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Heat and additive induced biochemical transitions in gluten from good and poor breadmaking quality wheats
Journal of Cereal Science 40 (2004) 245–256 www.elsevier.com/locate/jnlabr/yjcrs
Heat and additive induced biochemical transitions in gluten from good and poor breadmaking quality wheats Mehmet Haytaa,*, J. David Schofieldb a
Department of Food Engineering, Inonu University, Elazig Yolu 44069 Malatya, Turkey b Department of Food Science and Technology, Reading University, Reading, UK Received 26 February 2004; revised 7 June 2004; accepted 25 June 2004
Abstract Glutens from poor breadmaking quality wheat, cv. Riband, had a higher SDS extractability than glutens from good quality cv. Hereward. Heating of gluten, especially above 70 8C, caused a reduction in the amount of SDS-extractable gluten proteins. Treatment of gluten with redox additives (ascorbic acid, potassium bromate or glutathione) affected extractability, being highest for bromate treated glutens. The SH content of gluten was lower for poor breadmaking Riband and heating resulted in greater decrease in SH content of gluten from good breadmaking Hereward. Hereward gluten had a higher SS content than Riband. The alteration of SS content on heating was not significant and may indicate the heat-induced involvement of non-covalent interactions. SDS-PAGE revealed that oxidants, especially bromate, affect polypeptide composition leading to a more heat stable/tolerant protein structure. q 2004 Published by Elsevier Ltd. Keywords: Wheat gluten; Heating; Redox additives; Sulphydryl; Disulphide
1. Introduction Heat treatment can cause alterations in protein structure e.g. in conformation and molecular size (Phillips et al., 1994). During the baking process, gluten proteins are exposed to mechanical work as well as to heat conditions that might be expected to change their physicochemical properties significantly. Heating causes the gluten proteins to associate to form large protein aggregates that are less extractable (Booth et al., 1980; Schofield et al., 1983). Aggregation behaviour and extractability differences have been reported between glutens from good and poor baking quality flours, glutens from good baking quality flours being more highly aggregated and less extractable than those from flours of poor baking quality (Butaki and Dronzek, 1979; He and Hoseney, 1991). Abbreviations: AA, ascorbic acid; DHAA, dehydroascorbic acid; DTNB, dithiobis 2-nitrobenzoic acid; fwb, flour weight basis; GSH, glutathione; mw, molecular weight; SDS, sodium dodecyl sulphate; SH, sulphydryl; SS, disulphide; TGE, tris–glycine–EGTA. * Corresponding author. Tel.: C90 42234 10010; fax: C90 42234 10046. E-mail address: [email protected] (M. Hayta). 0733-5210/$ - see front matter q 2004 Published by Elsevier Ltd. doi:10.1016/j.jcs.2004.06.006
Redox agents modify the structure and functional properties of wheat gluten proteins (Bloksma and Bushuk, 1988). Reducing agents, such as sodium sulphite, mercaptoethanol, and dithiothreitol enhance the solubilization of wheat proteins (Kim and Bushuk, 1995). Their action cleaves SS bonds which results in a decrease in molecular weight, leading to an increase in extractability (Lavelli et al., 1996; Weegels et al., 1994). The extractability of proteins by SDS solutions gives a good indication of the degree of crosslinking. Extracting agents such as SDS produce their effects by altering the intermolecular bonding that is responsible for holding the protein chains together. As aggregation progresses, the SDS extractable fraction decreases and SDS unextractable fraction increases (Jeanjean et al., 1980; Schofield et al., 1983). Redox induced SH–SS exchange reactions cause alterations in the native structures of proteins leading to important physicochemical changes their properties. SH groups and SS bonds have a significant role in determining gluten properties and SS bond formation within gluten is important in protein network formation which affects the quality of the final product (Bloksma and Bushuk, 1988). Measurements of changes in SH groups suggest that heat
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setting is accompanied by changes in SS bond structure (Schofield et al., 1983; Weegels et al., 1994). SH/SS changes have been determined using amperometric procedures (Redman and Ewart, 1971), radiolabelling of SH groups with specific reagents (Schofield et al., 1983) and by direct amino acid analysis (Ewart, 1985). However, perhaps the most widely used method for SH/SS determination in proteins relies on reaction of DTNB (Ellman’s reagent) with SH groups. Electrophoretic patterns of proteins before and after heat treatment have been used to monitor changes in the aggregation behaviour of gluten polypeptides after heat treatment (Boye et al., 1997; Schofield et al., 1983). As gluten proteins are chiefly responsible for differences in baking quality among wheat flours, more information is required to clarify the changes that occur during the different steps in the breadmaking process. The objective of the present study was to gain information on heat-induced alterations in extractability, changes in free SH groups and SS bonds and in polypeptide composition of gluten in the absence and presence of redox additives.
with a pestle and mortar, the samples were milled in a micro-hammer mill (Glen Creston, setting 5) with a 1.0 mm screen. This resulting in a gluten powder with a particle size not exceeding 250 mm. 2.3. Heat treatment of gluten Freeze-dried gluten powder (20 g) and water or solutions of ascorbic acid (AA, 1000 ppm, gluten weight basis, gwb), potassium bromate (500 ppm, gwb) or reduced glutathione (GSH, 250 ppm, gwb) were hand mixed with a spatula, the volume of water or solution being such as to give a final moisture content of 65% (w/w). The gluten was allowed to hydrate for 2 h in a sealed plastic container. Hydrated glutens were placed between aluminium plates with a gap of 2 mm and rested for 15 min. The plates were immersed in a temperature controlled water bath and heated at 30, 50, 70 or 90 8C for 15 min. Thereafter, the plates were cooled immediately in ice water and the samples freeze-dried and ground as described in Section 2.2. 2.4. Analytical procedures
2. Experimental 2.1. Materials 2.1.1. Wheat grain and flour milling procedure Two bread wheat varieties, Hereward and Riband of good and poor breadmaking quality, respectively, were milled into straight-run white flours on a Bu¨hler experimental mill (Model MLU 202) after tempering for 24 h at 16% (w/w) moisture. The flours were stored at K4 8C. 2.2. Gluten isolation procedure The procedure was that described by Booth and Melvin (1979) with some modification. Flour (1 kg) and water or solutions of ascorbic acid (AA) (100 ppm, flour weight basis, fwb), potassium bromate (50 ppm, fwb) or reduced glutathione (GSH) (100 ppm, fwb) (1 l) were evenly mixed in a Hobart type hook mixer for 30 s at speed setting 1. The mixing speed was then increased to speed setting 3 and the batter mixed until a coherent mass formed i.e. when the dough pulled away from the side of the bowl as a coherent mass with a slapping noise. The mixer was then stopped and another 2 l water at 15 8C was added. Stirring was continued at speed setting 1 for 5 min. Normally gluten forms a cohesive mass that sticks to the mixer hook but this varies from flour to flour. Weak glutens may not behave as described and may break up into strands, which requires the use of a coarse metal sieve to separate them from starch milk. After removal of the starch milk, the gluten was returned to the mixer for additional washing at speed 1 until the wash water was clear, at which stage the gluten was frozen and freeze-dried. After coarse grinding the dry gluten
2.4.1. Moisture The moisture contents of flours and glutens (%, dry basis (db)) were determined by oven drying method at 130 8C for 1 h (Approved Methods of American Association of Cereal Chemist, 1995). 2.4.2. Protein The total protein contents of flours and glutens were determined using Kjeldahl (N!5.7) standard method. The protein contents of sodium dodecyl sulphate (SDS) or acetic acid extracts of glutens were determined using the bicinchoninic acid (BCA) protein assay kit (Sigma Chemical Co., Procedure No.: Tpro-562). This method is based on the reaction of BCA with protein-reduced copper (I) forming a purple complex with an absorbance maximum at 562 nm, which is proportional to protein concentration. The extracts (0.1 ml) were diluted with distilled water (0.9 ml). The diluted extracts (0.1 ml) were mixed with 2.0 ml of BCA reaction solution (50 part BCA plus 1 part copper (II) sulphate) and incubated at 37 8C in a water bath for 30 min. The absorbance values of reaction mixtures were measured at 562 nm against blanks (2% SDS or 0.05 M acetic acid plus BCA reaction solution) after cooling down to room temperature. Bovine serum albumin (Sigma, P-0914) was used as the protein standard. 2.4.3. Starch Total starch contents of glutens were determined by amyloglucosidasealpha-amylase (Megazyme Int. Ireland Ltd).
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2.4.4. Farinograph and mixograph mixing tests Farinograms of flours were obtained using a Brabender Farinograph (C W. Brabender Instruments, Inc., South Hackensack, NJ, USA) fitted with 300 g mixing bowl to assess water absorption values according to the standard method of Approved Methods of American Association of Cereal Chemist (1995). The Farinograms were used to obtain standard dough mixing characteristics, such as development time. Mixing tests were also performed on flour (2 g) samples with a 2 g direct drive Mixograph. Samples were weighed at 14% moisture basis and mixed at a speed of 88 rpm. Water was added according to the water absorption values determined by the Farinograph to give final moisture content of 63.3 and 57.6% (fwb) for flours of Hereward and Riband wheat, respectively. The dough mixing parameters were obtained using the software, Mixsmart (National Mfg, TMCO, Lincoln, NE, USA). 2.4.5. Gluten protein extractability in SDS solution To examine the effect of heat on extractability, gluten (0.2 g) heated at different temperatures was suspended in 20 ml of 2% (w/v) SDS and mixed with a magnetic stirrer for 30 min, 1, 2, 5, 10, 15, 20 h or overnight and centrifuged at 30,000g for 1 h. The protein contents of the supernatants were determined by the BCA method after filtration through filter paper (Whatman No. 541).
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2.4.7. SDS-polyacrylamide gel electrophoresis SDS-PAGE was performed on a Protean II (Bio-Rad) Vertical electrophoresis cell (160!160!1 mm) using a 12.5% polyacrylamide separating gel containing 1.35% bis-acrylamide cross-linker according to Laemmli (1970). 2.4.8. Data analysis Statistical analysis was performed using statistical analysis system (SAS) software. Determination of significant differences among the means was performed by variance analysis and least significant difference (LSD) method.
