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Journal of Cereal Science 47 (2008) 417–428

www.elsevier.com/locate/jcs

Dynamic viscoelastic, calorimetric and dielectriccharacteristics of wheat protein isolates

Jasim Ahmed�,1, Hosahalli S. Ramaswamy, Vijaya G.S. Raghavan

Department of Food Science & Agricultural Chemistry, Macdonald Campus, McGill University, Ste. Anne de Bellevue, PQ, Canada H9X 3V9

Received 31 October 2006; received in revised form 2 May 2007; accepted 10 May 2007

Abstract

Dynamic oscillatory rheology of two wheat protein isolate (Prolite 100 and Prolite 200) doughs (E48% moisture content, wet basis)

were studied over a frequency range of 0.1–10Hz during temperature sweep from 20 to 90 1C at a heating rate of 2 1C/min. Both doughs

behaved similarly during heating; showed a threshold value and increased sharply, thereafter. Prolite 200 dough had a higher elastic

modulus (G0) and lower phase angle (d) whereas Prolite 100 showed a distinct gel point at 52.2 1C followed by significant increase up to

90 1C. Rheological data of doughs after isothermal heating at 90 1C for 15min followed by cooling to 20 1C resulted in strong mechanical

strength. However, Prolite 100 dough showed more viscoelastic characteristics with significant transformation from liquid-like to solid-

like behavior after heating than Prolite 200. Thermal analysis of isolates indicated distinct endothermic peaks in wider temperature range

(50–130 1C) at various moisture levels. Lower temperatures could be associated with denaturation of various fractions of proteins

whereas higher temperature linked to glass transition temperature of isolates. SDS–PAGE did not show any clear distinction among

protein subunits between two isolates. Dielectric measurements of isolates at frequencies from 500 to 3000MHz and temperature range

between 30 and 80 1C indicated Prolite 200 had higher dielectric constant (e0) and loss factor (e00) than Prolite 100. Isolates showed

significant changes in dielectric properties above 50 1C indicating protein denaturation and supported rheological and calorimetric data.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Wheat protein isolate; Elastic modulus; Viscoelasticity; Solid-like property; Denaturation; Glass transition temperature; Dielectric loss factor;

Penetration depth

1. Introduction

Wheat proteins are the best known cereal proteins, andthe gluten complex plays the major role in determining thetechnological quality of wheat (MacRitchie, 1987; Shewryand Tatham, 1997; Shewry et al., 1995). Wheat glutenproteins have unique viscoelastic properties of three-dimensional structures formation. Gluten mainly consistsof two major storage protein fractions, namely gliadin andglutenin (about 65/45w/w) (Redl et al., 2003). Gliadin, thelow molecular weight (LMW) fraction of 30–50 kDa(Tatham et al., 1990) and glutenins with molecular massof 69–88 kDa based on SDS–PAGE (Anderson et al., 1988)

e front matter r 2007 Elsevier Ltd. All rights reserved.

s.2007.05.013

ing author. Tel.: +1514 398 8666; fax: +1 514 398 7977.

ess: jahmed2k@yahoo.com (J. Ahmed).

ress: Polymer Source Inc., 124 Avro St., Dorval, Quebec,

X8.

both contribute to rheological, structural and bakingcharacteristics of wheat. The high molecular weight(HMW) distribution of glutenins has been recognized asone of the major determinants of dough properties andbaking performance (Wieser, 2007).Wheat protein isolates (WPI) have been utilized com-

mercially as functional ingredients in food formulations(baked goods, extruded high-protein foods, nutritionalbars, etc.) for their high nutritional value and minimumcost. The protein isolates are currently of special interest toprocessors and consumers due to low fat content and as asubstitute for egg and dairy proteins. The major functionalproperties of wheat proteins are: hydration, foaming,improve sheeting properties of laminated dough, lightand floppy texture and clean flavor.Knowledge of the rheological behavior of wheat proteins

in aqueous system is very important to understandmechanical properties of the dough and control finished

ARTICLE IN PRESSJ. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428418

products. Small amplitude oscillatory shear (SAOS)measurements afford the measurement of dynamic rheo-logical functions, without altering the internal networkstructure of materials tested and are far more reliable thansteady shear measurement (Bistani and Kokini, 1983).Viscoelasticity of dough is directly related to nature offlour, water uptake, air incorporation and type of mixing(Savador et al., 2006). Viscoelastic characteristics of wheatdough and gluten subfraction (gliadin or glutenin) duringheat treatment have been studied extensively by severalresearchers (Cocero and Kokini, 1991; Madeka andKokini, 1994; Redl et al., 2003; Savador et al., 2006;Stathopoulos et al., 2006). Schofield et al. (1983) found thatthe thermal denaturation happened with rapid increase inthe number of rheologically active cross-links in gluten.Guerrieri et al. (1996) advocated that heating of gluten to90 1C leads to disulfide bond linked aggregates andconformational changes that affect mainly gliadins andLMW albumin and globulin. Rheological characterizationof WPIs, therefore, could provide better understandingand functionality during heat processing/baking. Westudied the effect of composition and temperature on therheological characteristics of two commercial proteinisolates.

