Heat and additive induced biochemical transitions in gluten

from good and poor breadmaking quality wheats

Mehmet Haytaa,*, J. David Schofieldb

aDepartment of Food Engineering, Inonu University, Elazig Yolu 44069 Malatya, TurkeybDepartment 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).

0733-5210/$ - see front matter q 2004 Published by Elsevier Ltd.

doi:10.1016/j.jcs.2004.06.006

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: mhayta@inonu.edu.tr (M. Hayta).

Redox agents modify the structure and functional

properties of wheat gluten proteins (Bloksma and Bushuk,

1988). Reducing agents, such as sodium sulphite, mercap-

toethanol, 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 altera-

tions 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

Journal of Cereal Science 40 (2004) 245–256

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M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256246

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 pro-

cedures (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.

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 Buhler experi-

mental 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

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

2.4.1. 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 Chemi-

cal 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).

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256 247

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).

Table 1

Proximate composition of flours and glutens from cultivars Hereward and

Riband

Hereward Riband

Flour

Protein contenta (%, dbb) 13.7 11.1

Moisture (%, w/w) 12.2 13.5

Wet gluten content (%, w/w) 28.5 21.5

Gluten

Protein content (%, db) 78.2 74.6

Moisture (%, w/w) 4.5 6.0

Starch content (%, db) 12.2 18.1

All values are means of at least duplicate determinations.a Kjeldahl protein (N!5.7).b db: dry matter basis.

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.

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 signifi-

cant 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 proper-

ties 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

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 2

Mixing properties of flours from cultivars Hereward and Riband

Hereward Riband

Farinograph

Water absorption (%) 63.3 57.6

Development time (min) 3.0 2.0

Stability (min) 9.0 3.5

Degree of softening (BUa) 100 140

Mixing tolerance index (BU) 90 180

Mixograph

Peak time (min) 3.18 2.62

Peak dough resistance (MUb) 71.6 50.7

Peak height 71.7 51.2

Width at peak 27.4 18.5

All values are means of at least duplicate determinations.a BU, Brabender unit.b MU, Mixograph unit.

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.

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256248

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 Extracted protein (%)

Flour

Hereward 87G1.7

Riband 91G2.3

Gluten

Hereward 59G0.9

Riband 68G1.5

Hereward and Riband, good and poor breadmaking quality wheats,

respectively. Extraction was for 30 min 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 extrac-

tability 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

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.

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256 249

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.

Fig. 3. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. Riba

ascorbic acid (AA) (100 ppm, flour weight basis, fwb), potassium bromate (50 pp

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

nd gluten washed out from dough treated with redox additives. Solutions of

m, fwb) or reduced glutathione (GSH) (100 ppm, fwb) were used.

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.

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256250

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

Fig. 5. Effects of heat on the extractability (2% (w/v) SDS) of protein from cv. R

ascorbic acid (1000 ppm, gluten weight basis, gwb), potassium bromate, (500 pp

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

iband gluten treated with redox additives. The solutions of additives were:

m, gwb) and reduced glutathione (250 ppm, gwb).

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

Temperature (8C)

30 SDa 50 SD 70 SD 90 SD

Hereward control 2.78 0.11 1.95 0.13 1.51 0.16 1.16 0.13

AAb 1.88 0.07 1.61 0.11 1.33 0.11 0.99 0.04

Bromatec 1.75 0.09 1.56 0.15 1.20 0.13 0.76 0.07

GSHd 2.49 0.15 2.48 0.13 2.38 0.14 2.27 0.05

Riband control 1.83 0.13 1.52 0.06 1.44 0.13 1.40 0.10

AA 1.79 0.04 1.35 0.12 1.13 0.04 0.83 0.02

Bromate 1.46 0.20 1.17 0.10 0.81 0.04 0.38 0.06

All values are the means of triplicate determinations.a Standard deviation.b

L-Ascorbic acid.c Potassium bromate.d Reduced glutathione.

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256 251

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 tempera-

ture 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.

Table 5

Effect of heat on the SH (mmol/g of protein) contents of glutens treated directly w

Wheat cultivar

treatment

Temperature (8C)

30 SDa 50 SD

Hereward control 2.56 0.10 1.70 0.18

AAb 1.89 0.12 1.23 0.09

Bromatec 1.27 0.14 1.22 0.13

GSHd 3.76 0.16 3.47 0.17

Riband control 2.45 0.12 1.86 0.14

AA 2.15 0.15 1.81 0.10

Bromate 1.87 0.19 1.53 0.09

GSH 3.64 0.11 3.34 0.09

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.

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

ith redox additives

70 SD 90 SD

1.35 0.08 1.23 0.02

0.94 0.12 0.75 0.05

0.52 0.11 0.29 0.12

2.99 0.14 2.85 0.10

1.57 0.13 1.22 0.09

1.28 0.10 1.05 0.07

0.77 0.12 0.22 0.05

3.28 0.05 3.16 0.14

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 36.47 0.58 36.11 0.07 35.92 0.95 34.70 0.78

AAb 38.88 0.30 35.59 0.10 38.08 0.30 37.39 0.24

Bromatec 33.80 0.61 34.61 0.66 34.26 0.49 37.51 0.43

GSHd 37.47 0.43 36.41 0.22 33.26 1.10 32.24 0.14

Riband control 33.53 0.14 34.28 0.28 33.84 0.18 29.14 0.89

AA 34.28 0.11 31.14 0.32 32.50 0.09 31.12 0.06

Bromate 30.59 0.97 30.64 0.15 31.34 0.30 31.47 0.36

All values are the means of triplicate determinations.a Standard deviation.b

L-Ascorbic acid.c Potassium bromate.d Reduced glutathione.

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256252

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.

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.

Table 7

Effect of heat on the SS (mmol/g of protein) contents of glutens treated with redo

Wheat cultivar

treatment

Temperature (8C)

30 SDa 50 SD

Hereward control 35.60 0.17 36.44 0.17

AAb 36.12 0.46 36.40 0.91

Bromatec 36.03 0.30 32.80 0.32

GSHd 36.78 0.15 34.55 0.58

Riband control 33.10 0.30 33.17 1.29

AA 33.83 0.50 34.24 1.07

Bromate 31.75 0.23 33.60 0.34

GSH 32.69 0.50 33.07 0.14

All values are the means of triplicate determinations.a Standard deviation.b

L-Ascorbic acid.c Potassium bromate.d Reduced glutathione.

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.

3.5. 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

x additives

70 SD 90 SD

33.54 0.86 34.03 0.16

37.67 0.85 35.68 1.36

34.74 0.76 36.84 0.26

36.66 0.43 36.43 0.15

35.13 0.85 32.39 0.30

32.69 0.29 32.86 0.14

32.86 0.80 32.74 0.19

33.83 0.19 33.27 0.21

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256 253

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

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 extrac-

tability 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 depolymer-

isation 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

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256254

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 dehy-

droascorbic 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 ampero-

metric 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).

M. Hayta, J.D. Schofield / Journal of Cereal Science 40 (2004) 245–256 255

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 SDS-

PAGE (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 heat-

induced SDS-extractability and the polypeptide compo-

sition of gluten proteins as determined SDS-PAGE analysis.

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