Mechanism of Heat and Shear Mediated Aggregation of Wheat Gluten

Biomacromolecules 2002, 3, 488-497

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Mechanism of Heat and Shear Mediated Aggregation of Wheat Gluten Protein upon Mixing Marie-He´ le` ne Morel,* Andre´ as Redl, and Ste´ phane Guilbert Unite´ de Technologie des Ce´ re´ ales et des Agropolyme` res, ENSA.M-INRA, 2 place Viala, 34060, Montpellier Cedex 01, France Received November 13, 2001; Revised Manuscript Received January 29, 2002

Changes in wheat gluten network structure upon mixing were studied from the biochemical analyses of gluten/glycerol blends mixed at 100 rpm with increasing times (up to 30 min) and temperatures of regulation (40, 60, and 80 °C). Whereas mixing induced protein solubility loss, the reduction of disulfide bonds restored protein extractability. But disulfide bond reduction became less efficient in promoting gluten extractability as mixing severity increased. This feature is consistent with the formation of a three-dimensional protein network stabilized by the formation of an increasing number of interchain disulfide bonds. Mixing induced a transient increase in free thiol groups while total thiol-equivalent groups dropped continuously. The changes were attributed to a shear-mediated scission of gluten disulfide bonds followed by oxidation of the thiyl radical moieties. Upon mixing, gluten solubility loss showed an Arrhenius-type temperature dependence with activation energy of 33.7 kJ‚mol-1 instead of the more than 100 kJ‚mol-1 reported for heat-induced gluten protein solubility loss. To explain this discrepancy, we postulated that during mixing, the disulfide interchange reactions are mediated by thiyl radicals in place of free thiol groups. A general model accounting for shear and temperature effects on gluten network structure is proposed. Introduction Among cereal proteins, wheat gluten is unique in its ability to form a dough exhibiting viscoelastic and gas barrier properties.1 Numerous studies have investigated the physicochemical characteristics of gluten in relation to the breadmaking potential of flour.2,3 Gluten, which consists up to 85% of protein, comprises monomers (gliadin) and polymers (glutenin) in a weight balance of approximately 65/45.4,5 The polymeric structure of glutenin arises from the connection of different types of polypeptides (named glutenin subunits) through disufide cross-linkings.6 The precise structure and composition of gluten polymers are rather unknown, but it is widely accepted that their sizes may extend up to millions.7 Molecular size distribution of glutenin polymers was found as one of the main factor responsible for variations in dough viscoelasticity among different wheat flour samples.8-10 Wheat storage proteins are characterized by their high contents in glutamine and proline and, conversely, by their low content in charged amino acid. In consequence they are insoluble in water, and ionic detergents, such as sodium dodecyl sulfate or cetyl amonium bromide, are needed to assist in their extractability. In addition, the large molecular size of glutenin polymer impairs its solubility and sonication and/or reduction of interchains disulfide bonds are usually applied to achieve total protein extraction.11-14 Gluten, owing to its viscoelastic properties and its low water solubility, offers very interesting characteristics for nonfood applications. Attempts to produce degradable “bio* Corresponding author. Telephone (33) 04 99 61 25 62. Fax (33) 04 67 52 20 94. E-mail: [email protected].

plastic” from gluten/glycerol blend by extrusion have revealed that temperature and shear rate strongly affected the structural characteristics of extrudates.15 In particular, gluten solubility loss (in 1% sodium dodecyl sulfate (SDS) buffer) was observed when high specific mechanical energy input was used during extrusion. It is well-known that the solubility of wheat flour protein decreases dramatically during extrusion processing of food products as the die temperature increases.16-18 Physicochemical modifications of gluten protein upon heating above 60-65 °C are largely documented and were recently reviewed by Weegels and Hamer.19 Heat treatments affect both biochemical and rheological charateristics. Whatever the parameter under consideration (rheological changes, solubility loss), activation energy for heat-induced aggregation of gluten ranged from 137 to 300 kJ/mol, higher values being reported for low moisture systems.20 It is still unclear whether the heat-induced gluten protein solubility loss in SDS-buffer results from an increased aggregation by weak forces, from protein crosslinking, or from a conformational change of the protein per se. Nevertheless several features indicate that protein solubility loss involves thiol and disulfide groups. Thus, ω-gliadins, which are devoid of cysteine residue, remain soluble even after exhaustive thermal treatment, whereas other gliadin polypeptides, which include two to four intrachain disulfide bonds became insoluble.21 On the other hand, alkylation of sulfhydryl groups with N-ethyl-maleimide suppresses the large increase in gluten elasticity normally observed upon heating, providing evidence for thiol groups involvement.22 Schoffield et al.23 have shown that oxidation of gluten sulfhydryl groups was not significant upon heating. In fact,

10.1021/bm015639p CCC: $22.00 © 2002 American Chemical Society Published on Web 03/06/2002

Biomacromolecules, Vol. 3, No. 3, 2002 489

Changes in Wheat Gluten Network Structure Table 1. Characteristics of Mixing Experiments and Experimental Samplings

Trega

FTb

40

78.5

60

89.2

80

103.5

peak1c torque tP1 (min) (N m) 2.26 ((0.21) 0.43 ((0.11) 0.87 ((0.02)

33.0 ((1.0) 30.9 ((0.9) 26.3 ((0.3)

peak2c torque tP2 (min) (N m) 6.00 ((0.3) 3.93 ((0.2) 3.06 ((0.1)

37.1 ((0.9) 31.8 ((1.2) 26.2 ((0.6)

experimental samplinge

Tp2d 77.3 ((1.3) 89.0 ((0.6) 99.0 ((1.8)

2 min (0.2, 57.0) 1 min (nd, 78.7)

4 min (1, 73.0) 2.5 min (0.6, 89.2) 1 min (nd, 92.4)

6 min (1.5, 77.5) 4 min (1.1, 89.0) 3 min (0.8, 99.4)

8 min (2.5, 76.1) 8 min (2.5, 87.1) 8 min (2.2, 97.4)

15 min (5.5, 75.6) 15 min (5.1, 88.1) 15 min (3.9, 100)