Denaturation is the most important transition occurringin proteins during the baking process. The proteindenaturation has been extensively studied by differentialscanning calorimetry (DSC) where denaturation is indi-cated as an endothermic peak in thermogram (Privalov andKhechinashvili, 1974). Protein fractions of wheat consist ofseveral proteins and, therefore, several denaturationendotherms are expected. However, DSC response towheat proteins (gliadin and glutenin) is limited (Ma,1990a, b). Most of the reported DSC data on glutendenaturation are contradictory. Eliasson and Hegg (1980)observed gluten denaturation endothermic peaks at 88 and101 1C with very small change in enthalpy while Erdogdu etal. (1995) did not observe any denaturation peak. Lackinga tertiary-ordered structure, gluten cannot be put into adisordered form by heat, which explains the absence ofDSC thermograms (Hoseney and Rogers, 1990). RecentlyLeon et al. (2003) found gliadin denaturation temperatureat 58 1C whereas two endothermic peaks at 64 and 84 1Cfor glutenins with high enthalpies. Those authors suggestedthat DSC protein denaturation peaks were a function ofsample water content. These results indicate that morestudies on DSC measurement of wheat proteins are neededto confirm denaturation as a function of moisture content.A reversible change of the proteins from a rigid or glassystate to a soft or rubbery state through a temperatureincrease and/or plasticizer addition was also observed.Wheat gluten is a glassy, amorphous and plasticizablepolymer, and water has a strong plasticizing effect on theglass transition temperature (Tg) of flexible biopolymerslike gluten (Hoseney et al., 1986). For example, increasingthe water content of a high molecular weight glutenin, from3 to 12% (w/w), depresses the calorimetric glass transition

from 100 to 25 1C (Noel et al., 1995). Similar ranges of Tg

(110–21 1C) were obtained by Cocero and Kokini (1991)while glutenin samples hydrated between 4% and 14%moisture content. The information on Tg of protein isolatescould be used to formulate and develop newer productsbased on WPI.Use of microwave energy in baking is of particular

interest due to the importance of the industry, and lowdensity of many baked products which allows morepenetration of microwave energy. Dielectric properties ofa material determine penetration of microwave power andlocal power absorption rates. Knowledge of dielectricproperties is essential for mathematical description oftemperature history in microwave or radio-frequencyheated foods and design of applicators. Various factorsinfluence the dielectric properties of food materials such asfrequency, ionic nature, moisture content, temperature,concentration, nature and constituents of food materials(Ahmed et al., 2007). Understanding the impact of heatingon the dielectric properties of proteins can potentially helpto develop online sensors and detect structural changesduring denaturation of protein. Dielectric data presentedby some researchers (Kent, 1987; Mellgren et al., 1988) forwheat-based products were either presented at considerablylower frequencies (107Hz) or at lower temperature(o35 1C) and those data could not be used for practicalpurposes. Goedeken et al. (1997) studied dielectric proper-ties of bread dough at 2450MHz as function of moisturecontent, temperature and salt content. Dielectric constant(e0) was found to be independent of salt content whileloss factor (e00) increased when salt was added. Bothdielectric parameters increased with temperature whilesamples contained 1% salt. However, available informa-tion on dielectric properties of wheat proteins at widerfrequency range during heat treatment is very scarce in theliterature.The present study intends to examine the technological

ability of two WPIs with different protein contents on thebasis of the rheological, thermal and dielectric behavior.Rheological and dielectric properties were studied asfunction of temperature keeping in view changes associatedwith protein denaturation at selected temperature range.This study would help baking and other food processingindustries to formulate, function and the possible use ofmicrowave baking of wheat protein-based food products.

2. Materials and methods

2.1. Materials

WPIs (Prolite 100 and 200) were provided by ArcherDaniels Midland Company (Decatur, IL, USA) for use inthis study. The analytical properties of these isolates asprovided by the supplier are reported in Table 1. Ninety-eight percent of the particles of both isolates went through#60 US Standard Screen.

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Table 1

Proximate composition of wheat protein isolates

Prolite 100 Prolite 200

Moisture (%) 6% maximum 6% maximum

Ash (%) 1% maximum 5% maximum

Protein

(N� 6.25) (%)

90% minimum 85% minimum

Fat (%) 6% maximum 6% maximum

Label statement Wheat gluten, lactic

acid, sulfite

Wheat gluten, phosphate, lactic

acid, sulfite

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428 419

2.2. Preparation of sample

Distilled water was added gradually to measured amountof WPIs (5 g) and stirred well to make dough. Theoptimum water absorption by each isolate was approxi-mately 4ml based on three replicates (sample towater ¼ 4:5 (w/w); moisture content E47.8% wet basis).Prolite 100 resulted as highly viscous dispersion anddifficult to handle whereas Prolite 200 produced smoothdough. Both WPI hydrated samples were kept for 30min atroom temperature (20 1C) for dough relaxation. Allmeasurements were conducted with the same lot of productfrom the same container. Each sample was prepared intriplicate.

2.3. Rheological measurements

SAOS measurements were performed on TA (AR 2000,TA Instruments, New Castle, DE, USA) controlled-stressrheometer. The plate–plate geometry was used with adiameter of 60mm and a gap between two plates of 1mm.The AR 2000 System is based on efficient Peltiertemperature control which efficiently monitored thetemperature during the experiments. For each test,approximately 2 g of well-mixed dough (WPI towater ¼ 5:4) was placed on the rheometer plate. Fortemperature sweep test, dough samples were heated from20 to 90 1C at a heating rate of 2 1C/min in situ of therheometer plate at 1Hz. Solvent trap was used duringrheological measurement at higher temperature. Frequencysweeps were carried out at 20 1C before and after heating to90 1C and additional holding for 15min. Frequency sweeptests were made between 0.1 and 10Hz at linear viscoelasticranges (LVR). In order to ensure that all the measurementsare carried out within the LVR, initially oscillation stresssweeps (at 1Hz) were tested for both WPI dough samplesindividually.