3. Results 3.1. Intrinsic properties of the flour and gluten The Proximate compositional analysis (Table 1) shows that the total protein contents of both flour and gluten from the good breadmaking quality wheat cv Hereward were typically higher than the protein content of the poor breadmaking quality wheat cv. Riband. The mixing properties of the two flours are shown in Table 2. The hard wheat cv. Hereward had higher Farinograph water absorption values than the soft wheat cv. Riband. 3.2. Extractability
2.4.6. SS/SH content of flours and glutens Free SH contents were determined by the method of Beveridge et al. (1974). Flour (80 mg) or gluten (20 mg) was suspended in 1 ml of 85.9 mM Tris, 91.9 mM Glycine, 3.2 mM EDTA, pH 8 (TGE) buffer containing 2.5% (w/v) SDS. After incubation for 60 min at room temperature, 15 ml of 10 mM 5.5 dithiobis 2-nitrobenzoic acid (DTNB) in dimethylformamide was added and the reaction was allowed to proceed for 30 min at room temperature. The suspensions were then centrifuged at 10,000g for 10 min. The absorbances of the diluted supernatants (1/10 with TGE plus SDS) were measured against buffer blank solutions (TGE plus SDS) and against reagent blanks (TGE plus SDS containing DTNB). Total SS contents of glutens were determined by the methods of Weegels et al. (1994) with some modifications. Flour and gluten samples (20 and 80 mg, respectively) were suspended in 0.4 ml of 1.5% (w/v) SDS, Tris/HCl, pH 8 and mixed with 0.4 ml of 6 M NaOH. After 45 min incubation, 0.8 ml of H3PO4 and 0.05 ml of 10 mM DTNB (in 0.2 M phosphate buffer, pH 7) were added and further incubated for 1 h. After centrifugation at 10,000g for 10 min, the absorbance values of the supernatants were read at 412 nm against buffer and sample blanks. The SH and SS contents were calculated using an extinction coefficient of 13,600 MK1 cmK1. SS contents were calculated as the differences between the total and the free SH contents.
Table 3 shows that the extractability of both flour and gluten proteins into 2% (w/v) SDS was higher for the poor breadmaking quality cv Riband, as compared with the good breadmaking quality cv. Hereward. As shown in Fig. 1, heating resulted, as expected, in a decrease in the extractability of the gluten proteins. The extractability of protein from gluten heated at 50 8C was slightly higher than unheated gluten up to 10 h. The proportion of total protein extracted from unheated gluten after 20 h extraction was 92.5%. However, after the same time only 54.4% of total protein from gluten heated at 80 8C was extractable with SDS. For all the samples, protein extractability had Table 1 Proximate composition of flours and glutens from cultivars Hereward and Riband
Flour Protein contenta (%, dbb) Moisture (%, w/w) Wet gluten content (%, w/w) Gluten Protein content (%, db) Moisture (%, w/w) Starch content (%, db)
Hereward
Riband
13.7 12.2 28.5
11.1 13.5 21.5
78.2 4.5 12.2
74.6 6.0 18.1
All values are means of at least duplicate determinations. a Kjeldahl protein (N!5.7). b db: dry matter basis.
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Table 2 Mixing properties of flours from cultivars Hereward and Riband
Farinograph Water absorption (%) Development time (min) Stability (min) Degree of softening (BUa) Mixing tolerance index (BU) Mixograph Peak time (min) Peak dough resistance (MUb) Peak height Width at peak
Hereward
Riband
63.3 3.0 9.0 100 90
57.6 2.0 3.5 140 180
3.18 71.6 71.7 27.4
2.62 50.7 51.2 18.5
All values are means of at least duplicate determinations. a BU, Brabender unit. b MU, Mixograph unit.
essentially reached a plateau value after 5 h, indicating that this time was adequate to extract gluten proteins into SDS. The flour or gluten proteins from poor breadmaking quality cv. Riband were more soluble in 2% (w/v) SDS than the proteins of good breadmaking quality cv. Hereward. 3.3. Effect of redox additives on the extractability of heat-treated glutens The effect of redox additives was tested in two ways. In the first, doughs were treated with different redox additives and the gluten washed out immediately using the Glutomatic apparatus or Hobart mixer. In the second, freeze-dried gluten was prepared by the batter technique in the absence of additives and then rehydrated by mixing to peak development in the 2 g Mixograph in the presence of different redox agents to a final moisture content of 65% (w/w). 3.3.1. Gluten freshly washed out from dough The SDS extractability of good quality Hereward gluten, washed out from optimally mixed dough containing ascorbic acid (AA, 100 ppm), potassium bromate (50 ppm) or reduced glutathione (GSH, 100 ppm) remained almost constant as the temperature increased from 30 to 70 8C. However, the SDS extractability of the protein of Hereward control gluten decreased significantly (P!0.05) Table 3 Extractability in SDS (2%, w/v) of the protein from flours and glutens from cvs Cultivar Flour Hereward Riband Gluten Hereward Riband
Extracted protein (%) 87G1.7 91G2.3 59G0.9 68G1.5
Hereward and Riband, good and poor breadmaking quality wheats, respectively. Extraction was for 30 min at room temperature.
Fig. 1. Extractability of protein from unheated (C) heated at 50 8C (,), 60 8C (6), 70 8C (x), 80 8C (B), glutens (cv. Hereward flour) as a function of time. Extractant was 2% (w/v) SDS at room temperature.
over this temperature range (Fig. 2). The extractability declined more strongly at 90 8C, such that approximately 58% of the protein of the control gluten of Hereward became insoluble with heating at 90 8C. A similar pattern was observed for Riband control gluten (Fig. 3), although the changes in extractability were smaller. Redox agents affected SDS-extractability of gluten protein differently depending on the nature of the additive and the temperature. The extractability of protein from AA treated Hereward gluten remained more or less constant from 30 to 70 8C, then decreased significantly (P!0.05) to 54.1% at 90 8C (Figs. 2 and 3). The extractability of the gluten from AA treated Riband dough was slightly higher than the control at each temperature up to 70 8C, but at 90 8C the extractability of the AA treated Riband gluten fell to below that of the control (56.0% compared with 60.4%). Interestingly the extractability of the bromate treated Hereward gluten was more or less constant up to 70 8C, but at 90 8C it was approximately 15% higher than the control (Fig. 2), which was significant (P!0.05). While the protein extractability of untreated Hereward gluten decreased by about 10% as a result of heating at 70 8C compared with the unheated control, treatment with GSH, like that for oxidising agents, maintained protein extractability up to 70 8C, whereas at 90 8C the extractability fell to a value similar to the control. Bromate treated Riband gluten showed a similar pattern to the GSH or bromate treated Hereward gluten in that protein extractability remained constant up to 70 8C then at 90 8C fell to a value similar to the control (Figs. 2 and 3). No gluten could be washed out from the Riband dough treated with GSH. Hereward gluten treatment with both oxidants (bromate and ascorbate) apparently maintained constant gluten protein extractability constant up to 70 8C, whereas a significant (P!0.05) fall was observed for the control. The reducing agent, GSH, had a similar effect over this temperature range. At 90 8C the protein extractability of the oxidant treated Hereward gluten was higher than that of the
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Fig. 2. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Hereward gluten washed out from doughs treated with redox additives. Solutions of ascorbic acid (AA) (100 ppm, flour weight basis, fwb), potassium bromate (50 ppm, fwb) or reduced glutathione (GSH) (100 ppm, fwb) were used.
control, especially for the bromate treated gluten, whereas the extractability of the GSH treated gluten had fallen to a similar value to the control. As with Riband glutens, the oxidants seemed to have only small effects on the heat induced changes in protein extractability, the behaviour of the oxidant treated glutens being more similar to that of the control than in the case of Hereward gluten.
3.3.2. Freeze-dried glutens treated directly with redox additives SDS protein extractabilities for glutens that had been rehydrated in the 2 g Mixograph were lower than the values for gluten prepared directly from dough (Figs. 4 and 5). Although similar extractabilities were observed at 30 and 50 8C, heat treatment at 70 8C caused a slight reduction in
Fig. 3. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Riband gluten washed out from dough treated with redox additives. Solutions of ascorbic acid (AA) (100 ppm, flour weight basis, fwb), potassium bromate (50 ppm, fwb) or reduced glutathione (GSH) (100 ppm, fwb) were used.
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Fig. 4. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Hereward gluten treated with redox additives. Solutions of ascorbic acid (1000 ppm, gluten weight basis, gwb), potassium bromate, (500 ppm, gwb) and reduced glutathione (GSH) (250 ppm, fwb) were used.
extractability. Further heating at 90 8C dramatically reduced the amount of SDS extractable protein except for the bromate treated glutens of both cultivars. The extractabilities of bromate treated glutens were significantly (P!0.05) high even after heat treatment at 90 8C (Figs. 4 and 5). Slightly
higher extractabilities were observed for poor quality Riband gluten irrespective of temperature and additive used compared with good quality Hereward gluten. There was relatively little difference between the control and AA treated glutens at any temperature. Interestingly, the GSH treated
Fig. 5. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Riband gluten treated with redox additives. The solutions of additives were: ascorbic acid (1000 ppm, gluten weight basis, gwb), potassium bromate, (500 ppm, gwb) and reduced glutathione (250 ppm, gwb).
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Table 4 Effect of heat on the SH (mmol/g of protein) contents of glutens prepared from doughs treated with redox additives Wheat cultivar treatment
30
Temperature (8C) SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate
2.78 1.88 1.75 2.49 1.83 1.79 1.46
0.11 0.07 0.09 0.15 0.13 0.04 0.20
1.95 1.61 1.56 2.48 1.52 1.35 1.17
0.13 0.11 0.15 0.13 0.06 0.12 0.10
1.51 1.33 1.20 2.38 1.44 1.13 0.81
0.16 0.11 0.13 0.14 0.13 0.04 0.04
1.16 0.99 0.76 2.27 1.40 0.83 0.38
0.13 0.04 0.07 0.05 0.10 0.02 0.06
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
The SH content of the gluten from the poor breadmaking quality cv. Riband was significantly (P!0.05) lower than that of the gluten from the good breadmaking quality cv. Hereward. Whereas heating at 90 8C reduced the SH content of Riband gluten by about 20%, whereas the reduction was 60% for the Hereward gluten.
gluten had a significantly (P!0.05) lower extractability after heating at 90 8C than the control unheated glutens for both wheat cvs. 3.4. SH and SS contents 3.4.1. SH content of gluten freshly washed out from dough The effect of heat treatment (30–90 8C) on the SH contents of glutens prepared from dough treated with or without redox additives is illustrated in Table 4. In general, the SH contents of control and oxidant treated glutens decreased significantly (P!0.05) as the temperature increased from 30 to 90 8C The SH content of control (untreated) gluten from the cv. Hereward heated at 90 8C was approximately 60% lower than that of gluten heated at 30 8C. While oxidant treatments (ascorbate and bromate) cause about a 50% reduction in SH content, treatment with the reducing agent, glutathione (GSH), resulted in less pronounced decrease in gluten SH content when the gluten was heated at 70 and 90 8C compared with 30 and 50 8C.