30 min (11, 78.5) 30 min (8.7, 89.2) 30 min (7.4, 103.5)

a Treg: regulation temperature of the mixing bowl (°C). b FT: sample temperature after 30 min mixing (°C). c Torque level and mean time (t ) at peaks p 1 and 2 (average value calculated from the different runs of the same Treg series). d Mean sample temperature recorded after peak2 (°C) and calculated e from the 8, 15, and 30 min runs. Numbers in parentheses correspond to the specific mechanical energy (kJ/g) and the sample temperature (°C), respectively.

gliadin and glutenin polymers are practically devoid of free sulfhydryl groups, all cysteine residues being oxidized in disulfide bonds. On the other hand, thiol reducing agents are very efficient in promoting protein extractability from heat-treated gluten. To account for these features, Schoffield et al.23 proposed that protein aggregation involved sulfhydryl/ disulfide interchange reactions that fixed the protein into the denaturated state. Since that pioneer work, several studies supported the role of sulfhydryl-disulfide interchange reaction in wheat protein aggregation during extrusion.18,24,25 During extrusion processing, sample temperature results from heat exchange with the environment (possibly with an external cooler or heater) and from heat generated by frictional forces. This last parameter is a function of mechanical work input, i.e., of shearing rate and sample viscosity. In this, temperature and shearing stress are dependent parameters during the extrusion process and their specific effects on gluten aggregation remained speculative. Shearing stress is likely to induce mechanical degradation of protein or starch polymers.26,27 Analysis of gluten/glycerol samples collected along the extrusion screw showed that proteins were depolymerized in the high-shear-rate zone of the screw, whereas aggregation occurred in the converging section of the die, where sample temperature sharply rose.15 During the extrusion process, the disruptive effect of shear is usually hidden by the rise of the sample temperature, which triggered protein aggregation. It is admitted that extrusion involves a complete restructuring mechanism with dissociation and unraveling of the macromolecules followed by their recombination into an oriented pattern.28 A comprehensive understanding of the gluten extrusion process would be gained from specific knowledge of the changes induced by shear stress, on one hand, and by temperature, on the other hand. To address the problem we had investigated the rheological and biochemical changes induced by mixing (up to 30 min, 100 rpm) gluten/glycerol blend at different temperatures of regulation of the mixing bowl (Treg ) 40, 60, and 80 °C).29 In any case, we observed aggregation of gluten proteins according to a mechanism which was strongly temperature dependent. Gluten aggregation was expected because during mixing the sample temperature rapidly rose above 60 °C and leveled off within a few minutes at 77, 89, and 99 °C according to Treg. Taking these temperatures into account, we calculated an activation energy of 33,7 kJ/mol for gluten aggregation upon mixing.29 Considering the reported activation energy for the heat-

induced aggregation of gluten (137-300 kJ/mol), such a low value was totally unexpected.20 The objective of the present work is to determine the underlying mechanisms responsible for this feature. Several biochemical parameters were examined. We studied the rate of changes in thiol and disulfide groups to assess for sulfhydryl/disulfide interchange upon mixing. To investigate the network connectivity, we studied the effect of stepwise reduction of disulfide bonds on protein extractability and on protein size distribution. Similar approaches have already been used to characterize the native glutenin polymeric structure in flour or dough but never to follow its alteration upon thermomechanical treatments.14,30 Kinetics of the biochemical changes involved during mixing were fitted according to first-order rate models. These approaches allowed us to propose a mechanistic model of the effects of temperature and mechanical shear on the changes in gluten protein network structure. Experimental Section Gluten was graciously obtained from Amylum (Aalst, Belgium). According to the manufacturer, the contents of protein, starch, lipid, and ash were 76.5%, 11.8%, 5.0%, and 0.8% (dry matter basis), respectively. Moisture content was 7.2%. Anhydrous glycerol was purchased from Fluka Chemie (Buchs, Switzerland) in p.a. quality. Chemicals for biochemical analysis of the samples were obtained from Sigma or Merck in p.a. quality. Mixing Process and Materials. Fifty grams of a gluten and glycerol blend (32.68/17.32) was mixed in a two-blade counter-rotating batch mixer turning at a 3:2 differential speed (Plasticorder W 50, Brabender, Duisburg, Germany) as described by Redl et al.29 Mixing speed was 100 rpm, and the mixing chamber was regulated at 40, 60, and 80 °C using a cryostat (Lauda RC 20) and water circulation in the double chamber of the mixer (75 g/s). Torque and temperature were continuously recorded during mixing. Typically, torque curves rose rapidly and then showed two consecutive and close maxima (peak1 and peak2) followed by a more or less stable phase. In contrast, increase in sample temperature was merely exponential and its leveling coincided to peak2. Sampling was performed from distinct mixing experiments, from 1 to 30 min periods. Table 1 presented the sampling and resumed the main characteristics of the recorded torque and temperature signals. Collected samples were grounded

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with dry powder of soluble starch (Merck) (1/5 g/g) and in the presence of liquid nitrogen by using a laboratory ball mill (Prolabo, France). Protein content (N × 5.7) of blends was determined by the Kjeldahl method.31 Thiol, Thiol Equivalent, and Disulfide Contents. Contents in thiol (SH), thiol equivalent (SHeq), and disulfide (SS) groups from samples were assayed as described elswere.31,32 Briefly, for SH determination, 60 mg of ground sample was mixed for 10 min with 1.3 mL of a propan-2-ol, Tris/HCl buffer (250 mM, pH 8.5), and 5,5′-dithiobis-2-nitrobenzoic acid (DNTB) (4 g/L, in ethanol) solution (1/1/0.2, v/v/v). After centrifugation, absorbency of the nitro-thiobenzoate anion was read at 412 nm ( ) 13 600 M-1‚cm-1). For dosage of thiol equivalent groups (SHeq ) 2SS + SH), the sample (30 mg) was exhaustively reduced with dithioerythritol (DTE) (40 mM in 80 mM Tris/HCl pH 8.5, 0.3 mL) for 2 h at 60 °C and then washed two times with 1.6 mL of glacial acetone including 100 mM acetic acid. The pellet was suspended in 3 mL of DNTB/propan-2-ol solution (see above) and vortexed for 15 min, the sample was centrifuged, and supernatant absorbency was read at 412 nm. Disulfide (SS) group content was calculated from SH and SHeq determinations. All results are expressed in micromoles per gram of protein. Thiol and disulfide contents of the native gluten were 1.6 ( 0.2 and 88.2 ( 2.3 µmol/g. Stepwise Reduction of Disulfide. Stepwise reduction with DTE was performed according to the method described by Morel and Bonicel.30 Typically a series of six samples (30 mg) were incubated for 0, 4, 8,12, 16, and 20 min with 300 µL of 2-morpholinoethanesulfonic acid/NaOH buffer (50 mM, pH 6.5) including DTE (at 2, 4, or 6 mM, depending on experiments). Sample reduction was stopped by adding 1.6 mL of glacial acetone/100 mM acetic acid. Extra DTE was removed by two washes with glacial acetone/100 mM acetic acid, and thiol content of the protein pellet was determined with DNTB as described in the former section. In any cases consumption of DTE was limited to 10%, so that we may assume that disulfide bond (SS) reduction proceeded according to a pseudo-first-order kinetic d(SS)/d(t) ) k[DTE0][SS]