All the experimental dynamic rheological data (elasticmodulus, G0, viscous modulus, G00; phase angle, d andcomplex viscosity, Z*) were obtained directly from the TARheology Advantage Data Analysis software V 5.1.42 (AR2000, TA Instruments, New Castle, DE, USA). Thedeviation did not exceed 5% between duplicate runs, asthe experiment was repeated. The average of the three runswas reported as the measured value.

2.4. Thermal analysis by DSC

The calorimetric properties of WPI dough samples weremeasured by a TA Instrument Q100 DSC (Newcastle, DE,USA) calibrated with indium for heat flow and tempera-ture. The instrument was coupled with refrigerated coolingsystem. Nitrogen was used as a purge gas at a flow rate of50ml/min. The WPI powdered samples were accuratelyweighed (3–5mg) onto polymer coated aluminium pansand different volumes of deionized water or 0.01Mphosphate buffer (pH 7.4) was added directly to pan usingmicropipette, hermetically sealed and allowed to equili-brate at the initial temperature for 2 h. The dried samplewithout water was considered as control one. The samplepans were heated from 30 to 160 1C at a heating rate of5 1C/min and further rescanned between 40 and 200 1C toidentify glass transition temperature (Tg). An empty panwas used as the reference. The DSC measurement was donein triplicate. The peak transition temperature (denaturationtemperature) and glass transition temperature (Tg) werecomputed from the thermograms by a Universal AnalysisSoftware (version 3.6C), which was provided with theinstrument (TA Instruments, Newcastle, DE). The glasstransition temperature was recorded from the inflexionpoint of the change in heat flow.

2.5. Electrophoretic analysis

SDS–PAGE was performed using the method of Flingand Gregerson (1986) using stacking gel and resolving gelof 4 and 12% (w/v) polyacrylamide. Slab gels (0.75-mmthickness) were run at a constant current of 15mA/gel; theelectrophoresis was performed with a Bio-Rad Mini Transblotting system following the method described earlier byAhmed et al. (2003) for egg protein.

2.6. Dielectric properties measurement

A vector network analyzer (Model: Agilent 8722ES,Agilent Technology, Palo Alto, CA) with an open-endedcoaxial cable (#8120-6192, Hewlett Packard) connected toa probe (85070C, Agilent Technology, Palo Alto, CA) inelectronic calibration module (E-cal, 4693A) was used tomeasure dielectric properties of WPI dough. The doughsample (WPI:water 1:1; approximately 25 g) was placed ina wide glass tube (50ml) and the open co-axial probe(probe diameter E2.4mm) was set into the tube. Thesample was heated from 30 to 80 1C and dielectricproperties were measured in the frequency range of500–3000MHz. The detailed procedure of the dielectricmeasurement was reported elsewhere (Ahmed et al., 2007).All the measurements were carried out in triplicate andwere reproducible to 75%. Mean values and standarderrors were calculated from three replicates.The penetration depth is an important parameter in

characterizing temperature distribution in microwaveheated foods. Penetration depth (Dp) is generally described

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0

4000

8000

12000

16000

1010.1

Frequency (Hz)

G',

G"

(Pa)

P100 G' P200 G'

P100 G'' P200 G''

Fig. 2. Rheograms of wheat protein isolates at 20 1C.

Table 2

Effect of heating and cooling on regression coefficients of equation lnG0

vs. lno and lnG00 vs. lno of wheat protein dough samples at 20 1C

Sample type ln G0 ¼ ln A0 þ n ln o ln G00 ¼ ln A00 þ n ln o

Slope R2 SE Slope R2 SE

Unheated P100 0.57 0.99 0.009 0.56 0.99 0.008

Heat–cool P100 0.11 0.99 0.015 0.11 0.99 0.008

Unheated P200 0.48 0.99 0.025 0.49 0.99 0.007

Heat–cool P200 0.24 0.99 0.008 0.28 0.99 0.007

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428420

as the distance from the surface of a dielectric materialwhere the incident power decreased to 1/e of the incidentpower. The magnitude of Dp was calculated using thedielectric constant and loss factor at frequencies of 915 and2450MHz for rice flour slurries (Datta, 2001) as

Dp ¼l0

2p

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi2�0

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þ ð�00=�0Þ2

q� 1

� �s , (1)

where l0 ¼ 0.328m at 915MHz and 0.122m at 2450MHz.

2.7. Data analysis

Microsoft Excel software package (Microsoft Corpora-tion, USA) was used to carry out the statistical analysisand linear regressions of experimental data.

3. Results and discussion

3.1. Rheological properties

3.1.1. Linear viscoelastic behavior

Both WPI dough samples were tested to determine LVRby varying oscillation stress at a constant frequency of1Hz. Fig. 1 illustrates LVR for both WPIs at 20 1C. Basedon these results, oscillation stresses for isolate doughsamples were selected. Oscillation stress values of 70 and50Pa were found to follow LVR for Prolite 100 and Prolite200 dough, respectively, and, therefore, all subsequentmeasurements were carriedout at those stress values.