3.4.2. SH content of freeze-dried glutens treated directly with redox additives Gluten isolated from flour was treated directly with redox additives in a comparison of their direct effects on gluten protein with their indirect effects when the gluten was freshly washed out from doughs treated with additives (Section 3.4.1). The addition of oxidants (ascorbate and bromate) directly to gluten resulted in a decrease in the SH content of both glutens (Table 5). Ascorbate treatment reduced the SH content by 26 and 12% in Hereward and Riband glutens, respectively. While bromate treatment reduced the SH content of unheated (30 8C) Hereward gluten by 50%, the decrease was 23% for poor quality Riband gluten at 30 8C. Heating at 90 8C enhanced
Table 5 Effect of heat on the SH (mmol/g of protein) contents of glutens treated directly with redox additives Wheat cultivar treatment
30
Temperature (8C) SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate GSH
2.56 1.89 1.27 3.76 2.45 2.15 1.87 3.64
0.10 0.12 0.14 0.16 0.12 0.15 0.19 0.11
1.70 1.23 1.22 3.47 1.86 1.81 1.53 3.34
0.18 0.09 0.13 0.17 0.14 0.10 0.09 0.09
1.35 0.94 0.52 2.99 1.57 1.28 0.77 3.28
0.08 0.12 0.11 0.14 0.13 0.10 0.12 0.05
1.23 0.75 0.29 2.85 1.22 1.05 0.22 3.16
0.02 0.05 0.12 0.10 0.09 0.07 0.05 0.14
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
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Table 6 Effect of heat on the SS (mmol/g of protein) contents of glutens prepared from doughs treated redox additives Wheat cultivar treatment
Temperature (8C) 30
SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate
36.47 38.88 33.80 37.47 33.53 34.28 30.59
0.58 0.30 0.61 0.43 0.14 0.11 0.97
36.11 35.59 34.61 36.41 34.28 31.14 30.64
0.07 0.10 0.66 0.22 0.28 0.32 0.15
35.92 38.08 34.26 33.26 33.84 32.50 31.34
0.95 0.30 0.49 1.10 0.18 0.09 0.30
34.70 37.39 37.51 32.24 29.14 31.12 31.47
0.78 0.24 0.43 0.14 0.89 0.06 0.36
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
the decrease in the presence of bromate for both cultivars. For example at 30 8C when bromate was used, the SH content was reduced by 50%. However, on heating at 90 8C a further 25% decrease in the SH content was observed. The SH contents of the GSH treated glutens were higher than those of the controls for both cultivars, and the SH contents decreased slightly on heating.
Such small changes are likely to be within the experimental error of the method used for SS determination. 3.4.4. SS content of gluten treated directly with redox additives The SS bond contents of glutens treated directly with redox additives were similar to those of glutens prepared from dough treated with redox additives (Table 7). Again there was no clear effect on SS bond content of redox additive treatment or of heat treatment.
3.4.3. SS content of gluten freshly washed out from dough The SS contents of control and glutens of Hereward and Riband treated with additives were similar (Table 6), although the good quality Hereward gluten had a slightly higher amount of SS than the poor quality Riband gluten throughout the temperature range studied The SS analysis provided little evidence that the decrease in SH content due to oxidant treatment or to heating resulted in increases in SS contents. However, since the changes in SH contents were only about 1 mmol of SH/g gluten at most, and since two SH groups are lost for every SS bond formed, the changes in SS bond contents are, in any case, very small compared with the total SS content of the control untreated gluten.
3.5. Polypeptide composition of gluten freshly washed out from dough Heating the glutens prepared from redox treated doughs at 30 and 50 8C had no apparent effect on polypeptide composition of the glutens when analysed by SDS-PAGE under reducing conditions (not shown). However, band intensities generally decreased after heating to 70 8C and the decrease became more pronounced as a result of heating to 90 8C (not shown). Bands corresponding to HMW subunits
Table 7 Effect of heat on the SS (mmol/g of protein) contents of glutens treated with redox additives Wheat cultivar treatment
Temperature (8C) 30
SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate GSH
35.60 36.12 36.03 36.78 33.10 33.83 31.75 32.69
0.17 0.46 0.30 0.15 0.30 0.50 0.23 0.50
36.44 36.40 32.80 34.55 33.17 34.24 33.60 33.07
0.17 0.91 0.32 0.58 1.29 1.07 0.34 0.14
33.54 37.67 34.74 36.66 35.13 32.69 32.86 33.83
0.86 0.85 0.76 0.43 0.85 0.29 0.80 0.19
34.03 35.68 36.84 36.43 32.39 32.86 32.74 33.27
0.16 1.36 0.26 0.15 0.30 0.14 0.19 0.21
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
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of glutenin had almost disappeared after heating to 70 and 90 8C, but interestingly the band intensities of HMW glutenin subunits in bromate treated glutens were partly retained when compared with untreated control gluten. Ascorbate treatment also showed a slight protective effect against heating. Additionally, the HMW glutenin subunits from the good quality Hereward gluten were affected to a greater extent by heating to 90 8C than were those of poor quality Riband gluten as evidenced by the band intensities. Almost all the protein that did not enter the separating gel under non-reducing condition (not shown) migrated into the gel as discrete identifiable bands under reducing conditions. 3.6. Polypeptide composition of gluten treated directly with redox additives Similar trends were observed in the electrophoretic patterns of SDS-extractable proteins of glutens treated directly with redox additives. Again the band intensities in HMW glutenin subunit region of bromate treated gluten from both varieties were partly preserved even after heat treatment at 90 8C.
4. Discussion 4.1. Intrinsic properties of the flours and glutens Total protein and gluten contents generally show a linear relationship with breadmaking performance as measured by loaf volume (Finney and Barmore, 1948). The starch damage and protein content of wheat are the main determinants of the water absorption of flour. The differences in endosperm texture results in variation in the ability to direct the mechanical energy to the starch granules during milling (Stevens, 1987). The degree of starch damage during milling is greater for hard wheats than for soft wheats, therefore, the water absorption capacity of hard wheats increases. Protein content is also generally higher in hard wheats than in soft wheats (Kent and Evers, 1994). As expected the dough development time and peak time measured by the Farinograph and Mixograph, respectively, were longer for Hereward flour than for the poor quality Riband flour. This implies that the hydration of protein and starch and the development of the viscoelastic structure to a certain consistency require more time and energy input for good quality flour. Starch, protein and their interactions have a strong influence on the mixing behaviour of flour (Preston and Kilborn, 1984). Again stability and mixing tolerance index values, which are influenced by interactions of flour components, especially starch and proteins, were greater for Hereward flour. The empirical data on mixing characteristics suggests that there are differences in physical and possibly molecular properties of flours from diverse breadmaking quality wheat. Thus these data gave assurance, that the flours chosen for
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this study had diverse physicochemical/functional properties. 4.2. SDS-extractability The extractability of flour and gluten proteins with SDS from good and poor breadmaking quality (cvs. Hereward and Riband, respectively) showed that gluten is less extractable from good than poor flours. This observation is in accordance with previous studies. Butaki and Dronzek (1979) found that stronger wheat flours had more acid insoluble (0.05 M acetic acid) gluten than weak flours. Similarly, He and Hoseney (1991) showed that gluten from poor quality flour had higher solubility in SDS solution than gluten from good quality flour. The difference in protein extractabilities between glutens was assumed to result from differences in molecular size and aggregation tendency. Differences in the aggregation behaviour (Arakawa and Yonezawa, 1975), molecular weight or interaction tendency (He and Hoseney, 1991) or conformational properties (Huang and Khan, 1996) have been suggested as possible factors responsible for the varying extractabilities of gluten proteins from wheats of diverse breadmaking quality. However, in respect to extractability in SDS solution, molecular weight is likely to be the dominant factor. As the solubility of heated proteins depends on their molecular weights (MW) (Pomeranz, 1988), the decrease in extractability into SDS solution after heat treatment, especially at higher temperatures, probably indicates an increase in MW, which might result from aggregation of proteins during heating. In agreement with the previous reports of Booth et al. (1980), Jeanjean et al. (1980) and Schofield et al. (1983), the results of the extractability of gluten proteins with SDS solution revealed that heat causes the gluten proteins to associate to form larger protein aggregates that are less extractable; as aggregation (crosslinking) progresses the SDS-extractable fraction decreases. The heat effect on gluten is explained on the basis of protein crosslinking through a SH/SS exchange mechanism. Redox agents affected SDS-extractability of gluten protein differently depending on the type of additive and the temperature. The effect of redox improvers is greater in good breadmaking flour than in poor breadmaking flour due to differences in the rate of reactions, which are lower in good quality flour (Mair and Grosch, 1979). One might have expected that oxidising agents would have caused a reduction in protein extractability by increasing protein crosslinking through SS bond formation, whereas reducing agents would have caused a depolymerisation of gluten by cleaving the SS bonds. However, improvers such as ascorbic acid and potassium bromate increase the solubilization of insoluble protein during dough mixing (Danno and Hoseney, 1982; Graveland et al., 1985; Sievert et al., 1991). Different explanations have been proposed involving physical forces, covalent and
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non-covalent bonds and conformational rearrangements. One proposed explanation is that oxidising agents cause crosslinking between glutenin fractions leading to increase in stiffness of dough and increased work input. The increased work input would lead to mechanical rupture of SS bonds that would increase protein extractability (Graveland et al., 1984). However, Eckert et al. (1993) proposed that oxidant effects result from conformational rearrangements rather than size reduction leading to a more extended structure and thus increased extractability. If the dough is allowed to rest after mixing, polymerisation of glutenin due to oxidation of SH groups (Graveland et al., 1985; Wang et al., 1992) or decrease in polymer surface due to structural relaxation, results in a decrease in protein extractability (Eckert et al., 1993). In the presence of oxidants (ascorbic acid and potassium bromate), the enhancement of the changes in glutathione (GSH, GSSG, PSSG) during dough mixing (Chen, 1994), such as shortening of the dough development time and narrowing of Farinograph band width in rheological tests (Frater et al., 1960; Ikoeza and Tipples, 1968) are consistent with the results of the present study. One of the postulates is that oxidants enhance protein depolymerization and dough breakdown (Chen, 1994). However, it is also known that oxidation favours polymerisation of gluten proteins. The higher extractability of additive treated, especially AA and bromate treated glutens, after heat treatment at higher temperatures (70 and 90 8C) suggest that redox additives alter the protein structure in such a way that heat induced changes in SS bonding are modified and gluten protein extractability is maintained. The rate of oven rise for oxidant treated doughs was observed to be higher than that of untreated doughs implying a delay in the denaturation temperature of gluten network (Yamada and Preston, 1992). This appears to be consistent with the present observations on the effects of heat on the SDS extractability of glutens from oxidant treated doughs. We observed an increase in extractability of good breadmaking quality Hereward glutens washed from oxidant treated dough. The action of AA is thought to involve oxidation of endogenous flour GSH to GSSG (Grosch and Wieser, 1999). Although this reaction has not been experimentally demonstrated, it is speculated that AA (a reducing agent) must first be converted by oxidation with molecular oxygen to dehydroascorbic acid (DHAA, an oxidising agent). If the mixing environment does not incorporate enough oxygen into the dough, the conversion of AA to DHAA may not occur efficiently. Therefore, AA may act as a reducing agent by breaking SS bonds under conditions of oxygen insufficiency (Mauseth et al., 1967). The increase in protein extractability in SDS caused by oxidants is likely to result from redox modification of SH groups such that interchain SS bonding among gluten polymers was decreased. This is not to say that hydrophobic interactions do not play a role in the non-covalent aggregation of gluten proteins during heating that may