(1)

where k is rate constant, DTE0 is the initial concentration in DTE, and SS is gluten disulfide content. Accordingly, data are plotted as a function of time-per-DTE concentration (in min‚M). Size Exclusion High-Performance Liquid Chromatography Analysis of the Size Distribution of Protein. Exhaustive presentation of the size exclusion high-performance liquid chromatography (SE-HPLC) method is given in Redl et al.33 Briefly, ground samples (160 mg) was dispersed at 60 °C for 80 min with 20 mL of 1% sodium dodecyl sulfate (SDS), 0.1 M sodium phosphate buffer (pH 6.9). The SDS-soluble protein extract was recovered by centrifugation (30 min at 37000g and 20 °C). The pellet was vortexed with 10 mL of SDS-phosphate buffer containing 20 mM DTE and sonicated (Vibra Cell sonificator, 20 kHz) for 3 min at 30% power setting, to extract SDS-insoluble protein. Samples having undergone stepwise or exhaustive reduction with DTE (see above) were also analyzed. Fol-

Figure 1. Time course of disulfide bonds reduction for native gluten and two mixed samples: native gluten (open circle); sample mixed at 40 °C Treg for 6 min (gray box); sample mixed at 60 °C Treg for 30 min (solid box).

lowing the last acetone/100 mM acetic acid washing step, those samples were extracted with 1.6 mL of 0.1 M phosphate/NaOH buffer, 1% sodium dodecyl sulfate (SDS) by 10 min of shaking at ambient temperature. All types of extracts were applied on the columns as 20 µL of sample, without being filtered. The SE-HPLC apparatus was a Waters model (LC Module1 plus) controlled by Millenium software (Waters). A TSK G4000-SW (TosoHaas) size exclusion analytical column (7.5 × 300 mm) was used with a TSK G3000-SW (TosoHaas) guard column (7.5 × 75 mm). The columns were eluted at ambient temperature with 0.1 M sodium phosphate buffer (pH 6.9) containing 0.1% SDS. The flow rate was 0.7 mL/min, and protein was recorded at 214 nm. The apparent molecular weight of proteins was estimated by calibrating the column with protein standards.33 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of samples was performed as indicated by Redaelli et al.34 A sample (100 mg) was extracted with 1 mL of Tris/HCL buffer (62.5 mM, pH 6.8), 2% SDS, 40% glycerol, and 10% β-mercaptoethanol for 1 h at ambient temperature under vigorous vortexing before being boiled for 2 min and 30 s in a water bath. After centrifugation, an aliquot of 5 µL was loaded onto the gel. Molecular weight markers (Pharmacia, Biotech) including phosphorylase b (Mr 94000), bovine serum albumin (Mr 67000), ovalbumin (Mr 43000), carbonic anhydrase (Mr 30000), soybean trypsin inhibitor (Mr 20100), and R-lactalbumin (Mr 14400) were loaded onto the first track. Results Sensitivity of Disulfide Groups to Reduction. Sensitivity of disulfide bond toward reduction with dithioerythritol (DTE) was investigated using various concentrations of DTE and reaction times. Figure 1 shows the reduction profiles as a function of time-per-DTE concentration (in min‚M) measured from native gluten and two mixed samples. The exponential rise would correspond to the reduction of the more sensitive bonds, whereas the steady increase above 0.08

Changes in Wheat Gluten Network Structure

Figure 2. Change in the amount of disulfide bonds reduced after 20 min of incubation with 2 mM DTE, as a function of mixing time and temperature. Samples were mixed at a regulation temperature of 40 °C (open symbols), 60 °C (gray symbols), and 80 °C (solid symbols). Data were fitted according to the following first-order kinetic expression: % reduced disulfide bonds ) R + β(1 - exp(-tA0 exp(-33700/ RTp2)); with (R + β ) 100) as constraint and where t is the mixing time, A0 is the frequency factor for the reaction leading to the increasing sensitivity of the protein disulfide bond toward DTE reduction, R is the universal gas constant (8.314 J/mol), and Tp2 is the leveling temperature of the mixing series from which data derive.

min‚M would mainly be due to the reduction of the less sensitive bonds. For native gluten, during the exponential rise approximately 20-23% of the total disulfide bonds were reduced. This content increased significantly for mixed samples, whereas the following steady rises remained almost parallel for the three samples. In the absence of chemical denaturing agent (urea or SDS) intrachain disulfide bonds from wheat proteins have been shown to be protected from chemical reduction.30,32,35 Indeed, it is well documented that (i) disulfide bond sensitivity toward chemical reduction depends on their accessibility and (ii) in contrast to the interchains disulfide bonds, intrachain bonds are likely to be buried into the core of the native molecule. Increase in the amount of disulfide bonds sensitive to DTE would therefore indicate the formation of new interchain disulfide bonds and/or the intrachain disulfide bonds exposure following the alteration in protein conformation. For a comparison purpose, all samples were reduced at 0.04 min‚M (2 mM DTE for 20 min). Results showed that longer mixing times and higher regulation temperatures resulted in increasing amounts in reduced disulfide bonds, suggesting the occurrence of a heat-activated reaction. We have previously shown that gluten protein solubility loss during mixing was temperature dependent, and according to Arrhenius law, an activation energy of 33.7 kJ‚mol-1 has been calculated.29 We hypothesized that both types of biochemical events were related, so that the same activation energy of 33.7 kJ‚mol-1 could be applied. Figure 2 shows that activation energy of 33.7 kJ‚mol-1 was successfully applied to collapse all the data into one single-exponential progress curve (r2 ) 0.95), irrespective of the mixing temperature. Thus, we can consider that increase in disulfide susceptibility to reduction, upon mixing, was related to the mechanism leading to protein solubility loss.


