.6 ml 0.5 M NaCl, 0.3 ml 0.5 M NaCl, 0.6 ml 70% ethanol, 0.6 ml 50% 1-propanol, 0.3 ml 50% 1-propanol þ 1% 1,4-dithio-DL-threitol (DTT), (7) 0.6 ml 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid,
(1) (2) (3) (4) (5) (6)
(8) 0.6 ml 0.5% sodium dodecyl sulphate (SDS) þ 1% DTT, (9) 0.6 ml 6 M urea þ 0.5% SDS þ 1% DTT. The extracting materials were 100 mg of dough and ultracentrifuged gluten phase. After each extraction, the supernatant was collected and a new buffer was added to the pellet. The sample was suspended in the buffer, stirred for 30 min at 2000 rpm and centrifuged for 30 min at 10,000 g to obtain the supernatant. Extractions (2) and (3) were performed at 4 8C, (6) and (7) at 60 8C and for extractions (8) and (9) the samples were heated to 100 8C for 5 min. The other extractions were made at room temperature. The samples were rinsed with H2O between extractions (3) and (4) in order to remove the salt.
2.5.2. Protein separation by RP-HPLC After the protein extractions (9 steps) from the gluten phases and flour samples, protein fractionation was carried out using reversed phase chromatography (RP-HPLC) (Kuktaite et al., 2000). Three replicates of each gluten phase were analysed and at least two replicates of the flour samples were used for the investigation of protein composition.
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3. Results 3.1. Percentages of large UPP and total UPP in flours The flours studied in this work exhibited a variation in percentage of large UPP and total UPP (Fig. 2a). Statistically significant higher percentages of large UPP
Fig. 2. Percentages of (a) large UPP and total UPP in different flour types, (b) large UPP in gluten phase of different flours after different mixing times, and (c) total UPP of different flours after different mixing times. Bars indicate standard deviations.
and total UPP were found for the strong flour compared to the rest of the flours. Rye flour had, significantly, the lowest percentages of large UPP and total UPP (Fig. 2a). 3.2. Protein interactions during dough mixing Compared to flour, the percentages of large UPP and total UPP in gluten phases increased at Min mixing for the three flours investigated (Fig. 2a –c). The influences of mixing on percentage of large UPP and total UPP in gluten phases were similar for all flours investigated. Percentages of large UPP and total UPP in gluten phases were not influenced from Min to Opt, but were decreased after overmixing (Fig. 2b and c). The percentage of large UPP and total UPP was lower for biscuit flour compared to standard and strong flour at Min and Opt mixing. The decrease from Opt to overmixing in percentage of large UPP and total UPP was lower for biscuit flour compared to standard and strong flour. Significantly higher relative amounts of SDS-extractable proteins were found for the biscuit flour compared to the standard and strong flours during Min and Opt mixing (Fig. 3). From opt to overmixing, the percentages of SDSextractable proteins increased (Fig. 3) and the percentages of SDS-unextractable proteins decreased for all flours. The changes were smaller for the biscuit flour compared to the strong and standard flour. The composition of SDS-unextractable large monomeric proteins (large UMP) did not differ significantly between the flours studied (Fig. 4a). A higher percentage of large UMP was found in the gluten phases compared to the flours (Fig. 4). No significant changes in percentage of large UMP were found between Min and Opt mixing for any of the flours, but there was a decrease in percentage of large UMP
Fig. 3. Percentage of SDS-extractable protein (calculated from areas of SEHPLC chromatograms) in gluten phases of different flours mixed at different mixing times. Bars indicate standard deviations.
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For the gluten phases from all the flours and mixing times, a relatively lower amount of gluten proteins extracted in step 4 and 5 were found compared to the flours (Fig. 6b– d). Relatively higher amounts of glutenin proteins extracted with the buffers 6– 9 were found for all gluten phases, independent of flour type and mixing time, compared to flours (Fig. 6). No significant differences in extractability of proteins were detected between gluten phases of different flours or mixing times.
4. Discussion
Fig. 4. Percentage of large UMP in different (a) flours and (b) gluten phases. Bars indicate standard deviations.
at Overmixing. The largest changes in percentage of large UMP were found for strong flour (Fig. 4b). 3.3. Extractability of protein from flours Differences in extractability of gluten proteins were found between the different flours (Fig. 5a). The differences were mainly seen in extraction steps 6 – 8, in which the glutenin subunits were extracted. For biscuit flour, the highest amount of the glutenin subunits was extracted in extraction step 6 (Fig. 5a). In extraction step 7, the highest extracted amounts of glutenin subunits were found for standard flour compared to the other flours. A significantly higher amount of extracted glutenins was found for strong flour compared to the other flours during extraction step 8 (Fig. 5a). 3.4. Extractability of protein from dough Proteins extractable with all the types of extraction buffers used were found in the gluten phases (results not shown). A comparison of the amount of gluten proteins extracted in extraction steps 4– 9 showed large differences between the flour and the gluten phases (Fig. 6).