facilitate subsequent oxidation of SH groups to SS bonds or the rearrangement of SS bonds through SH/SS exchange reactions, but that heat-induced changes in hydrophobic interactions themselves do not lead to differences in SDS extractability. With other non-denaturing solvents, however, such interactions could well affect protein solubility. 4.3. SH and SS contents The actions of oxidising and reducing agents on dough properties occur at different stages of breadmaking (mixing, proofing, baking) and consequently there are also changes in protein structure at different stages of breadmaking (Fitchett and Frazer, 1986). There are contradictory reports of the effects of heat and oxidant on the SH content of doughs. Although a significant decrease in the SH content of bromate treated and baked dough was reported by Tsen (1968), the finding of a similar reduction in SH groups during heating of both bromate treated and untreated doughs does not support the mechanism of bromate oxidation (Andrews et al., 1995). Notwithstanding an extremely high level of oxidant, compared with commercial practice, was used, the SH content of oxidant treated (ascorbate and bromate, 1200 ppm) doughs mixed in the Do-Corder and heated from 35 to 75 or 85 8C decreased and the decrease in SH content of bromate treated dough was higher than that of ascorbate treated dough (Nagao et al., 1981). The concentrations of SH groups, analysed by amperometric titration, by Matsuo and McCalla (1964) for glutens from hard, soft and durum wheat varieties were 3.8, 4.0 and 4.4 mequiv/g of protein, respectively. The values were approximately half those obtained in the present study although the differences in wheat cultivars and method of determination would obviously affect the results. There are various reports on the relationship between SH and SS contents and wheat variety. For example, Tsen and Bushuk (1968) reported that total SH content increases with decreasing flour strength. Although total SS content decreased with decreasing strength, the number of reactive SS bonds increased. It was also found that there was no relationship between protein and SH contents (Axford et al., 1962, Tsen and Anderson, 1963). SH content of doughs differing in their response to bromate showed that the decline in the SH content of bromate responsive flours was larger than that for non-responsive flours (Andrews et al., 1995). The literature regarding the SS contents of flours is somewhat contradictory. Total SS content showed an inverse relationship with various flour quality parameters and also varied independently among very strong, strong and weak wheat flours (Axford et al., 1962). In another study neither total SH nor SS contents of flours showed correlations with baking quality (Graveland et al., 1978).
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4.4. Polypeptide composition In wheat, the glutenin protein components of gluten are polymers stabilised by intra- and intermolecular SS bonds and the gliadins are monomers with either no SS bonds (u-gliadins) or intramolecular bonds (a- and b-type) (Tatham et al., 1990). During heating the native protein structure is destabilised and unfolding may facilitate SH/SS interchange and oxidation together with hydrophobic interactions (Li and Lee, 1998). The appearance of the HMW glutenin subunit region in the reduced gels even after high temperature (90 8C) heat treatment of gluten in the presence of bromate suggests that the addition of bromate could reduce the decrease in protein extractability caused by heat treatment. It is also likely that bromate favours oxidation to the disulphide and higher oxidation states. This observation is consistent with the direct measurements of protein extractability for glutens heated to different temperatures. This may occur by weakening the extensive crosslinking of gluten. However, this finding contrary to previous reports in which SDSPAGE (Atanassova and Popova, 1977) and SE-HPLC (Bekes et al., 1996) of flour proteins showed that the high molecular weight fraction (glutenin aggregates) increased as a result of bromate treatment. A similar, but less pronounced, effect of ascorbate was also observed. In a study by Veraverbeke et al. (1997), it was found that addition of the oxidant, potassium iodate, to dough resulted in higher levels of SDS-extractable protein from bread. This is consistent with the findings of the present study on heatinduced SDS-extractability and the polypeptide composition of gluten proteins as determined SDS-PAGE analysis.
References Andrews, D.C., Caldwell, R.A., Quail, K.J., 1995. Sulfhydryl analysis.II Free sulfydryl content of heated doughs from two wheat cultivars and effect of potassium bromate. Cereal Chemistry 72, 330–333. Approved Methods of American Association of Cereal Chemist. 1995. St Paul. MN. Arakawa, T., Yonezawa, D., 1975. Compositional difference of wheat flour glutens in relation to their aggregation behaviour. Agricultural and Biological Chemistry 39, 2123–2128. Atanassova, E., Popova, V. (1977). Effects of oxidising agents on the proteins and lipids in wheat flour. In: Internationale Probleme der Modernen Getreideverarbeitung und Getreidechemie.7th Symp. pp 11-16. Axford, D.W.E., Campbell, J.D., Elton, G.A.H., 1962. Disulphide groups in flour proteins. Journal of the Science of Food and Agriculture 13, 73–78. Bekes, F., Panozzo, J.F., Gupta, R.B., Ronald, J.A., Wrigley, C.W., 1996. Bromate and baking test: changes in proteins and lipids. In: Proceedings of the 46th Australian Cereal Chemistry Conferance. RACI, Melbourne. pp. 313–316. Beveridge, T., Toma, S.J., Nakai, S., 1974. Determination of SH and SS groups in some food proteins using Ellman’s reagent. Journal of Food Science 39, 49–51.
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Bloksma, A.H., Bushuk, W., 1988. Rheology and chemistry of dough, in: Pomeranz, Y. (Ed.), Wheat Chemistry and Technology, third ed, pp. 131–152. Booth, M.R., Melvin, M.A., 1979. Factors responsible for the poor breadmaking quality of high yielding European wheat. Journal of the Science of Food and Agriculture 30, 1057–1064. Booth, M.R., Bottomley, R.C., Ellis, J.R.S., Malloch, G., Schofield, J.D., Timms, M.F., 1980. Annales de Technologie Agricole 29, 399–408. Boye, J.I., Ma, C.Y., Harwalkar, V.R., 1997. Thermal denaturation and coagulation of proteins, In: Damodaran, S., Paraf, A. (Eds.), Food Proteins and Their Applications. Marcel Dekker, New York, pp. 25–44. Butaki, R.C., Dronzek, B., 1979. Effect of protein content and wheat variety on relative viscosity, solubility and electrophoretic properties of gluten proteins. Cereal Chemistry 56, 162. Chen, X., 1994. Glutathione in wheat, PhD Thesis, King’s College, London. Danno, G., Hoseney, R.C., 1982. Changes in flour proteins during dough mixing. Cereal Chemistry 59, 249–253. Eckert, B., Amend, T., Belitz, H.D., 1993. The course of SDS and zeleny sedimentation tests for gluten quality and related phenomena studied using the light microscope. Zeitschrift fu¨r Lebensmittel-Untersuchung und-Forschung. 196, 122–125. Ewart, J.A.D., 1985. Blocked thiols in glutenin and protein quality. Journal of the Science of Food and Agriculture 36, 101–112. Finney, K.F., Barmore, M.A., 1948. Loaf volume and protein content of hard winter and spring wheats. Cereal Chemistry 25, 291–312. Fitchett, C.S., Frazer, P.J., 1986. Action of oxidants and other improvers,In: Blanshard, J.M.V, Frazier, P.J., Galliard, T. (Eds.), Chemistry and Physics of Baking Special Publication No. 56, 56. The Royal Society of Chemistry, London, pp. 179–198. Frater, R., Hird, F.J.R., Moss, H.J., Yates, J.R., 1960. A role for thiol and disulfide groups in determining the rheological properties of dough made from wheaten flour. Nature. 186, 451–454. Graveland, A., Bosveld, P., Marseille, J.P., 1978. Determination of thiol groups and disulphide bonds in wheat flour and dough. Journal of the Science of Food and Agriculture 29, 53–61. Graveland, A., Bosveld, P., Lichtendonk, W.J., Moonen, J.H.E., 1984. Structure of glutenins and their breakdown during dough mixing by a complex oxidation-reduction system, In: Graveland, A., Moonen, J.H.E. (Eds.), Proceedings of 2nd International Workshop: Gluten Proteins. Waganingen, Netherland. Graveland, A., Bosveld, P., Lichtendonk, W.J., Marsseille, J.P., Moonen, J.H.E., Scheepstra, A., 1985. A model for the molecular structure of the glutenins from wheat flour. Journal of Cereal Science 4, 1117–1128. Grosch, W., Wieser, H., 1999. Redox reactions in wheat dough as affected by ascorbic acid. Journal of Cereal Science 29, 1–16. He, H., Hoseney, R.C., 1991. Gas retention in bread dough during baking. Cereal Chemistry 68, 521–525. Huang, D.Y., Khan, K., 1996. Unexpected solubility changes in wheat proteins during fermentation and oven stages of the breadmaking process. Cereal Chemistry 73, 512–513. Ikoeza, K., Tipples, K.H., 1968. Farinoraph studies on the effect of various oxidising agents in the sponge and dough system. Cereal Science Today 13, 327–330 (see also p. 358). Jeanjean, M.F., Damidaux, R., Feillet, P., 1980. Effect of heat treatment on protein solubility and viscoelastic properties of wheat gluten. Cereal Chemistry 57, 325–331. Kent, N.L., Evers, A.D., 1994. Tecnology of Cereals, fourth ed. Pergamon Press, Oxford, UK. Kim, H.R., Bushuk, W., 1995. Changes in some physicochemical properties of flour proteins due to partial reduction with dithiothreitol. Cereal Chemistry 72, 450–456. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
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Lavelli, V., Guerrieri, N., Cerletti, P., 1996. Controlled reduction study of modifications induced by gradual heating in gluten proteins. Journal of the Science of Food and Agriculture 44, 2549–2555. Li, M., Lee, T.C., 1998. Effect of cysteine on the molecular weight distribution and the disulfide crosslink of wheat flour proteins in extrudates. Journal of the Science of Food and Agriculture 46, 846–853. Mair, G., Grosch, W.J., 1979. Changes in glutathione content (reduced and oxidised form) and the effect of ascorbic acid and potasium bromate on glutathione oxidation during dough mixing. Journal of the Science of Food and Agriculture 30, 914–920. Matsuo, R.R., McCalla, A.G., 1964. Physicochemical properties of gluten from three types of wheat. Canadian Journal Biochemistry 42, 1487– 1498. Mauseth, R.E., Nees, J.L., Chamberlain, L.M., Johnston, W.R., 1967. Oxidising and reducing effects in the continuous dough process. Cereal Science Today 12, 390–396. Nagao, S., Endo, S., Tanaka, K., 1981. Scanning electron microscopy studies of wheat protein fractions from doughs mixed with oxidants at high temperature. Journal of the Science of Food and Agriculture 32, 235–242. Phillips, L.G., Whitehead, D.M., Kinsella, J.E., 1994. Structure-Function Properties of Food Proteins. Academic Press, San Diego. Pomeranz, Y., 1988. Composition and functionality of wheat flour components,In: Pomeranz, Y. (Ed.), Wheat Chemistry and Technology, vol. II. AACC, St Paul, MN. Preston, K.R., Kilborn, R.H., 1984. Dough rheology and Farinograph, In: D’Appolonia, B.L., Kunerth, W.H. (Eds.), Farinograph Handbook, third ed. AACC, St Paul., MN. Redman, H., Ewart, J.A.D., 1971. Measurement of disulphide bonds in cereal glutelins. Journal of the Science of Food and Agriculture 22, 19–21. Schofield, J.D., Bottomley, R.C., Timms, M.F., Booth, M.R., 1983. The effect of heat on wheat gluten and the involvement of sulphydryldisulphide interchange reactions. Journal of Cereal Science 1, 241–253.