Figure 3. Size exclusion distribution profiles from native (parts a and b, solid line), fully reduced (part a, dotted line), and stepwise reduced (part b, dotted and dashed lines) SDS-soluble gluten protein samples. Stepwise reduction was performed with 2 mM DTE at pH 6.5 for 4 min (part b,dotted line) and 20 min (part b, dashed line). Fractions F1 to F5 respectively included glutenin polymers above 680000 (F1) and ranging from 95000 to 680000 (F2), gliadin monomers ranging from 55000 to 95000 (F3) and from 20000 to 55000 (F4), and salinosoluble proteins from 6000 to 20000 (F5).

Increase in Protein Extractability as a Function of Disulfide Bond Reduction. Typical SE-HPLC profiles from native gluten, before (plain line) and after (dotted line) exhaustive DTE reduction, are shown in Figure 3a. In the native gluten SE-HPLC profile, fractions F1 and F2 consist of glutenin polymer and fractions F3 to F5 consist mainly of gliadin monomers of decreasing size (ω, γ, β, and R) and of some remaining soluble proteins. Total reduction of disulfide bonds alters the size distribution of protein, which eluted in three peaks. The first one, which coincided with fraction F3 of native gluten, would consist of high-molecularweight glutenin subunits (HMW-GS) and ω-gliadin (Mr ≈ 55000-95000). The following fractions would consist of reduced gliadin and low-molecular-weight glutenin subunits (LMW-GS). Compared with native gluten, we noticed a shift (Figure 3a, F4b f F4a) toward shorter retention times for fraction F4. This suggested that gliadin monomers unfolded, a consequence of intrachain disulfide bond reduction. Upon stepwise reduction, (0, 4, and 20 min with 2 mM DTE at pH 6.5) the release of LMW-GS was evidenced as the growing F4a shoulder while HMW-GS eluted within F3 (Figure 3b). Also a transient increase in fractions F1 and F2 was registered at the beginning. Initially reduction brought SDS-insoluble glutenin polymers into solution as large polymers which were in turn reduced into smaller ones. Typical samples were treated with various combinations of time and DTE. Experiments were conducted on duplicate samples in order to determine, from one sample, the size distribution profile of the extractable protein and, from the other, the extent in disulfide bond reduction. Figure 4 shows

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Figure 4. Increase in protein extractability upon disulfide bonds reduction from native and mixed gluten samples: gluten (O); gluten sample mixed at 40 °C for 2 (1), 4 (2), 6 ([), 8 (b), and 15 (9) min; gluten samples mixed 30 min at 60 °C (gray square) and 80 °C (gray triangle).

Figure 5. Size exclusion distribution profiles from nonreduced (plain line) or partially reduced (4 and 20 min with DTE 2 mM, dotted and dashed lines) mixed gluten samples (4 min at 40 °C).

that reduction of disulfide bonds increased protein solubility in SDS-phosphate buffer. For native gluten, almost total protein was brought into solution after the reduction of less than 10% of the disulfide bonds. In contrast, increase in solubility was slower for mixed samples, especially for the more severe mixing conditions. Thus, for a sample mixed for 30 min at 60 °C Treg, reduction of more than 30% of the total disulfide bonds did not lead to very significant increase in protein extractability. Whereas mixing increased the sensitivity of disulfide bonds to DTE reduction, less and less protein was brought into solution when the bonds were broken. Figure 5 shows the typical changes in SE-HPLC profiles upon stepwise reduction, from a mixed sample (4 min at 40 °C Treg). Reduction conditions (0, 4, and 20 min at 2 mM DTE, pH 6.5) were comparable with those for native gluten (Figure 3b). In contrast with native gluten, protein from the mixed samples was mostly brought into solution as monomers, whereas changes within fractions F1 and F2 remained small. This trend was verified for all mixed samples at the exception of the less mixed sample (2 min at 40 °C Treg). Figure 6 shows that for native gluten, the reduction of 6% of the total disulfide bonds results in a transient increase in F1 up to 9%. In the case of the sample mixed 4 min at 40 °C Treg, the transient increase in F1 was smaller (4%) and delayed until the reduction of 25%-30% of total disulfide bonds. Examination of the other mixed samples showed that

Morel et al.

Figure 6. Change in fraction F1 (in % of total protein) upon disulfide bond reduction from native and mixed gluten samples: gluten (O); gluten sample mixed at 40 °C for 2 (1), 4 (2), 6 ([), 8 (b), and 15 (9) min; gluten samples mixed 30 min at 60 °C (gray square) and 80 °C (gray triangle).