3.1.2. Frequency sweeps

Fig. 2 illustrates changes in elastic (G0) and viscousmodulus (G00) as a function of frequency for WPI doughsamples at 20 1C. Both elastic and viscous modulusexhibited frequency dependency and the magnitude ofmodulii increased as frequency was increased. A closerlook on the rheogram indicates that both samples behavedas true viscoelastic fluid at the studied frequency rangethough mechanical spectra of samples were dissimilar from

100

1000

10000

100101

Oscillation stress (Pa)

G' (

Pa)

10

100

δ (°

)

P 100 G'P 200 G'P 100 deltaP 200 delta

Fig. 1. Linear viscoelastic range of Prolite 100 and Prolite 200 at 20 1C.

each other. The dynamic rheological data of lno vs. lnG0

and lnG00 (Eq. (2)) were subjected to linear regression andthe calculated slopes are presented in Table 2. The slopesfor Prolite 100 were 0.57 and 0.56 while correspondingslopes for Prolite 200 were 0.48 and 0.49, respectively.These results infer that both dough samples are very similarin nature and both elastic and viscous characteristics areequally contributing towards rheological properties. Ourresults are not close in agreement with the earlier onesreported by Janssen et al. (1996) for hydrated gluten wherethe researchers found pre-dominating viscous property(G00). However, the difference could be attributed bymoisture content (55%) and nature of protein subfractions.

G0 or G00 ¼ Aon

Linear form : ln G0 or ln G00 ¼ ln Aþ n ln o, ð2Þ

where n is the slope (dimensionless) and A is the intercept(Pa sn).

3.1.3. Temperature sweep

Temperature sweep of wheat isolate dough was per-formed at 2 1C/min and is presented in Fig. 3. Time–temperature profile of this kinetically controlled reactionindicates the behavior of dynamic rheological parameters.As the temperature increased, both G0 and G00 started to

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100

1000

10000

0 20 40 60 80 100

Temperature (°C)

G',

G''

(Pa)

0

20

40

60

δ (°

)

P100 G' P200 G'P100 G'' P200 G''P100 delta P200 delta

Transition zoneGel pt.

(52.2C)

Fig. 3. Effect of heating on mechanical properties of wheat protein

isolates during temperature ramp.

1000

10000

100000

1010.1

Frequency (Hz)

G',

G''

(Pa)

P100 G' P200 G'

P100 G'' P200 G'

Fig. 4. Effect of heating–cooling on dough wheat protein dough rheology

measured at 20 1C.

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428 421

decrease gradually, until a critical temperature wasreached. The critical temperatures for Prolite 100 andProlite 200 were 52.2 and 64.6 1C, respectively. Thisobservation is close in agreement with earlier work ofCocero and Kokini (1991) on thermorheology of glutenin.During heating, a distinct gel point (cross-over of G0 andG00) was observed for Prolite 100 at 52.2 1C and, thereafter,G0 increased sharply. On the other hand, Prolite 200exhibited equal magnitude of G0 and G00 at the beginningand gradually reached a peak at 90 1C with substantialincrease in its solid-like property. The sharp increase in theelastic modulus was attributed to cross-linking/aggregationreactions happening among gliadin molecules resulting inthe formation of a network structure (Madeka and Kokini,1994). The viscous modulus (G00) of Prolite 100 and 200decreased during heating and reached a minimum value(64.6 and 70.7 1C). As the temperature was furtherincreased from 70 to 90 1C the G00 values superimposed(P40.05) with each other indicating similar liquid-likeproperty. Phase angle (d, which is an index of viscoelasticproperty of food materials) data clearly demonstratedsharp transformation of wheat dough in the transitionregion. Phase angle data inside the circle were very muchsimilar to onset temperature (Fig. 3) of glass transitiontemperature.

Our observations on heating behavior of wheat proteindoughs are close in agreement with reported values(Attenburrow et al., 1990; Kokini et al., 1994, 1995;Savador et al., 2006). Attenburrow et al. (1990) observedthat the G0 of heated hydrated-gluten decreased graduallyup to about 60 1C while it began to increase, and at about90 1C it increased significantly. The mechanical spectrareported by Savador et al. (2006) for wheat flour dough(E14% protein content) were similar to our observation.Interestingly, G0min of Prolite 100 was close in agreementwith their reported value. However, those authors de-scribed the increase in rheological parameters wereattributed to starch gelatinization and protein denatura-tion, respectively. Our observation demonstrated thatprotein denaturation played major role in rheological

changes of dough during heating confirming earlierobservation of Eliasson (1986) where the author reportedthat protein content played significant role in heat inducedrheological characteristics of bread dough. A large increasein elastic modulus for wheat protein dough was reported byKokini et al. (1994, 1995) while working on heating ofgluten, gliadin or glutenin dough in temperature rangebetween 70 and 80 1C. Higher G0 of Prolite 200 could becontributed by high molecular weight glutenin as reportedearlier by Janssen et al. (1996) where researchers havefound effect of higher glutenin to gliadin ratio significantlyincreased dynamic modulii.