Sievert, D., Sapirstein, H.D., Bushuk, W., 1991. Changes in electrophoretic patterns of acetic acid-insoluble wheat proteins during dough mixing. Journal of Cereal Science 14, 243–256. Stevens, D.J., 1987. Water absorption of flour, In: Morton, I.D. (Ed.), Cereals in European Context. VCH. Weinhem and Ellis Horwood Ltd, Chichester, pp. 273–284. Tatham, A.S., Shewry, P.R., Belton, P.S., 1990. Structural studies of cereal prolamins including wheat gluten. Advences in Cereal Science and Technology 10, 1–78. Tsen, C.C., 1968. Oxidation of sulfhydryl groups of flour by bromate under various conditions and during the breadmaking process. Cereal Chemistry 45, 531. Tsen, C.C., Anderson, J.A., 1963. Determination of Sulfhydryl and disulfide groups in floor and their relation to wheat quality. Cereal Chemistry 40, 314–323. Tsen, C.C., Bushuk, W., 1968. Reactive and total sulfhydryl and disulfide contents of flours of different mixing properties. Cereal Chemistry 45, 58–62. Veraverbeke, W.S., Roels, S.P., Delcour, J.A., 1997. Heat-induced changes in sodium dodecyl sulphate-sedimentation volume and functionality of vital wheat gluten. Journal of Cereal Science 26, 177–181. Wang, G.I.J., Faubion, J.M., Hoseney, R.C., 1992. Studies of bread making and reformation of SDS insoluble glutenin proteins with dough mixing and resting. Lebensmıttel-Wıssenschaft und-Technologie-Food Science and Technology 25, 228–231. Weegels, P.L., Verhoek, J.A., de Groot, A.M., Hamer, R.J., 1994. Effects on gluten of heating at different moisture contents. I. Changes in functional properties. Journal of Cereal Science 19, 31–38. Yamada, Y., Preston, K.R., 1992. Effects of individual oxidants and oven rise and bread properties of Canadian short process bread. Journal of Cereal Science 15, 237–244.
Heat and additive induced biochemical transitions in gluten from good and poor breadmaking quality wheats Mehmet Haytaa,*, J. David Schofieldb a
Department of Food Engineering, Inonu University, Elazig Yolu 44069 Malatya, Turkey b Department of Food Science and Technology, Reading University, Reading, UK Received 26 February 2004; revised 7 June 2004; accepted 25 June 2004
Abstract Glutens from poor breadmaking quality wheat, cv. Riband, had a higher SDS extractability than glutens from good quality cv. Hereward. Heating of gluten, especially above 70 8C, caused a reduction in the amount of SDS-extractable gluten proteins. Treatment of gluten with redox additives (ascorbic acid, potassium bromate or glutathione) affected extractability, being highest for bromate treated glutens. The SH content of gluten was lower for poor breadmaking Riband and heating resulted in greater decrease in SH content of gluten from good breadmaking Hereward. Hereward gluten had a higher SS content than Riband. The alteration of SS content on heating was not significant and may indicate the heat-induced involvement of non-covalent interactions. SDS-PAGE revealed that oxidants, especially bromate, affect polypeptide composition leading to a more heat stable/tolerant protein structure. q 2004 Published by Elsevier Ltd. Keywords: Wheat gluten; Heating; Redox additives; Sulphydryl; Disulphide
1. Introduction Heat treatment can cause alterations in protein structure e.g. in conformation and molecular size (Phillips et al., 1994). During the baking process, gluten proteins are exposed to mechanical work as well as to heat conditions that might be expected to change their physicochemical properties significantly. Heating causes the gluten proteins to associate to form large protein aggregates that are less extractable (Booth et al., 1980; Schofield et al., 1983). Aggregation behaviour and extractability differences have been reported between glutens from good and poor baking quality flours, glutens from good baking quality flours being more highly aggregated and less extractable than those from flours of poor baking quality (Butaki and Dronzek, 1979; He and Hoseney, 1991). Abbreviations: AA, ascorbic acid; DHAA, dehydroascorbic acid; DTNB, dithiobis 2-nitrobenzoic acid; fwb, flour weight basis; GSH, glutathione; mw, molecular weight; SDS, sodium dodecyl sulphate; SH, sulphydryl; SS, disulphide; TGE, tris–glycine–EGTA. * Corresponding author. Tel.: C90 42234 10010; fax: C90 42234 10046. E-mail address: [email protected] (M. Hayta). 0733-5210/$ - see front matter q 2004 Published by Elsevier Ltd. doi:10.1016/j.jcs.2004.06.006
Redox agents modify the structure and functional properties of wheat gluten proteins (Bloksma and Bushuk, 1988). Reducing agents, such as sodium sulphite, mercaptoethanol, and dithiothreitol enhance the solubilization of wheat proteins (Kim and Bushuk, 1995). Their action cleaves SS bonds which results in a decrease in molecular weight, leading to an increase in extractability (Lavelli et al., 1996; Weegels et al., 1994). The extractability of proteins by SDS solutions gives a good indication of the degree of crosslinking. Extracting agents such as SDS produce their effects by altering the intermolecular bonding that is responsible for holding the protein chains together. As aggregation progresses, the SDS extractable fraction decreases and SDS unextractable fraction increases (Jeanjean et al., 1980; Schofield et al., 1983). Redox induced SH–SS exchange reactions cause alterations in the native structures of proteins leading to important physicochemical changes their properties. SH groups and SS bonds have a significant role in determining gluten properties and SS bond formation within gluten is important in protein network formation which affects the quality of the final product (Bloksma and Bushuk, 1988). Measurements of changes in SH groups suggest that heat
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setting is accompanied by changes in SS bond structure (Schofield et al., 1983; Weegels et al., 1994). SH/SS changes have been determined using amperometric procedures (Redman and Ewart, 1971), radiolabelling of SH groups with specific reagents (Schofield et al., 1983) and by direct amino acid analysis (Ewart, 1985). However, perhaps the most widely used method for SH/SS determination in proteins relies on reaction of DTNB (Ellman’s reagent) with SH groups. Electrophoretic patterns of proteins before and after heat treatment have been used to monitor changes in the aggregation behaviour of gluten polypeptides after heat treatment (Boye et al., 1997; Schofield et al., 1983). As gluten proteins are chiefly responsible for differences in baking quality among wheat flours, more information is required to clarify the changes that occur during the different steps in the breadmaking process. The objective of the present study was to gain information on heat-induced alterations in extractability, changes in free SH groups and SS bonds and in polypeptide composition of gluten in the absence and presence of redox additives.