release of polymers was more and more delayed as mixing severity increased. Also less and less polymer seemed to be released upon reduction. SE-HPLC and SDS-PAGE Analysis of Fully Reduced Protein. Total reduction of gluten disulfide bonds was carried out in Tris/HCl buffer at pH 8.5, with 40 mM DTE for 120 min at 60 °C. For all samples, total reduction of disulfide bonds leads to total protein extractability in SDS-buffer, as attested from the constancy of total SE-HPLC areas, from native gluten to harshly mixed samples. SE-HPLC chromatograms revealed that even after total disulfide bond reduction, protein polymers (eluted within fractions F1 and F2) were still present in some mixed samples, in contrast with native gluten. Increasing the reduction time up to 4 h at 60 °C or adding 2% SDS to guarantee protein unfolding did not lead to any change in SE-HPLC profiles of samples. Apart from the formation of new interchain disulfide bonds, mixing would induce the formation of intermolecular isopeptide bonds responsible for the accumulation of nonreducible polymers. Accumulation of nonreducible polymers, estimated from the sum areas of fractions F1 and F2, was shown consistently related to the activation energy accounting for protein solubility loss (Figure 7). A source of DTE resistant cross-links could be the random recombination of radical moieties produced by peptide bond scission, upon mixing. Schaich and Rebello recently showed by use of electron paramagnetic resonance (EPR) that shear and temperature (over 150 °C) were involved in the production of carbon and nitrogen centered radicals.36 Nevertheless, in the same work, the shear-mediated scission of disulfide bonds was also strongly supported by EPR detection of sulfur and persulfide radicals (RS• and RSS•). Shear-mediated scission of the C-S bond of cystine residue might proceed similarly to a β-elimination reaction inducing one persulfide and one dehydroalanine residue.37 Reaction of the dehydroalanine residue with one lysine residue would also induce a nonreducible covalent bond. To assess for the nature of polypeptide fragmentation, proteins were analyzed after total disulfide bond reduction,



Figure 7. Increase in nonreducible polymers (fractions F1 + F2, from SE-HPLC profiles of fully reduced samples) upon mixing time and temperature. Samples mixed at a regulation temperature of 40 °C (open symbols), 60 °C (gray symbols), and 80 °C (solid symbols). Data were fitted according to the following first-order kinetic expression: % nonreducible polymers ) R + β(1 - exp(-tA0 exp(-33700/ RTp2)), with (R + β e 100) as constraint and where t is the mixing time, A0 is frequency factor for the accumulation of nonreducible polymers, R is the universal gas constant (8.314 J/mol), and Tp2 is the leveling temperature of the mixing series from which data derive.

Figure 9. Changes in free thiol (a) and disulfide (b) groups upon mixing time and according to Treg: 40 °C (coarse stripes); 60 °C (empty); 80 °C (fine stripes).

Figure 8. SDS-PAGE pattern from fully reduced, native, and mixed gluten samples (at Treg 40 °C): MWM, molecular weight markers (see Materials and Methods for details); G, native gluten. Minute mixing is given at the top of the figure.

by SDS-PAGE analysis. We expected that peptide bond scission, which is likely to be random along the amino acid chain, would result in protein bands fading and formation of small degradation products. Conversely C-S scission would not alter the primary structure of the protein chain. It would lead to the formation of new protein complexes because of the dehydroalanine reactivity, together of course with protein bands fading. A typical result obtained from samples mixed at 40 °C Treg is presented in Figure 8. We observed a gradual weakening of the high molecular weight glutenin subunit bands (HMW-GS) and an increasing smearing of the upper part of the gel. The gliadin pattern, especially the ω-gliadin, did not change significantly. This specific involvement of HMW-GS was relevant to a specific mechanical fragmentation of the glutenin polymer by mechanical shearing. In contrast, Rebello and Schaich38 showed that for wheat flour extrudates, protein fragmentation upon exposure to very high temperature (over 150 °C) concerned all protein classes, including gliadin.

There was no clear evidence of peptide bond scission since the lower part of the gel remained unchanged. On the other hand, enhancement of the upper gel background, rather than formation of new discrete protein bands, supports that if C-S scission occurred, the dehydroalanine residues reacted rapidly and at random. Of course, if in the case of peptide bond scission the radical moieties were also reacting immediately and at random, no smearing would have been observed in the lower part of the gel. So it was not possible to conclude definitely whether peptide or C-S bonds were cleaved. Changes in Thiol and Disufide Groups during Mixing. Samples from mixing series performed at 40, 60, and 80 °C temperature of regulation were analyzed for their accessible thiol (free SH) and total disulfide (SS) contents. Figure 9a shows that during mixing, content in free SH increased in a transient way. The SH maximun dropped with increasing temperatures of regulation while the decay phase occurred earlier. Transient increase in SH groups might originate from reduction or scission of disulfide bonds and should be balanced by a decrease in disulfide content. The lack of accuracy of SS dosage impaired definitive conclusion (Figure 9b). While mixing continues, a net decrease in SS groups which was not balanced by free SH increase was recorded. Decay seemed faster at higher Treg and losses reached 8, 10, and 16 µmol‚g-1 after 30 min of mixing at 40, 60, and 80 °C temperature of regulation, respectively. This result indicated an irreversible loss in thiol equivalent groups (SHeq ) 2SS + SH), which exhibited a time-temperature dependence. A similar finding was reported by Anderson and Ng, who noticed that flour extrudates showed a net decrease in SHeq by 16% relative to the native flour sample.39 Rebello and Schaich to account for such behavior suggested that some sulfur-containing volatile or sulfoxyl compounds were produced during extrusion.38 Transient increase in sulfhydryl groups might originate from disulfide bond scission upon shear force. In that respect,

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Figure 10. Putative reaction pathways for free thiol groups transient increase and disulfide groups loss upon mixing.

our samples were mixed at 0.2 to 11 kJ/g of specific mechanical energy input compared to the 0.35-1 kJ/g range, used by Schaich and Rebello, who found evidence of radical formation.36 Figure 10 presents some reactions that might occur during mixing. Mechanical scission of the SS bond would lead to thiyl radicals (S•) that may recombine into disulfide (path 1), be converted into nonradical species(S-) (path 2), or oxidize into sulfenyl or sulfinyl radicals (path 4). The latter reaction might account for part of SHeq loss. Nevertheless considering bond enthalpies, the C-S bond is almost as weak as the S-S bond (259 kJ‚mol-1 against 226 kJ‚mol-1).40 Cleavage of the S-C bond according to the β-elimination mechanism would result in persulfide and dehydroalanine residues (path 1′). So, irreversible loss in SHeq might also occur through elimination of sulfur atom from persulfide anion (path 5). On its side, dehydroalanine might react with the -H2N group of lysine residue, leading to the formation of isopeptide bonds between or within protein chains (path 6). We postulated that SHeq loss showed the same activation energy of 33.7 kJ‚mol-1 that had been found for protein solubility loss during mixing.29 Accordingly, data from the three mixing experiments were arbitrarily fitted together by using SHeq ) β + R[exp(-Kt i

exp(-Ea/RTP2i)]