In the current investigation, the different flours of various gluten strengths exposed differences in percentage of large UPP and total UPP, and in extractability of glutenin subunits. The different flours did not show any statistical differences in percentage of large UMP. Several authors have reported the positive relationship between percentage of large UPP and total UPP and gluten strength (Johansson et al., 2001; Singh and MacRitchie, 2001). In this study, as well as in previous studies (Johansson et al., 2001), no relationship between percentage of monomeric proteins (e.g. large UMP) and gluten strength has been found. Quality varies considerably as a result of environmental conditions and genetic composition of wheat cultivars (Dupont and Altenbach, 2003; Johansson and Svensson, 1998, 1999). The composition of proteins and protein subunits is genetically determined (Payne, 1987). The protein composition of the flours used in our study varied, with biscuit wheat containing HMW glutenin subunits 2 þ 12, while standard and strong flours contained subunits 8 þ 10. One of the cultivars, Tarso, included in the standard flour, also contained a 1BL/1RS rye translocation (Johansson et al., 2002). Thus, the gluten strength of the flours reflected well the protein composition of the cultivars, with HMW gluten subunits 5 þ 10 implying strong gluten properties, and 2 þ 12 and the 1BL/1RS rye translocation resulting in weak gluten properties (Lee et al., 1995; Payne et al., 1987). Variations in flour strength are governed by a shifting of the polymeric protein (PP) fraction from SDSsoluble to SDS-insoluble large polymeric proteins (LPP), leading to changed percentages of large UPP and total UPP (Johansson et al., 2001). This shifting was seen in the wheat flours in our study, and is most likely related to the protein composition. The influence of the protein composition was also evident in the results from the extractability investigations performed in the present study. While it was possible to extract the largest part of the glutenin subunits with propanol and DTT in the biscuit flour, acetic acid was also needed for the standard flour. Reducing pH with acetic acid influenced the charged amino acids in the proteins, causing reductions of disulphide bonds of the polymeric proteins (Horton et al., 1993). However, the selective cleavage of inter-molecular bonds over intra-molecular bonds at low reducing agent conditions (i.e. DTT concentration) results
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Fig. 5. Relative amounts of extractable proteins (calculated from areas of RP-HPLC chromatograms) in (a) different flours and gluten phases after (b) min mixing, (c) opt mixing and (d) overmixing. Extraction buffers used during the different extraction steps were: 4 ¼ 70% ethanol, 5 ¼ 50% 1-propanol, 6 ¼ 50% 1-propanol þ 1% 1,4-dithio-DL-threitol (DTT), 7 ¼ 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid, 8 ¼ 0.5% sodium dodecyl sulphate (SDS) þ 1% DTT, 9 ¼ 6 M urea þ 0.5% SDS þ 1% DTT. Bars indicate standard deviations.
in subunits being released from the gluten polymer before they are fully reduced (Lindsay and Skerritt, 1998), as for the flours at the extraction step 6 (50% 1-propanol þ 1% DTT). For the strong flour, SDS was needed to reduce the amount of hydrophobic interactions within the protein polymers, leading to conformational rearrangements (unfolding) of the protein structure. It is known that the composition of the polymeric protein results in differences in reduction of individual subunits from the polymer, particularly between flours of different HMW-gs composition (Lindsay and Skerritt, 1998). This means that the different extractability of the strong flour proteins, compared to the other flours, might be related to a higher number of disulphide bonds between protein subunits, strength of bonds, and/or branching patterns of the subunits. Thus, the disulphide bonds between particular polypeptides of strong flour were not more susceptible for the DTT reduction until after SDS treatment. Protein composition and gluten strength of the cultivars thereby reflect well the polymeric structure of protein in the flour, with more hidden and harder to reach disulphide bonds in the cultivars with higher gluten strength. Dough mixing results in changes in gluten structure (Lindsay et al., 2000; Skerritt et al., 1999a,b; Weegels et al., 1997). As the structure of gluten changes, functionally
induced alterations in the protein composition take place (Bekes et al., 2001), and cross-links between tyrosine residues of HMW-gs (Tilley et al., 2001) and disulphide bonds between cystein residues (Shewry and Tatham, 1997) are formed. In the present investigation, the observed increase in the percentage of large UPP, total UPP and large UMP when mixing began, indicated such an effect on gluten structure. However, contrary to earlier findings showing that the amount of HMW glutenins increased with progressive stages of dough mixing (Lee et al., 2002), the percentages of large UPP, total UPP and large UMP did not increase significantly from Min to Opt mixing time. After Overmixing, percentages of large UPP, total UPP and large UMP decreased below the level in the respective wheat flours. This is in accordance with earlier findings (Skerritt et al., 1999a,b) of overmixing leading to a breakdown of disulphide bonds and a disruption of the protein matrix. In earlier investigations, it was indicated that the size and the amount of HMW glutenins are related to the strength of the dough and the amount of protein matrix present in the dough (Lee et al., 2002). A higher percentage of large UPP, total UPP and large UMP were also found for the standard and strong flour after Min and Opt mixing in the present investigation. However, the differences in percentage of large UPP and total UPP were recorded in the flours already