with a pestle and mortar, the samples were milled in a micro-hammer mill (Glen Creston, setting 5) with a 1.0 mm screen. This resulting in a gluten powder with a particle size not exceeding 250 mm. 2.3. Heat treatment of gluten Freeze-dried gluten powder (20 g) and water or solutions of ascorbic acid (AA, 1000 ppm, gluten weight basis, gwb), potassium bromate (500 ppm, gwb) or reduced glutathione (GSH, 250 ppm, gwb) were hand mixed with a spatula, the volume of water or solution being such as to give a final moisture content of 65% (w/w). The gluten was allowed to hydrate for 2 h in a sealed plastic container. Hydrated glutens were placed between aluminium plates with a gap of 2 mm and rested for 15 min. The plates were immersed in a temperature controlled water bath and heated at 30, 50, 70 or 90 8C for 15 min. Thereafter, the plates were cooled immediately in ice water and the samples freeze-dried and ground as described in Section 2.2. 2.4. Analytical procedures
2. Experimental 2.1. Materials 2.1.1. Wheat grain and flour milling procedure Two bread wheat varieties, Hereward and Riband of good and poor breadmaking quality, respectively, were milled into straight-run white flours on a Bu¨hler experimental mill (Model MLU 202) after tempering for 24 h at 16% (w/w) moisture. The flours were stored at K4 8C. 2.2. Gluten isolation procedure The procedure was that described by Booth and Melvin (1979) with some modification. Flour (1 kg) and water or solutions of ascorbic acid (AA) (100 ppm, flour weight basis, fwb), potassium bromate (50 ppm, fwb) or reduced glutathione (GSH) (100 ppm, fwb) (1 l) were evenly mixed in a Hobart type hook mixer for 30 s at speed setting 1. The mixing speed was then increased to speed setting 3 and the batter mixed until a coherent mass formed i.e. when the dough pulled away from the side of the bowl as a coherent mass with a slapping noise. The mixer was then stopped and another 2 l water at 15 8C was added. Stirring was continued at speed setting 1 for 5 min. Normally gluten forms a cohesive mass that sticks to the mixer hook but this varies from flour to flour. Weak glutens may not behave as described and may break up into strands, which requires the use of a coarse metal sieve to separate them from starch milk. After removal of the starch milk, the gluten was returned to the mixer for additional washing at speed 1 until the wash water was clear, at which stage the gluten was frozen and freeze-dried. After coarse grinding the dry gluten
2.4.1. Moisture The moisture contents of flours and glutens (%, dry basis (db)) were determined by oven drying method at 130 8C for 1 h (Approved Methods of American Association of Cereal Chemist, 1995). 2.4.2. Protein The total protein contents of flours and glutens were determined using Kjeldahl (N!5.7) standard method. The protein contents of sodium dodecyl sulphate (SDS) or acetic acid extracts of glutens were determined using the bicinchoninic acid (BCA) protein assay kit (Sigma Chemical Co., Procedure No.: Tpro-562). This method is based on the reaction of BCA with protein-reduced copper (I) forming a purple complex with an absorbance maximum at 562 nm, which is proportional to protein concentration. The extracts (0.1 ml) were diluted with distilled water (0.9 ml). The diluted extracts (0.1 ml) were mixed with 2.0 ml of BCA reaction solution (50 part BCA plus 1 part copper (II) sulphate) and incubated at 37 8C in a water bath for 30 min. The absorbance values of reaction mixtures were measured at 562 nm against blanks (2% SDS or 0.05 M acetic acid plus BCA reaction solution) after cooling down to room temperature. Bovine serum albumin (Sigma, P-0914) was used as the protein standard. 2.4.3. Starch Total starch contents of glutens were determined by amyloglucosidasealpha-amylase (Megazyme Int. Ireland Ltd).
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2.4.4. Farinograph and mixograph mixing tests Farinograms of flours were obtained using a Brabender Farinograph (C W. Brabender Instruments, Inc., South Hackensack, NJ, USA) fitted with 300 g mixing bowl to assess water absorption values according to the standard method of Approved Methods of American Association of Cereal Chemist (1995). The Farinograms were used to obtain standard dough mixing characteristics, such as development time. Mixing tests were also performed on flour (2 g) samples with a 2 g direct drive Mixograph. Samples were weighed at 14% moisture basis and mixed at a speed of 88 rpm. Water was added according to the water absorption values determined by the Farinograph to give final moisture content of 63.3 and 57.6% (fwb) for flours of Hereward and Riband wheat, respectively. The dough mixing parameters were obtained using the software, Mixsmart (National Mfg, TMCO, Lincoln, NE, USA). 2.4.5. Gluten protein extractability in SDS solution To examine the effect of heat on extractability, gluten (0.2 g) heated at different temperatures was suspended in 20 ml of 2% (w/v) SDS and mixed with a magnetic stirrer for 30 min, 1, 2, 5, 10, 15, 20 h or overnight and centrifuged at 30,000g for 1 h. The protein contents of the supernatants were determined by the BCA method after filtration through filter paper (Whatman No. 541).
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2.4.7. SDS-polyacrylamide gel electrophoresis SDS-PAGE was performed on a Protean II (Bio-Rad) Vertical electrophoresis cell (160!160!1 mm) using a 12.5% polyacrylamide separating gel containing 1.35% bis-acrylamide cross-linker according to Laemmli (1970). 2.4.8. Data analysis Statistical analysis was performed using statistical analysis system (SAS) software. Determination of significant differences among the means was performed by variance analysis and least significant difference (LSD) method.
3. Results 3.1. Intrinsic properties of the flour and gluten The Proximate compositional analysis (Table 1) shows that the total protein contents of both flour and gluten from the good breadmaking quality wheat cv Hereward were typically higher than the protein content of the poor breadmaking quality wheat cv. Riband. The mixing properties of the two flours are shown in Table 2. The hard wheat cv. Hereward had higher Farinograph water absorption values than the soft wheat cv. Riband. 3.2. Extractability
2.4.6. SS/SH content of flours and glutens Free SH contents were determined by the method of Beveridge et al. (1974). Flour (80 mg) or gluten (20 mg) was suspended in 1 ml of 85.9 mM Tris, 91.9 mM Glycine, 3.2 mM EDTA, pH 8 (TGE) buffer containing 2.5% (w/v) SDS. After incubation for 60 min at room temperature, 15 ml of 10 mM 5.5 dithiobis 2-nitrobenzoic acid (DTNB) in dimethylformamide was added and the reaction was allowed to proceed for 30 min at room temperature. The suspensions were then centrifuged at 10,000g for 10 min. The absorbances of the diluted supernatants (1/10 with TGE plus SDS) were measured against buffer blank solutions (TGE plus SDS) and against reagent blanks (TGE plus SDS containing DTNB). Total SS contents of glutens were determined by the methods of Weegels et al. (1994) with some modifications. Flour and gluten samples (20 and 80 mg, respectively) were suspended in 0.4 ml of 1.5% (w/v) SDS, Tris/HCl, pH 8 and mixed with 0.4 ml of 6 M NaOH. After 45 min incubation, 0.8 ml of H3PO4 and 0.05 ml of 10 mM DTNB (in 0.2 M phosphate buffer, pH 7) were added and further incubated for 1 h. After centrifugation at 10,000g for 10 min, the absorbance values of the supernatants were read at 412 nm against buffer and sample blanks. The SH and SS contents were calculated using an extinction coefficient of 13,600 MK1 cmK1. SS contents were calculated as the differences between the total and the free SH contents.
Table 3 shows that the extractability of both flour and gluten proteins into 2% (w/v) SDS was higher for the poor breadmaking quality cv Riband, as compared with the good breadmaking quality cv. Hereward. As shown in Fig. 1, heating resulted, as expected, in a decrease in the extractability of the gluten proteins. The extractability of protein from gluten heated at 50 8C was slightly higher than unheated gluten up to 10 h. The proportion of total protein extracted from unheated gluten after 20 h extraction was 92.5%. However, after the same time only 54.4% of total protein from gluten heated at 80 8C was extractable with SDS. For all the samples, protein extractability had Table 1 Proximate composition of flours and glutens from cultivars Hereward and Riband
Flour Protein contenta (%, dbb) Moisture (%, w/w) Wet gluten content (%, w/w) Gluten Protein content (%, db) Moisture (%, w/w) Starch content (%, db)
Hereward
Riband
13.7 12.2 28.5
11.1 13.5 21.5
78.2 4.5 12.2
74.6 6.0 18.1
All values are means of at least duplicate determinations. a Kjeldahl protein (N!5.7). b db: dry matter basis.
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Table 2 Mixing properties of flours from cultivars Hereward and Riband
Farinograph Water absorption (%) Development time (min) Stability (min) Degree of softening (BUa) Mixing tolerance index (BU) Mixograph Peak time (min) Peak dough resistance (MUb) Peak height Width at peak
Hereward
Riband
63.3 3.0 9.0 100 90
57.6 2.0 3.5 140 180
3.18 71.6 71.7 27.4
2.62 50.7 51.2 18.5
All values are means of at least duplicate determinations. a BU, Brabender unit. b MU, Mixograph unit.
essentially reached a plateau value after 5 h, indicating that this time was adequate to extract gluten proteins into SDS. The flour or gluten proteins from poor breadmaking quality cv. Riband were more soluble in 2% (w/v) SDS than the proteins of good breadmaking quality cv. Hereward. 3.3. Effect of redox additives on the extractability of heat-treated glutens The effect of redox additives was tested in two ways. In the first, doughs were treated with different redox additives and the gluten washed out immediately using the Glutomatic apparatus or Hobart mixer. In the second, freeze-dried gluten was prepared by the batter technique in the absence of additives and then rehydrated by mixing to peak development in the 2 g Mixograph in the presence of different redox agents to a final moisture content of 65% (w/w). 3.3.1. Gluten freshly washed out from dough The SDS extractability of good quality Hereward gluten, washed out from optimally mixed dough containing ascorbic acid (AA, 100 ppm), potassium bromate (50 ppm) or reduced glutathione (GSH, 100 ppm) remained almost constant as the temperature increased from 30 to 70 8C. However, the SDS extractability of the protein of Hereward control gluten decreased significantly (P!0.05) Table 3 Extractability in SDS (2%, w/v) of the protein from flours and glutens from cvs Cultivar Flour Hereward Riband Gluten Hereward Riband
Extracted protein (%) 87G1.7 91G2.3 59G0.9 68G1.5
Hereward and Riband, good and poor breadmaking quality wheats, respectively. Extraction was for 30 min at room temperature.