3.1.4. Effect of heating–cooling on dough rheology

Wheat protein dough samples exhibited 6–10-foldincrease (Po0.05) in dynamic modulii while heating wasisothermal at 90 1C for 15min followed by immediatecooling to 20 1C (Fig. 4). Both G0 and G00 increased linearlywith frequency on the log–log scale and Prolite 200 showedrelatively higher values. Changes in mechanical property ofdough samples after heat treatment are presented inTable 2. The slopes of lnG0 vs. lno (Eq. (2)) of theregression equations decreased from 0.57 to 0.11 and 0.48to 0.24 (R2

¼ 0.99) for Prolite 100 and 200, respectively.The substantial decrease of slope indicated gradual changeof entangled polymer flow to more rubbery structureduring heating–cooling process. A perfect rubbery materialis characterized by the slope value approaching to zero(Ferry, 1980).The phase angle (d) and complex viscosity (Z*) data

before and after heating–cooling are shown in Fig. 5.Frequency sweep demonstrated a reduction in the magni-tude of d after heating–cooling as compared to unheatedsample suggesting increase in solid-like (rubbery) property.This is evidence of permanently cross-linked density;however it is still a ‘weak viscoelastic gel’ system(Apichartsrangkoon et al., 1999). Heat–cool process

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10

100

1000

10000

100000

1010.1

Frequency (Hz)

η* (

Pa.s

)

1

10

100

δ (°

)

P100 C |n*| P100HC |n*| P200 C |n*| P200HC |n*| P100 C delta P100HC delta P200 C delta P200HC delta

Fig. 5. Comparison of viscoelastic properties of wheat protein isolates

before and after heating at 20 1C.

134.5

67.7

127.5

71.6

–0.6

–0.5

–0.4

–0.3

–0.2

–0.1

0

25 50 75 100 125 150 175 200

Temperature (°C)

Hea

t flo

w (

W/g

)

P 100P 200

Fig. 6. Thermograms of wheat protein isolates in powder form.

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428422

resulted in a very strong dependence of Z* on frequency asobserved by Madeka and Kokini for gliadin at highertemperature. Such behavior is common for highly cross-linked materials (Richardsons and Ross-Murphy, 1981).

The large impact on G0 during holding period of wheatprotein dough at 90 1C was attributed by thermaldenaturation of proteins; completion of protein–proteinaggregation reaction and formation of highly cross-linkednetwork. This is supported by earlier publications wherenetwork formation of gluten was well described (Cuq et al.,2000; Jeanjean et al., 1980; Schofield et al., 1984). Thesealterations may result from less ordered random structureand polymerization by SHSS interchange reactions (Cuqet al., 2000). Higher temperature favors cross-link ofgliadin and cross-link through disulfide bonds of glutenins(Levine and Slade, 1988, 1990). Protein fractions contain-ing high molecular weight glutenin polymers appeared tobe less affected by heat as shown by Prolite 200 in our case(higher elastic modulus that increased with molecularweight and less change in slope of Eq. (1), whereas theextent of change increased for the smaller polymersespecially which contained relatively higher gliadins(Stathopoulos et al., 2006). Transition behavior of Prolite100 after heat–cool process suggests the presence of lowmolecular weight polymers. Stathopoulos et al. (2006) alsoreported dramatic rheological changes of LMW fractioncompared to HMW protein fractions.

Some researchers (Kokini et al., 1994; Levine and Slade,1990) advocated that the increasing trend of G0 continuesup to 130–135 1C where the aggregation reaction appearedto be completed and highly cross-linked network formed.However, G0 was less pronounced (data not shown) duringa holding time period of 30min at 90 1C in our study. Thisis in agreement with the heat treatment studies ofApichartsrangkoon (2002) with the wheat gluten for 6 h.It infers that heating up to 90 1C and holding for additional15min resulted in similar mechanical strength (viscoelasticproperty) and completion of protein–protein aggregationand rearrangement of disulfide bonds as reported by othersat 135 1C.

3.2. Thermal characteristics

Fig. 6 shows the DSC trace obtained, at a heating rate of5 1C/min, for dried wheat protein samples (moisturecontent 6%; wet basis). It clearly demonstrated two distinctthermal denature transitions. The transition temperatureswere 67.7 and 134.5 1C for Prolite 100 and, the correspond-ing values were 71.6 and 127.5 1C for Prolite 200. Theenthalpies were found to be 2.6 and 19.3 J/g; and 2.0 and20.0 J/g for Prolite 100 and 200, respectively. Similarthermograms have been recorded in numerous previousstudies of wheat proteins. While working with variousprotein fractions, Leon et al. (2003) found two thermaltransitions for glutenins (4% moisture content) at 64.3 and84.2 1C with enthalpy of 11.5 and 14.4 J/g, respectively. Thefirst endothermic peak in our study (67.7 and 71.6 1C) forboth samples can be assigned to low molecular weightglutenins since the values were very close to reported valuesof Leon et al. (2003). The second thermal peaks weresignificantly higher than the literature value and possiblycould be HMW glutenins. Several researchers (Arntfieldand Murray, 1981; Ma, 1990a, b) have reported that nodenaturation peak was detected during thermal scanning ofwheat proteins. The possible reason behind lack of non-traceability of denaturation peak in other studies was thewater content of the sample used for DSC studies (Leonet al., 2003).The denaturation temperature decreased as water con-

tent increased. Thermal denaturation temperatures asfunction of water are reported in Table 3. At highermoisture content (54%), Prolite 100 did not show anydistinct transition (except 109.2 with high enthalpy value)whereas two distinct thermal transitions were noticed at 69and 84 1C for Prolite 200. Inconsistent results and variationof thermal denaturation peaks (2–4) are reported in theliterature (Eliasson and Hegg, 1980; Falcao-Rodrigueset al., 2005; Harwalkar and Ma, 1987; Leon et al., 2003).Falcao-Rodrigues et al. (2005) detected thermal transitions(37.3–49.7 1C) of wheat proteins at about 50% moisturecontent. Representative thermograms of wheat isolates