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Fig. 6. SDS-PAGE of gliadins and glutenins extracted in steps with different extraction buffers of gluten phase (a) proteins extracted with extraction buffer 7 ¼ 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid; (b) proteins extracted with extraction buffer 8 ¼ 0.5% SDS þ 1% DTT; (c) proteins extracted with extraction buffer 9 ¼ 6 M urea þ 0.5% SDS þ 1% DTT) and flour (d) proteins extracted with extraction buffer 1 ¼ H2O; (e) proteins extracted with extraction buffer 2 ¼ NaCl; (f) proteins extracted with extraction buffer 4 ¼ 70% ethanol; (g) proteins extracted with extraction buffer 5 ¼ 50% 1-propanol; (h) proteins extracted with extraction buffer 6 ¼ 50% 1-propanol þ 1% DTT; (i) proteins extracted with extraction buffer 7 ¼ 50% 1-propanol þ 1% DTT þ 1% glacial acetic acid; (j) proteins extracted with extraction buffer 8 ¼ 0.5% SDS þ 1% DTT; (k) proteins extracted with extraction buffer 9 ¼ 6 M urea þ 0.5% SDS þ 1% DTT). The parenthesis in slots a, b, c, and g mark some omega gliadins. The parenthesis in slot k mark HMW albumins (Gupta et al., 1991).
before mixing started, indicating that the differences in protein matrix are already determined genetically or environmentally (Johansson et al., 2003) in the wheat grain. Earlier findings (Lee et al., 2002) have shown that the amount of gliadins decreased with progressive stages of dough development. In the present investigation large UMP increased when dough mixing started compared to in the flour, similarly to large UPP and total UPP. This indicates that the amount of gliadins unextractable with SDS increases during min and opt mixing. This might be related to the entanglements and specific non-covalent interactions in the gluten network during dough mixing. Large differences in glutenin macropolymer (GMP) extractability with acetic acid have been observed between flours during mixing. Weaker flours have higher rates of extractability compared to strong flours (Weegels et al., 1996) and the extractability of proteins from the GMP of doughs changes after different dough resting times (Weegels et al., 1997). Directly after mixing the extractability of HMW-gs from GMP is much lower compared to flour but after resting the amount of extractable HMW-gs from GMP again reaches a similar level to that in flour, although the composition changes (Weegels et al., 1997).
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In the present investigation, the composition of extractable gluten proteins changed dramatically in the gluten phases compared to the flours, although the doughs were allowed to rest before the ultracentrifugation (Kuktaite et al., 2003; Larsson and Eliasson, 1996). In the wheat flours, about 25% of the gluten proteins were extracted in step 4 and 5, i.e. with 70% ethanol and 50% propanol, extracting mainly all the gliadins in wheat flour. These are roughly similar to amounts found in an earlier investigation, covering four bread wheat cultivars grown during three different years (Johansson, unpublished results). In the gluten phases, irrespective of flour and mixing time used, only about 5% of the gluten proteins were extracted at step 4 and 5. However between 35– 40% of the gluten proteins were extracted at extraction step 7, i.e. with 50% propanol, 1% DTT and 1% acetic acid. The influence of the acetic acid, lowering the pH of the readily extractable proteins, indicates strong ionic interactions within the polymeric proteins in the gluten phase compared to the flour. These ionic interactions seem to involve both the glutenins and gliadins, mainly the omega gliadins, since the gliadins are not extracted at extraction steps 4 and 5. In addition, a comparison of the protein composition, revealed by SDSPAGE, at extraction steps 7, 8, 9 of flour and gluten phases, shows the differences in gliadin composition between the flour and the gluten phases (Fig. 6).
5. Conclusions The amount and size distribution of polymeric protein in the flour sample, genetically and environmentally determined, form the basis for the build-up (cross-linking) of protein polymers during dough formation. The pattern of cross-links and breakdown of the protein polymer during dough formation is similarly independent of flour type, although is not as pronounced in weak flours as in stronger flours. Glutenins, and particularly omega-gliadins, seem to take part in polymer formation during dough processing. The omega-gliadins interact with the glutenins during dough processing through non-covalent interactions. During dough formation the protein polymer seems to have hydrophilic residues orientated on the outside, so they can interact with the aqueous environment, and the hydrophobic part inside. This explains the differences in protein extractability between flour and gluten, i.e. it is difficult to extract gliadins inside the protein polymer due to non-covalent interactions.
Acknowledgements The study was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, the Royal Physiographical Society
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and the Swedish University of Agricultural Sciences. We thank Maria-Luisa Prieto Linde for technical assistance.
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