Fig. 1. Extractability of protein from unheated (C) heated at 50 8C (,), 60 8C (6), 70 8C (x), 80 8C (B), glutens (cv. Hereward flour) as a function of time. Extractant was 2% (w/v) SDS at room temperature.
over this temperature range (Fig. 2). The extractability declined more strongly at 90 8C, such that approximately 58% of the protein of the control gluten of Hereward became insoluble with heating at 90 8C. A similar pattern was observed for Riband control gluten (Fig. 3), although the changes in extractability were smaller. Redox agents affected SDS-extractability of gluten protein differently depending on the nature of the additive and the temperature. The extractability of protein from AA treated Hereward gluten remained more or less constant from 30 to 70 8C, then decreased significantly (P!0.05) to 54.1% at 90 8C (Figs. 2 and 3). The extractability of the gluten from AA treated Riband dough was slightly higher than the control at each temperature up to 70 8C, but at 90 8C the extractability of the AA treated Riband gluten fell to below that of the control (56.0% compared with 60.4%). Interestingly the extractability of the bromate treated Hereward gluten was more or less constant up to 70 8C, but at 90 8C it was approximately 15% higher than the control (Fig. 2), which was significant (P!0.05). While the protein extractability of untreated Hereward gluten decreased by about 10% as a result of heating at 70 8C compared with the unheated control, treatment with GSH, like that for oxidising agents, maintained protein extractability up to 70 8C, whereas at 90 8C the extractability fell to a value similar to the control. Bromate treated Riband gluten showed a similar pattern to the GSH or bromate treated Hereward gluten in that protein extractability remained constant up to 70 8C then at 90 8C fell to a value similar to the control (Figs. 2 and 3). No gluten could be washed out from the Riband dough treated with GSH. Hereward gluten treatment with both oxidants (bromate and ascorbate) apparently maintained constant gluten protein extractability constant up to 70 8C, whereas a significant (P!0.05) fall was observed for the control. The reducing agent, GSH, had a similar effect over this temperature range. At 90 8C the protein extractability of the oxidant treated Hereward gluten was higher than that of the
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Fig. 2. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Hereward gluten washed out from doughs treated with redox additives. Solutions of ascorbic acid (AA) (100 ppm, flour weight basis, fwb), potassium bromate (50 ppm, fwb) or reduced glutathione (GSH) (100 ppm, fwb) were used.
control, especially for the bromate treated gluten, whereas the extractability of the GSH treated gluten had fallen to a similar value to the control. As with Riband glutens, the oxidants seemed to have only small effects on the heat induced changes in protein extractability, the behaviour of the oxidant treated glutens being more similar to that of the control than in the case of Hereward gluten.
3.3.2. Freeze-dried glutens treated directly with redox additives SDS protein extractabilities for glutens that had been rehydrated in the 2 g Mixograph were lower than the values for gluten prepared directly from dough (Figs. 4 and 5). Although similar extractabilities were observed at 30 and 50 8C, heat treatment at 70 8C caused a slight reduction in
Fig. 3. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Riband gluten washed out from dough treated with redox additives. Solutions of ascorbic acid (AA) (100 ppm, flour weight basis, fwb), potassium bromate (50 ppm, fwb) or reduced glutathione (GSH) (100 ppm, fwb) were used.
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Fig. 4. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Hereward gluten treated with redox additives. Solutions of ascorbic acid (1000 ppm, gluten weight basis, gwb), potassium bromate, (500 ppm, gwb) and reduced glutathione (GSH) (250 ppm, fwb) were used.
extractability. Further heating at 90 8C dramatically reduced the amount of SDS extractable protein except for the bromate treated glutens of both cultivars. The extractabilities of bromate treated glutens were significantly (P!0.05) high even after heat treatment at 90 8C (Figs. 4 and 5). Slightly
higher extractabilities were observed for poor quality Riband gluten irrespective of temperature and additive used compared with good quality Hereward gluten. There was relatively little difference between the control and AA treated glutens at any temperature. Interestingly, the GSH treated
Fig. 5. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Riband gluten treated with redox additives. The solutions of additives were: ascorbic acid (1000 ppm, gluten weight basis, gwb), potassium bromate, (500 ppm, gwb) and reduced glutathione (250 ppm, gwb).
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Table 4 Effect of heat on the SH (mmol/g of protein) contents of glutens prepared from doughs treated with redox additives Wheat cultivar treatment
30
Temperature (8C) SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate
2.78 1.88 1.75 2.49 1.83 1.79 1.46
0.11 0.07 0.09 0.15 0.13 0.04 0.20
1.95 1.61 1.56 2.48 1.52 1.35 1.17
0.13 0.11 0.15 0.13 0.06 0.12 0.10
1.51 1.33 1.20 2.38 1.44 1.13 0.81
0.16 0.11 0.13 0.14 0.13 0.04 0.04
1.16 0.99 0.76 2.27 1.40 0.83 0.38
0.13 0.04 0.07 0.05 0.10 0.02 0.06
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
The SH content of the gluten from the poor breadmaking quality cv. Riband was significantly (P!0.05) lower than that of the gluten from the good breadmaking quality cv. Hereward. Whereas heating at 90 8C reduced the SH content of Riband gluten by about 20%, whereas the reduction was 60% for the Hereward gluten.
gluten had a significantly (P!0.05) lower extractability after heating at 90 8C than the control unheated glutens for both wheat cvs. 3.4. SH and SS contents 3.4.1. SH content of gluten freshly washed out from dough The effect of heat treatment (30–90 8C) on the SH contents of glutens prepared from dough treated with or without redox additives is illustrated in Table 4. In general, the SH contents of control and oxidant treated glutens decreased significantly (P!0.05) as the temperature increased from 30 to 90 8C The SH content of control (untreated) gluten from the cv. Hereward heated at 90 8C was approximately 60% lower than that of gluten heated at 30 8C. While oxidant treatments (ascorbate and bromate) cause about a 50% reduction in SH content, treatment with the reducing agent, glutathione (GSH), resulted in less pronounced decrease in gluten SH content when the gluten was heated at 70 and 90 8C compared with 30 and 50 8C.
3.4.2. SH content of freeze-dried glutens treated directly with redox additives Gluten isolated from flour was treated directly with redox additives in a comparison of their direct effects on gluten protein with their indirect effects when the gluten was freshly washed out from doughs treated with additives (Section 3.4.1). The addition of oxidants (ascorbate and bromate) directly to gluten resulted in a decrease in the SH content of both glutens (Table 5). Ascorbate treatment reduced the SH content by 26 and 12% in Hereward and Riband glutens, respectively. While bromate treatment reduced the SH content of unheated (30 8C) Hereward gluten by 50%, the decrease was 23% for poor quality Riband gluten at 30 8C. Heating at 90 8C enhanced
Table 5 Effect of heat on the SH (mmol/g of protein) contents of glutens treated directly with redox additives Wheat cultivar treatment
30
Temperature (8C) SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate GSH
2.56 1.89 1.27 3.76 2.45 2.15 1.87 3.64
0.10 0.12 0.14 0.16 0.12 0.15 0.19 0.11
1.70 1.23 1.22 3.47 1.86 1.81 1.53 3.34
0.18 0.09 0.13 0.17 0.14 0.10 0.09 0.09
1.35 0.94 0.52 2.99 1.57 1.28 0.77 3.28
0.08 0.12 0.11 0.14 0.13 0.10 0.12 0.05
1.23 0.75 0.29 2.85 1.22 1.05 0.22 3.16
0.02 0.05 0.12 0.10 0.09 0.07 0.05 0.14
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
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Table 6 Effect of heat on the SS (mmol/g of protein) contents of glutens prepared from doughs treated redox additives Wheat cultivar treatment
Temperature (8C) 30
SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate
36.47 38.88 33.80 37.47 33.53 34.28 30.59
0.58 0.30 0.61 0.43 0.14 0.11 0.97
36.11 35.59 34.61 36.41 34.28 31.14 30.64
0.07 0.10 0.66 0.22 0.28 0.32 0.15
35.92 38.08 34.26 33.26 33.84 32.50 31.34
0.95 0.30 0.49 1.10 0.18 0.09 0.30
34.70 37.39 37.51 32.24 29.14 31.12 31.47
0.78 0.24 0.43 0.14 0.89 0.06 0.36
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
the decrease in the presence of bromate for both cultivars. For example at 30 8C when bromate was used, the SH content was reduced by 50%. However, on heating at 90 8C a further 25% decrease in the SH content was observed. The SH contents of the GSH treated glutens were higher than those of the controls for both cultivars, and the SH contents decreased slightly on heating.
Such small changes are likely to be within the experimental error of the method used for SS determination. 3.4.4. SS content of gluten treated directly with redox additives The SS bond contents of glutens treated directly with redox additives were similar to those of glutens prepared from dough treated with redox additives (Table 7). Again there was no clear effect on SS bond content of redox additive treatment or of heat treatment.
3.4.3. SS content of gluten freshly washed out from dough The SS contents of control and glutens of Hereward and Riband treated with additives were similar (Table 6), although the good quality Hereward gluten had a slightly higher amount of SS than the poor quality Riband gluten throughout the temperature range studied The SS analysis provided little evidence that the decrease in SH content due to oxidant treatment or to heating resulted in increases in SS contents. However, since the changes in SH contents were only about 1 mmol of SH/g gluten at most, and since two SH groups are lost for every SS bond formed, the changes in SS bond contents are, in any case, very small compared with the total SS content of the control untreated gluten.
3.5. Polypeptide composition of gluten freshly washed out from dough Heating the glutens prepared from redox treated doughs at 30 and 50 8C had no apparent effect on polypeptide composition of the glutens when analysed by SDS-PAGE under reducing conditions (not shown). However, band intensities generally decreased after heating to 70 8C and the decrease became more pronounced as a result of heating to 90 8C (not shown). Bands corresponding to HMW subunits
Table 7 Effect of heat on the SS (mmol/g of protein) contents of glutens treated with redox additives Wheat cultivar treatment
Temperature (8C) 30
SDa
50
SD
70
SD
90
SD
Hereward control AAb Bromatec GSHd Riband control AA Bromate GSH
35.60 36.12 36.03 36.78 33.10 33.83 31.75 32.69
0.17 0.46 0.30 0.15 0.30 0.50 0.23 0.50
36.44 36.40 32.80 34.55 33.17 34.24 33.60 33.07
0.17 0.91 0.32 0.58 1.29 1.07 0.34 0.14
33.54 37.67 34.74 36.66 35.13 32.69 32.86 33.83
0.86 0.85 0.76 0.43 0.85 0.29 0.80 0.19
34.03 35.68 36.84 36.43 32.39 32.86 32.74 33.27
0.16 1.36 0.26 0.15 0.30 0.14 0.19 0.21
All values are the means of triplicate determinations. a Standard deviation. b L-Ascorbic acid. c Potassium bromate. d Reduced glutathione.