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Table 3

Thermal transitions range for wheat protein samples as a function of water content

Moisture content (% wet basis) Prolite 100 Prolite 200

First transition (1C) Second transition (1C) First transition (1C) Second transition (1C)

6 67.7 (2.60 J/g) 134.5 (19.3 J/g) 70.16 (3.2 J/g) 135.1 (28.4 J/g)

71.6 (1.96 J/g) 127.5 (20.9 J/g) 76.5 (1.5 J/g)

48 78.2 (0.06 J/g) 136.5a 64.1 (5.6 J/g) 90.8 (0.2 J/g)

54 109.2a 68.9a 84a

Phosphate buffer (pH 7.4) 51.8 (3.2 J/g) 94.3 (4.4 J/g) 65.6 (47.4 J/g) 82.3a

57.0 (11.8 J/g) 112.9a 74.9 (4.2 J/g)

aHigh enthalpy values.

Prolite 100

Prolite 200

94.3

51.9C

57.4C

–1

–0.8

–0.6

–0.4

–0.2

40 50 60 70 80 90 100

Temperature (°C)

Hea

t flo

w (

W/g

)

90.9 C

64.4 C

–0.35

–0.3

–0.25

–0.2

40 60 80 100

Temperature (°C)

Hea

t flo

w (

W/g

)

Fig. 7. Thermograms of wheat protein isolates dough (Prolite 100 with

phosphate buffer 48% m.c. and Prolite 200 with 54% m.c).

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428 423

(Prolite 100 in phosphate buffer and Prolite 200 with 54%moisture content) are presented in Fig. 7. In presence ofphosphate buffer (4.4mg sample and 5 ml buffer solutionresulted about 56% moisture) three thermal transitions(51.8, 57, 94.3 and 112.9 1C) were detected (112. 9 1C is notshown) for Prolite 100 sample whereas DSC thermogramof Prolite 200 was well represented by two distinct peaks at64.4 and 90.9 1C. These thermal transition values indicatedthat wheat proteins have definite denaturation temperaturewhich can be detected by DSC and the transitions are clearin presence of water or phosphate buffer solutions. Thenon-traceability of DSC transitions of wheat doughsamples could be due to interference of starch since

transition temperatures for both wheat starch and proteinsare in the similar range.The glass transition phenomenon, studied for amor-

phous polymeric systems, has been observed for WPIsusing DSC. During first scanning, endothermic transitiondue to denaturation of ordered structure of the protein wasobserved. The endothermic relaxation peaks were result ofsecondary molecular ordering undergone by amorphouspolymers in the glassy state (Roos, 1995). Since the glasstransition is a reversible phenomenon, the first heating ofsample eliminates the relaxation endotherms while thesecond scanning results in the Tg value (Noel et al., 1995).On heating of wheat isolates (second and even third scans),a glass transition occurs at the temperature when thesample undergoes change from solid-like character toliquid-like behavior during experimentation time. The Tg

range of dry WPI was found to be close to one another.The Tg values for Prolite 100 were 139.471.1 1C (onset),152.471.6 1C (midpoint) and 162.870.9 1C (end) whereasthe corresponding values for Prolite 200 were 136.471.4,149.371.3 and 161.371.8 1C, respectively. The glasstransition in dry wheat proteins is found to be reversibleto what we observed during second and third scan and theobservation is close in agreement with Kokini et al. (1995).While comparing Tg of protein fractions, our observedvalues were comparatively lower than the reported Tg ofnative gluten (175 1C) at similar moisture levels (Micardand Guilbert, 2000) but within the range of Noel et al.(1995). The factors affecting the Tg value are compactnessor aggregation of the protein structure, its molecularweight and interactions with other components (Micardand Guilbert, 2000).

3.3. Electrophoresis

The protein pattern in an SDS–PAGE gel of Prolite 100(lane 1) and Prolite 200 (lane 2) is illustrated in Fig. 8.Major protein bands in lanes 1 and 2 include five majorstorage proteins. The individual subunits present in WPIswere at molecular mass (based on band intensity andwidth) 25, 28, 41, 85 and 110 kDa. Comparison of lanes 1and 2 demonstrated that almost similar subunits were

ARTICLE IN PRESS

KDa

200116

97

66

45

31

21

14

7

F1 = 110

F2 = 85

F3 = 28

F4 = 25

F5 = 10

M 1 2

Fig. 8. SDS–PAGE profile of wheat protein isolates; lane (M) standard,

1: Prolite 100 and 2: Prolite 200.

Prolite 100

10

20

30

40

500 1000 1500 2000 2500 3000

Frequency (MHz)

ε'

30C 50C

70C 80C

Prolite 200

20

30

40

50

60

500 1000 1500 2000 2500 3000

Frequency (MHz)

ε'

30C 50C

70C 80C

Fig. 9. Frequency and temperature dependence of the dielectric constant

for wheat protein isolates.