(2)

where i specifies the mixing series according to Treg, R and β are constants whose sum amounts for the total SHeq content of native gluten, K is the frequency factor of the reaction, Ea is activation energy (33.7 kJ‚mol-1), R is the universal gas constant (R ) 8.3144 J‚mol-1‚K-1), and TP2i is the mean sample temperature (in kelvin) registered above Peak2 (see Table 1). Figure 11 shows that reasonably good adjustment of the data was obtained by considering the activation energy of 33.7 kJ‚mol-1 (r2 ) 0.92) and the first-order kinetic model. Maximum SHeq loss (R) and frequency factor K were fitted as 47.6 µmol‚g-1 and 2202 min-1, respectively. According to whether the SHeq losses resulted from radical scission of S-S or C-S bonds, scission would involve up to 24 or 48 µmol of disulfide bond per gram of gluten. Modeling of transient increase in free SH proved difficult. Several parameters needed to be considered. At first lag times of 2.26, 0.87, and 0.43 min were taken into account for mixing experiments at 40, 60, and 80 °C Treg, respectively. The delays corresponded to the mean time needed to reach

Figure 11. Decrease in equivalent thiol groups (SHeq) as a function of mixing time and temperature. Samples mixed at a regulation temperature of 40 °C (open symbols), 60 °C (gray symbols), and 80 °C (solid symbols). Data were fitted according to the following firstorder kinetic expression: SHeq ) R + β exp(-tA0 exp(-33700/RTp2), where t is the mixing time, A0 is frequency factor for SHeq loss, R is the universal gas constant (8.314 J/mol), and Tp2 is the leveling temperature of the mixing series from which data derive.

Peak1 on torque curve. In adiabatic mixing conditions, Peak1 was shown to correspond to a sample temperature of 60 °C, irrespective of mixing speed and glycerol content.33 We suggest that below 60 °C no change in free SH would occur. Assuming arbitrarily first-order kinetics for SH formation and decrease: k1

k2

SS 98 2SH 98 2SX (X ) O•, OO•)

(3)

Transient increase in SH can be expressed for modeling purpose as d(SH)/d(t) ) 2k1(SS) - k2(SH)(X) ) 2k1SS0 exp(-k1(t - tP1)) - k2(SH)(X) (4) with k1 and k2 rate constants for SH formation and decay, respectively. By neglecting the time-concentration dependency of the oxidative compound X in eq 4 k2(SH)(X) ) k2′(SH)

(5)

Equation 4 becomes d(SH)/d(t) ) 2k1SS0 exp(-k1(t - tP1i)) - k2′(SH) (6) Integration of eq 6 leads to SH ) 2SS0 k1[exp(-k1(t - tP1)) - exp(-k2′(t - tP1))]/ (k2′ - k1) (7) To account for the free SH content of native gluten, eq 7 was modified as SH ) 2SS0 k1[exp(-k1(t - tP1)) - exp(-k2′(t - tP1))]/ (k2′ - k1) + SH0 exp(k2′(t - tP1)) (8) where tP1 is the lag time which corresponds to time needed to reach Peak1 (mean value calculated from the 8, 15, and



k ) PZ0 exp(-Eaη/RT) exp(-Ea/RT)

(10)

In support to this hypothesis the sum of activation energies accounting for temperature dependency of viscosity (Eaη) and for time-temperature dependency of protein solubility loss (Ea) (21.9 and 33.7 kJ‚mol-1, respectively)29 is found within the range of the energy of activation calculated for SH decay (47.8 ( 9.0 kJ‚mol-1). Discussion

Figure 12. Modeling of free thiol groups decay with mixing time according to Treg. Samples were mixed at a regulation temperature of 40 °C (open symbols), 60 °C (gray symbols), and 80 °C (solid symbols). Data were fitted upon first-order kinetics for the formation and decay of an intermediate component using eq 8. Mixing time to peak 1 is given in Table 1.

30 min runs), SH0 is the free thiol content of gluten (1.46 µmol‚g-1), SS0 is the disulfide content at the origin of SH formation, and k1 and k2′ are the rate constants for SH formation and decay, respectively. Taking into account the maximun fitted SHeq loss (see above), we arbitrarily decided that SS0 should not exceed 48 µmol‚g-1. Figure 12 shows the fitted curve obtained for each mixing series, when using a common value of 7.5 µmol‚g-1 for SS0, which was found to give the best results. The values fitted for the rate constant k1 were rather similar, irrespective of Treg (0.085, 0.095, and 0.072 min-1 for mixing at 40, 60, and 80 °C Treg, respectively), and thus appeared to be independent of temperature. So, we might imagine that the mechanical scission of the disulfide bond at the origin of free thiol groups depends on mechanical work input, i.e., on shear rate and sample viscosity. Increase in viscosity should favor disulfide scission but in the same time should support radical recombination into the original disulfide. Thus formation of free thiol would be rather insensitive to the temperature dependency of viscosity. Fitted rate constants for SH decay were 0.079, 0.116, and 0.210 min-1 for mixing at Treg 40, 60, and 80 °C, respectively. Plotting the data on Arrhenius representation allowed calculation of an energy of activation of 47.8 ( 9.0 kJ‚mol-1 (r2 ) 0.96). This value differs from the energy of activation relevant for protein solubility loss (33.7 ( 2.8 kJ‚mol-1). According to the collision theory of chemical reactions, the relationship between temperature (T) and rate constant (k) is k ) PZ exp(-Ea /RT) SH

(9)

where P is the probability of the reaction when molecules collide, Z is the number of collisions per second per unit volume (collision rate), and EaSH is the energy of activation found for the SH decay phase (47.8 ( 9.0 kJ‚mol-1). We hypothesise that increase in viscosity might lower the diffusion rate of molecular oxygen within the plasticized gluten mass. So eq 9 could be writen as