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of glutenin had almost disappeared after heating to 70 and 90 8C, but interestingly the band intensities of HMW glutenin subunits in bromate treated glutens were partly retained when compared with untreated control gluten. Ascorbate treatment also showed a slight protective effect against heating. Additionally, the HMW glutenin subunits from the good quality Hereward gluten were affected to a greater extent by heating to 90 8C than were those of poor quality Riband gluten as evidenced by the band intensities. Almost all the protein that did not enter the separating gel under non-reducing condition (not shown) migrated into the gel as discrete identifiable bands under reducing conditions. 3.6. Polypeptide composition of gluten treated directly with redox additives Similar trends were observed in the electrophoretic patterns of SDS-extractable proteins of glutens treated directly with redox additives. Again the band intensities in HMW glutenin subunit region of bromate treated gluten from both varieties were partly preserved even after heat treatment at 90 8C.
4. Discussion 4.1. Intrinsic properties of the flours and glutens Total protein and gluten contents generally show a linear relationship with breadmaking performance as measured by loaf volume (Finney and Barmore, 1948). The starch damage and protein content of wheat are the main determinants of the water absorption of flour. The differences in endosperm texture results in variation in the ability to direct the mechanical energy to the starch granules during milling (Stevens, 1987). The degree of starch damage during milling is greater for hard wheats than for soft wheats, therefore, the water absorption capacity of hard wheats increases. Protein content is also generally higher in hard wheats than in soft wheats (Kent and Evers, 1994). As expected the dough development time and peak time measured by the Farinograph and Mixograph, respectively, were longer for Hereward flour than for the poor quality Riband flour. This implies that the hydration of protein and starch and the development of the viscoelastic structure to a certain consistency require more time and energy input for good quality flour. Starch, protein and their interactions have a strong influence on the mixing behaviour of flour (Preston and Kilborn, 1984). Again stability and mixing tolerance index values, which are influenced by interactions of flour components, especially starch and proteins, were greater for Hereward flour. The empirical data on mixing characteristics suggests that there are differences in physical and possibly molecular properties of flours from diverse breadmaking quality wheat. Thus these data gave assurance, that the flours chosen for
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this study had diverse physicochemical/functional properties. 4.2. SDS-extractability The extractability of flour and gluten proteins with SDS from good and poor breadmaking quality (cvs. Hereward and Riband, respectively) showed that gluten is less extractable from good than poor flours. This observation is in accordance with previous studies. Butaki and Dronzek (1979) found that stronger wheat flours had more acid insoluble (0.05 M acetic acid) gluten than weak flours. Similarly, He and Hoseney (1991) showed that gluten from poor quality flour had higher solubility in SDS solution than gluten from good quality flour. The difference in protein extractabilities between glutens was assumed to result from differences in molecular size and aggregation tendency. Differences in the aggregation behaviour (Arakawa and Yonezawa, 1975), molecular weight or interaction tendency (He and Hoseney, 1991) or conformational properties (Huang and Khan, 1996) have been suggested as possible factors responsible for the varying extractabilities of gluten proteins from wheats of diverse breadmaking quality. However, in respect to extractability in SDS solution, molecular weight is likely to be the dominant factor. As the solubility of heated proteins depends on their molecular weights (MW) (Pomeranz, 1988), the decrease in extractability into SDS solution after heat treatment, especially at higher temperatures, probably indicates an increase in MW, which might result from aggregation of proteins during heating. In agreement with the previous reports of Booth et al. (1980), Jeanjean et al. (1980) and Schofield et al. (1983), the results of the extractability of gluten proteins with SDS solution revealed that heat causes the gluten proteins to associate to form larger protein aggregates that are less extractable; as aggregation (crosslinking) progresses the SDS-extractable fraction decreases. The heat effect on gluten is explained on the basis of protein crosslinking through a SH/SS exchange mechanism. Redox agents affected SDS-extractability of gluten protein differently depending on the type of additive and the temperature. The effect of redox improvers is greater in good breadmaking flour than in poor breadmaking flour due to differences in the rate of reactions, which are lower in good quality flour (Mair and Grosch, 1979). One might have expected that oxidising agents would have caused a reduction in protein extractability by increasing protein crosslinking through SS bond formation, whereas reducing agents would have caused a depolymerisation of gluten by cleaving the SS bonds. However, improvers such as ascorbic acid and potassium bromate increase the solubilization of insoluble protein during dough mixing (Danno and Hoseney, 1982; Graveland et al., 1985; Sievert et al., 1991). Different explanations have been proposed involving physical forces, covalent and
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non-covalent bonds and conformational rearrangements. One proposed explanation is that oxidising agents cause crosslinking between glutenin fractions leading to increase in stiffness of dough and increased work input. The increased work input would lead to mechanical rupture of SS bonds that would increase protein extractability (Graveland et al., 1984). However, Eckert et al. (1993) proposed that oxidant effects result from conformational rearrangements rather than size reduction leading to a more extended structure and thus increased extractability. If the dough is allowed to rest after mixing, polymerisation of glutenin due to oxidation of SH groups (Graveland et al., 1985; Wang et al., 1992) or decrease in polymer surface due to structural relaxation, results in a decrease in protein extractability (Eckert et al., 1993). In the presence of oxidants (ascorbic acid and potassium bromate), the enhancement of the changes in glutathione (GSH, GSSG, PSSG) during dough mixing (Chen, 1994), such as shortening of the dough development time and narrowing of Farinograph band width in rheological tests (Frater et al., 1960; Ikoeza and Tipples, 1968) are consistent with the results of the present study. One of the postulates is that oxidants enhance protein depolymerization and dough breakdown (Chen, 1994). However, it is also known that oxidation favours polymerisation of gluten proteins. The higher extractability of additive treated, especially AA and bromate treated glutens, after heat treatment at higher temperatures (70 and 90 8C) suggest that redox additives alter the protein structure in such a way that heat induced changes in SS bonding are modified and gluten protein extractability is maintained. The rate of oven rise for oxidant treated doughs was observed to be higher than that of untreated doughs implying a delay in the denaturation temperature of gluten network (Yamada and Preston, 1992). This appears to be consistent with the present observations on the effects of heat on the SDS extractability of glutens from oxidant treated doughs. We observed an increase in extractability of good breadmaking quality Hereward glutens washed from oxidant treated dough. The action of AA is thought to involve oxidation of endogenous flour GSH to GSSG (Grosch and Wieser, 1999). Although this reaction has not been experimentally demonstrated, it is speculated that AA (a reducing agent) must first be converted by oxidation with molecular oxygen to dehydroascorbic acid (DHAA, an oxidising agent). If the mixing environment does not incorporate enough oxygen into the dough, the conversion of AA to DHAA may not occur efficiently. Therefore, AA may act as a reducing agent by breaking SS bonds under conditions of oxygen insufficiency (Mauseth et al., 1967). The increase in protein extractability in SDS caused by oxidants is likely to result from redox modification of SH groups such that interchain SS bonding among gluten polymers was decreased. This is not to say that hydrophobic interactions do not play a role in the non-covalent aggregation of gluten proteins during heating that may
facilitate subsequent oxidation of SH groups to SS bonds or the rearrangement of SS bonds through SH/SS exchange reactions, but that heat-induced changes in hydrophobic interactions themselves do not lead to differences in SDS extractability. With other non-denaturing solvents, however, such interactions could well affect protein solubility. 4.3. SH and SS contents The actions of oxidising and reducing agents on dough properties occur at different stages of breadmaking (mixing, proofing, baking) and consequently there are also changes in protein structure at different stages of breadmaking (Fitchett and Frazer, 1986). There are contradictory reports of the effects of heat and oxidant on the SH content of doughs. Although a significant decrease in the SH content of bromate treated and baked dough was reported by Tsen (1968), the finding of a similar reduction in SH groups during heating of both bromate treated and untreated doughs does not support the mechanism of bromate oxidation (Andrews et al., 1995). Notwithstanding an extremely high level of oxidant, compared with commercial practice, was used, the SH content of oxidant treated (ascorbate and bromate, 1200 ppm) doughs mixed in the Do-Corder and heated from 35 to 75 or 85 8C decreased and the decrease in SH content of bromate treated dough was higher than that of ascorbate treated dough (Nagao et al., 1981). The concentrations of SH groups, analysed by amperometric titration, by Matsuo and McCalla (1964) for glutens from hard, soft and durum wheat varieties were 3.8, 4.0 and 4.4 mequiv/g of protein, respectively. The values were approximately half those obtained in the present study although the differences in wheat cultivars and method of determination would obviously affect the results. There are various reports on the relationship between SH and SS contents and wheat variety. For example, Tsen and Bushuk (1968) reported that total SH content increases with decreasing flour strength. Although total SS content decreased with decreasing strength, the number of reactive SS bonds increased. It was also found that there was no relationship between protein and SH contents (Axford et al., 1962, Tsen and Anderson, 1963). SH content of doughs differing in their response to bromate showed that the decline in the SH content of bromate responsive flours was larger than that for non-responsive flours (Andrews et al., 1995). The literature regarding the SS contents of flours is somewhat contradictory. Total SS content showed an inverse relationship with various flour quality parameters and also varied independently among very strong, strong and weak wheat flours (Axford et al., 1962). In another study neither total SH nor SS contents of flours showed correlations with baking quality (Graveland et al., 1978).
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4.4. Polypeptide composition In wheat, the glutenin protein components of gluten are polymers stabilised by intra- and intermolecular SS bonds and the gliadins are monomers with either no SS bonds (u-gliadins) or intramolecular bonds (a- and b-type) (Tatham et al., 1990). During heating the native protein structure is destabilised and unfolding may facilitate SH/SS interchange and oxidation together with hydrophobic interactions (Li and Lee, 1998). The appearance of the HMW glutenin subunit region in the reduced gels even after high temperature (90 8C) heat treatment of gluten in the presence of bromate suggests that the addition of bromate could reduce the decrease in protein extractability caused by heat treatment. It is also likely that bromate favours oxidation to the disulphide and higher oxidation states. This observation is consistent with the direct measurements of protein extractability for glutens heated to different temperatures. This may occur by weakening the extensive crosslinking of gluten. However, this finding contrary to previous reports in which SDSPAGE (Atanassova and Popova, 1977) and SE-HPLC (Bekes et al., 1996) of flour proteins showed that the high molecular weight fraction (glutenin aggregates) increased as a result of bromate treatment. A similar, but less pronounced, effect of ascorbate was also observed. In a study by Veraverbeke et al. (1997), it was found that addition of the oxidant, potassium iodate, to dough resulted in higher levels of SDS-extractable protein from bread. This is consistent with the findings of the present study on heatinduced SDS-extractability and the polypeptide composition of gluten proteins as determined SDS-PAGE analysis.
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