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428424

present except small shape change in the subunit ofmolecular mass 25 kDa. The higher molecular weights(85–110 kDa) are closer to high molecular weight glutenins(HMM-GS) while lower MW (10–28 kDa) could berepresented by a series of protein fractions includinggliadins (o, a and g) and low molecular weight glutenin(LMM-GS) (DuPont et al., 2005).

3.4. Dielectric properties

Dielectric constant and loss factor of Prolite 100 and200 were studied as function of frequency and temperature(Figs. 9 and 10). Both dielectric constant (e0) and lossfactor (e00) decreased as frequency increased. This observa-tion is in close agreement with dielectric behavior ofprotein foods (Everard et al., 2006). The temperaturedependence of e0 and e00 indicated opposite trend in theselected frequency range (500–3000MHz). The e0 ofProlite 100 dough suddenly increased at 50 1C; thendecreased significantly at 70 1C, and finally leveled at80 1C (P40.05). This observation is close in agreementwith Goedeken et al. (1997) for bread dough. At similarmoisture level, Prolite 200 had higher e0 values thanProlite 100 dough. The e0 values for Prolite 200 doughdecreased gradually with temperature in the range of30–70 1C under studied frequency range and increasedsuddenly at 80 1C.

Following similar trends of e0 with temperature, as theProlite 100 was heated above 50 1C, a large increase(Po0.05) in loss factor was noticed. As the temperaturewas further increased, a reduction in e00 was observed.Statistically, values of e00 in the temperature range from 30to 70 1C were significantly different (Po0.05) from eachother whereas e00 data did not show any significantdifferences between 70 and 80 1C (P40.05). The e00 valuefor Prolite 200 was higher compared to Prolite 100 andbehaved differently as function of temperature. The e00

decreased gradually as temperature increased from 30 to

50 1C. The magnitude of e00 showed a small increase at70 1C, followed by a significant increase at 80 1C.In the temperature range of 50–80 1C, sudden changes in

e0 and e00 could be associated with protein denaturationcaused by the cooking action associated with exposure ofthe dough to high temperature. Both increasing anddecreasing trends of dielectric parameters of food materialsas a function of temperature have been reported in theliterature (Goedeken et al., 1997; Sipahioglu and Barringer,2003; Wang et al., 2003). The wide variation of e0 and e00

between two isolates at similar condition (moisturecontent, temperature and frequency) might be attributedby composition and protein subfractions. The higher e00 ofProlite 200 could be attributed by higher amount of ashcontent (E6% compared to 1% of Prolite 100). Ashincreases the e00 by increasing the ionic component of the e00.This observation is close in agreement with literaturevalues (Sipahioglu and Barringer, 2003). There are twomechanisms involved to explain the change of e00 of foodmaterials: dipole–dipole interaction and ionic. Dipole–dipole interaction produces the loss due to friction amongpolar molecule like water whereas loss also happened dueto heat dissipation during electrical conduction in thepresence of ionic substances (Metaxas and Meredith,

ARTICLE IN PRESS

Prolite 100

5

10

15

500 1000 1500 2000 2500 3000

Frequency (MHz)

ε''

30C 50C

70C 80C

Prolite 200

10

20

30

40

50

60

500 1000 1500 2000 2500 3000

Frequency (MHz)

ε''

30C 50C

70C 80C

Fig. 10. Frequency and temperature dependence of the loss factor for

wheat protein isolates.

Table 4

Dielectric parameters of WPI dough samples at selected temperatures

Temperature

(1C)

Sample type 915MHz 2450MHz

e0 e00 e0 e00

20 Prolite 100 25.871.6 8.570.5. 22.170.9 7.870.3

Prolite 200 34.171.8 20.371.2 28.570.9 14.470.8

30 Prolite 100 27.370.8 8.670.5 24.270.6 8.170.6

Prolite 200 34.871.6 19.270.9 29.470.5 14.170.7

40 Prolite 100 27.971.3 8.770.3 24.770.9 8.270.5

Prolite 200 33.271.1 17.270.6 29.770.5 12.970.6

50 Prolite 100 31.470.8 10.170.7 27.770.8 9.070.4

Prolite 200 32.971.4 17.670.9 28.370.5 12.470.8

60 Prolite 100 25.570.6 8.570.7 22.170.6 7. 470.5

Prolite 200 36.471.5 23.270.7 29.971.2 14.370.9

70 Prolite 100 25.570.7 8.670.2 21.970.8 7.370.3

Prolite 200 33.471.1 23.970.7 27.671.1 14.570.8

80 Prolite 100 25.671.1 8.970.4 21.970.9 7.570.5

Prolite 200 40.671.8 33.370.6 33.471.2 19.270.7

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428 425

1983). Dielectric loss factor of Prolite 200 samplesexhibited significantly (Po0.05) higher values due to theadditional d.c. conductivity in presence of more ashcontent. In addition, e00 consistently increased withtemperature while Prolite 100 exhibited mixed responseduring heating at similar condition.