Time, temperature, and shear are the driving variables of change in gluten structure upon mixing. The present investigations attempted to clarify the involvement of each variable on several biochemical characteristics. Table 2 resumed the kinetics parameters for the biochemical and rheological changes determined here and previously.29 For comparison and simplification purposes first-order kinetics were always used to fit the data. All the physical and biochemical alterations characterized (except modeling of transient SH increase) showed the same temperature dependency. Indeed the use of 33.7 kJ‚mol-1 as activation energy was found valid since data collapsed into single progress curves, irrespective of the temperatures at which the samples were mixed. From the frequency factors reported in Table 2, it appears that one of the first noticeable events upon mixing is the drop of protein solubility in SDS. Concurrently, Me, the molecular weight of network strands between entanglements, which had been calculated from rheological measurement,29 and the disulfide bond susceptibility to DTE reduction, decreases and increases, respectively. These three events progress at the same rate and could be causally related. Solubility drop and Me decrease both indicate a growing connectivity between polypeptide chains. We showed that the amount but also the structure of insoluble protein changed upon mixing. Indeed, for mixed samples and in contrast with native gluten, limited reduction of disulfide bonds did not allow the extraction of large polymers. Glutenin polymer structure is still under discussion, but the most favored model is that of a linear backbone of glutenin subunits end-to-tail disulfide bonded.7,11 For linear polymers, effective reduction in size can be expected after only a few scissions along the chain. Conversely, if polymer chains are connected into a three-dimensional network structure, random scissions along chains will be rather ineffective at the beginning. After a delay, which will Table 2. Frequency Factors of Biochemical or Rheological Changes upon Mixing biochemical or rheological events increase in insoluble proteinb decrease in Meb increase in SS sensitivity toward reduction with DTE loss in totSHeq increase in nonreducible polymers

frequency factora (min-1) 5738 ((1104) 5529 ((1995) 4978 ((1733) 2202 ((1136) 871 ((555)

a Frequency factors for first-order reaction kinetics. b From Redl et al. (previous issue). Me is the molecular weight of network strands between entanglements (g/mol).

496


increase with the density of connections between chains, monomer units or small polymeric fragments will be released into solution. So, our results indicated that mixing induced the buildup of a three-dimensional connected structure. Hydrophobic, disulfide, or isopeptide bonds could be involved in the growing connectivity of gluten protein. Indeed, a delayed response upon disulfide reduction may be expected from a three-dimensional network stabilized by any one of those types of bonds. Nevertheless the isopeptide bond role would be negligible, otherwise reduction of total disulfide bonds would not have brought total protein into solution. Gluten protein comprises cysteine residues supposed to be involved in either intra- or interchain disulfide bonds, without free sulfhydryl groups available.1,4 Thus, additional interchain disulfide bonds would be formed at the expense of intrachain bonds via interchange reactions. Conversion of intrachain disulfide bonds into interchain ones might be significant. Mixing (30 min at 80 °C) induced the irreversible loss of 16 µmol.g-1 of disulfide (i.e., 32 µmol‚g-1 of SHeq). Such disulfide loss would amount to about twice of the estimated gluten interchain disulfide bonds.41 We postulated that SHeq loss would arise from the shear-mediated scission of interchain disulfide bonds followed by one of the oxidation mechanisms proposed in Figure 10. So, it is likely that mixing ensures at least the continuous renewal of the broken interchain disulfide bonds otherwise loss in SHeq could never have reached such a high level. Free sulfhydryl groups are obligatory intermediates of disulfide interchanges. Due to their low concentration they could even be the limiting factor of protein solubility loss. Nevertheless the kinetics of protein solubility loss seemed totally disconnected from transient SH formation. This finding suggests that free thiol content might not be the driving variable of disulfide interchanges. Table 2 shows that frequency factors related to the increase in nonreducible polymers and decrease in totSHeq were within the same range. Radical residues originating from scission of glutenin polymers might be at the origin of both biochemical changes. Thiyl radicals (S•) could act as very efficient catalysts for SS interchanges. Rapid exchange between radical forms (N•, C•, S•) would ensure similar reaction rates for the formation of isopeptide and disulfidemediated protein cross-links. Mechanical shearing would be the driving forces lowering the need for thermal energy, explaining why a common activation energy of 33.7 kJ‚mol-1 was found for all the investigated biochemical changes. Work input (i.e., specific mechanical energy) for its part would account for the thermal history of the mixed sample, through the viscous dissipation of mechanical energy. The thiyl-radical-mediated SS interchange reaction might be at the origin of the lower activation energy observed upon gluten mixing. However it cannot be excluded that the accumulation of elastic energy plays a significant role in lowering the energy barrier that controls the change in molecular conformation from the stable to the activated state. Whether shear rate induced S-S, C-S, or C-N scissions remains to be seen. We showed that changes in SDS-PAGE

Morel et al.

patterns could support C-N as well as C-S scission if rapid and random recombination were presumed. Definitive invalidation of a β-elimination mechanism should be gained by following changes in lysine and lysinoalanine content. Conclusion Mixing of gluten plasticized with glycerol induced marked changes in the physicochemical properties of gluten proteins. In addition to the previously reported loss in protein extractability in SDS-buffer, mixing induced drastic change in thiol and disulfide contents, in disulfide bonds sensitivity toward DTE reduction, and in SDS-PAGE patterns and SEHPLC profiles of fully reduced proteins.29 In all cases the activation energy for those changes was 33.7 kJ/g, indicating that they were highly related. From the comparative analysis of the rate of changes, protein solubility loss was assumed to derive from the increasing connectivity of gluten protein upon mixing. The increasing degree of gluten protein polymerization would predominantly be due to the formation of additional intermolecular disulfide bonds and/or to increasing hydrophobic interactions between gluten protein. Formation of nonreducible polymers cross-linked by isopeptide bonds would play a minor role in the strengthening of the gluten network. In this work, indirect evidence of radical scission of peptide and disulfide bonds was gained through transient increase in free thiol, loss in thiol equivalents (SHeq), and accumulation of nonreducible polymers. Mixing as well as extrusion would to be a source of radical compounds.36 It is proposed that radical compounds were the catalysts of the increasing connectivity of the gluten network. Thiyl radicals, instead of free thiol groups, would mediate interchange reaction between intramolecular disulfide bonds, which would account for the formation of additional intermolecular disulfide bonds and/or for the stabilization of hydrophobic interactions between gluten proteins. Thiyl, carbon, and nitrogen radicals would interchange rapidly so that all types of bonds accumulated at the same rate. By this way shear stress would be able to decrease the activation energy for protein polymerization from more than 100 kJ/mol in a static system to 33.7 kJ/mol in a shear stressed system. References and Notes (1) MacRitchie, F. AdV. Food Nutr. Res. 1992, 36, 1-87. (2) Payne, P. I. Annu. ReV. Plant Physiol. 1987, 38, 141-153. (3) Gupta, R. B.; Bekes, F.; Wrigley, C. W. Cereal Chem. 1991, 68, 328-333. (4) Shewry, P. R.; Tatham, A. S. J. Cereal Sci. 1997, 25, 207-227. (5) Shewry, P. R.; Napier, J. A.; Tatham A. S The Plant Cell 1995, 7, 945-956. (6) Kasarda, D. D. Cereal Foods World 1999, 44 (8), 566-571. (7) Wrigley, C. W. Nature 1996, 381, 738. (8) Popineau, Y.; Cornec, M.; Lefebvre, J.; Marchylo, J. Cereal Sci. 1994, 19, 231-241. (9) Gupta, R. B.; Popineau, Y.; Lefebvre, J.; Cornec, M.; Lawrence, J. G.; MacRitchie, F. J. Cereal Sci. 1995, 21, 103-116. (10) Tsiami, A. A.; Bot, A.; Agterof, W. G. M.; Groot, R. D. J. Cereal Sci. 1997, 26, 15-27. (11) Graveland, A.; Bosveld, P.; Lichtendonk, W. J.; Marseille, J. P.; Moonen, J. H. E.; Scheepstra, A. J. Cereal Sci. 1985, 3, 1-16. (12) Weegels, P. Cereal Chem. 1994, 71, 308-309. (13) Singh, N. K.; Donovan, G. R.; Batey, I. L.; MacRitchie, F. Cereal Chem. 1990, 67, 150-161.

Changes in Wheat Gluten Network Structure (14) Skerritt, J. H. J. Agric. Food Chem. 1998, 46, 3447-3457. (15) Redl, A.; Vergnes, B.; Morel, M.-H.; Bonicel, J.; Guilbert, S. Cereal Chem. 1999, 76, 361-370. (16) Li, M.; Lee, T.-C. J. Agric. Food Chem. 1996, 44, 763-768. (17) Li, M.; Lee, T.-C. J. Agric. Food Chem. 1997, 45, 2711-2717. (18) Strecker, T. D.; Cavalieri, R. P.; Zollars, R. L.; Pomerantz, Y. J. Food Sci. 1995, 60, 532-537. (19) Weegels, P. L.; Hamer, R. J. Temperature-induced changes of wheat products. In Interaction: the keys to cereal quality; Hamer R. J., Hoseney R. C., Eds.; American Association of Cereal Chemists: St. Paul MN, 1998; pp 95-123. (20) Becker, H. A.; Sallans, H. R. Cereal Chem. 1956, 33, 254-265. (21) Wrigley, C. W.; du Cros, D. L.; Archer, M. J.; Downie, P. G.; Roxburgh, C. M. Aust. J. Plant Physiol. 1980, 7, 755-766. (22) Lefebvre, J.; Popineau, Y.; Deshayes, G.; Lavenant, L. Cereal Chem. 2000, 77, 193-201. (23) Schofield, J. D.; Bottoley, R. C.; Timms, M. F.; Booth, M. R. J. Cereal Sci. 1983, 1, 241-253. (24) Li, M.; Lee, T.-C. J. Agric. Food Chem. 1998, 46, 846-853. (25) Kohn B. K.; Karwe, M. V.; Schaichn, K. M. Cereal Chem. 1996, 73, 115-122. (26) Davidson, V. J.; Paton, D.; Diosady, L. L.; Rubin, L. J. J. Food Sci. 1984, 49, 1154-1157. (27) MacRitchie, F. J. Polym. Sci. Symp 1975, 49, 85-???. (28) Kinsella, J. E. CRC Crit. ReV. Food Sci. Nutr. 1978, 10, 147-207. (29) Redl, A.; Morel, M.-H.; Guilbert, S. Submitted to Polymer.

Biomacromolecules, Vol. 3, No. 3, 2002 497 (30) Morel, M.-H.; Bonicel, J. New investigations of disulfide bonds of wheat proteins by dithioerythritol (DTE) reduction. In Gluten ‘96, Proceedings of the Sixth International Gluten Workshop; Wrigley, C. W., Ed.; Royal Australian Chemical Institute: Melbourne, Australia, 1996; pp 257-261. (31) American Association of Cereal Chemists. ApproVed Methods of the AACC, 9th ed.; Method 46-11A, approved October 1976; American Association of Cereal Chemists: St. Paul MN, 1995. (32) Morel, M.-H.; Bonicel, J.; Micard, V.; Guilbert, S. J. Agric. Food Chem. 2000, 48, 186-192. (33) Redl, A.; Morel, M.-H.; Bonicel, J.; Guilbert, S.; Vergnes, B. Rheol. Acta 1999, 38, 311-320. (34) Redaelli, R.; Morel, M.-H.; Autran, J. C.; Pogna, N. E. J. Cereal Sci. 1995, 21, 5-13. (35) Kawamura, Y.; Matsumura, Y.; Matoba, T.; Yonezawa, D.; Kito, M. Cereal Chem. 1985, 62, 279-. (36) Schaich, K. M.; Rebello, C. A. Cereal. Chem. 1999, 76, 748-755. (37) Friedman, M. J. Agric. Food Chem. 1999, 47, 1295-1319. (38) Rebello, C. A.; Schaich, K. M. Cereal. Chem. 1999, 76, 756-763. (39) Anderson, A. K.; Ng, P. K. W. Cereal Chem. 2000, 77, 354-359. (40) Masterton, W. L.; Hurley, C. N. Covalent bond properties. In Chemistry principles and reactions; Saunders College Publishing: Philadelphia, PA, 1987; p 267. (41) Grosch, W.; Wieser, H. J. Cereal Sci. 1999, 29, 1-16.

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Mechanism of Heat and Shear Mediated Aggregation of Wheat Gluten

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