The dielectric parameters (e0 and e00) of wheat proteindough (E48% moisture w.b.) at selected temperatures andthe two commonly used microwave frequencies (915 and2450MHz) are presented in Table 4. As discussed earlier,both e0 and e00 of Prolite 100 exhibited peak value at 50 1Cwhile the corresponding peak value was found at 80 1C forProlite 200. DSC data (Table 3) supported substantialchange in dielectric data at specified temperature. Sig-nificant changes in dielectric parameters are associated withdenaturation of protein (Bircan and Barringer, 2002). Thedielectric properties measure mobility of water and ions insolution, so the increased mobility of the molecules in theprotein system during denaturation causes an increase inthe dielectric properties of the system. Temperaturedependence of e0 and e00 of Prolite 100 and 200 atmicrowave frequency range was described well by thesecond-order polynomial equations (Table 5A and B).

Penetration depth (Dp) of food materials indicates thedepth to which heat is dissipated uniformly. An increase inpenetration depth will increase the temperature uniformityin microwave heating. Temperature dependence of pene-tration depth is complex, because ionic and dielectric lossesinteract and, therefore, no generalized trend was observedbetween Dp and temperature of WPI samples (Fig. 11). Thepenetration depth of dough at 915MHz was highercompared to sample measured at 2450MHz. This is inclose agreement with earlier observation of Wang et al.(2003) and Everard et al. (2006). At 915MHz, the Dp

decreased between 30 and 50 1C and then became constantor decreased at higher temperature ranges for bothsamples. In contrast, the changes in Dp with temperatureat 2450MHz were minimal and not significant (P40.05).Frequency and temperature dependent changes in penetra-tion depth were expected in this study because of theirinfluence on dielectric properties. Temperature dependenceof Dp values of wheat protein samples at microwavefrequency range was described well by the followingsecond-order polynomial equations. Penetration depthdata of Prolite 100 did not fit well with temperaturechange as evidenced from the R2 values whereas Prolite 200data fitted adequately (R240.92). It infers that change ofDp values of Prolite 200 followed a regular trend withtemperature and it showed almost frequency-independentbehavior at 2450MHz.

Prolite 100 at 915MHz:

Dp ¼ 0:0002T2 � 0:030T þ 3:99; R2 ¼ 0:58,

Prolite 200 at 915MHz:

Dp ¼ �0:0002T2 þ 0:009T þ 1:62; R2 ¼ 0:92,

ARTICLE IN PRESS

Table 5

Regression equations correlating temperature dependency of dielectric parameters of wheat protein isolate dough sample

Temperature Dielectric constant (e0) R2 Loss factor (e00) R2

(A) Prolite 100

30 �0 ¼ 1E � 06T2 � 0:007T þ 33:3 0.93 �00 ¼ 2E � 06T2 � 0:008T þ 14:5 0.71

40 �0 ¼ 2E � 06T2 � 0:009T þ 33:5 0.95 �00 ¼ 3E � 06T2 � 0:013T þ 20:5 0.82

50 �0 ¼ 2E � 06T2 � 0:009T þ 38:9 0.94 �00 ¼ 3E � 06T2 � 0:013T þ 20:9 0.79

60 �0 ¼ 1E � 06T2 � 0:008T þ 32:3 0.94 �00 ¼ 2E � 06T2 � 0:010T þ 17:2 0.80

70 �0 ¼ 2E � 06T2 � 0:008T þ 32:6 0.94 �00 ¼ 3E � 06T2 � 0:011T þ 17:8 0.80

80 �0 ¼ 2E � 06T2 � 0:008T þ 33:0 0.94 �00 ¼ 3E � 06T2 � 0:012T þ 18:8 0.80

(B) Prolite 200

30 �0 ¼ 3E � 06T2 � 0:017T þ 48:9 0.90 �00 ¼ 7E � 06T2 � 0:030T þ 44:0 0.85

40 �0 ¼ 2E � 06T2 � 0:009T þ 41:9 0.94 �00 ¼ 3E � 06T2 � 0:011T þ 19:3 0.76

50 �0 ¼ 4E � 06T2 � 0:017T þ 43:8 0.90 �00 ¼ 9E � 06T2 � 0:038T þ 52:3 0.87

60 �0 ¼ 4E � 06T2 � 0:019T þ 52:8 0.92 �00 ¼ 1E � 05T2 � 0:041T þ 57:8 0.88

70 �0 ¼ 4E � 06T2 � 0:018T þ 48:6 0.90 �00 ¼ 1E � 05T2 � 0:045T þ 61:8 0.87

80 �0 ¼ 5E � 06T2 � 0:024T þ 60:6 0.90 �00 ¼ 1E � 05T2 � 0:066T þ 88:0 0.87

0

1

2

3

4

20 40 60 80 100

Temperature (°C)

Dp

(mm

)

915 MHz P100 915 MHz P200

2450 MHz P100

2450 MHz P200

Fig. 11. Effect of temperature on penetration depth of wheat protein

isolates at selected frequencies.

J. Ahmed et al. / Journal of Cereal Science 47 (2008) 417–428426

Prolite 100 at 2450MHz:

Dp ¼ �6E � 06T2 þ 0:002T þ 1:13; R2 ¼ 0:34,

Prolite 200 at 2450MHz:

Dp ¼ �0:0001T2 þ 0:013T þ 5:00; R2 ¼ 0:96.

4. Conclusions

Dynamic rheological measurements exhibited that theviscoelastic behavior of the two WPIs under investigationdiffered widely during heat treatment. The most significantchange of dough sample occurred between 50 and 80 1Cdue to protein denaturation. Prolite 200 had a highermechanical property with substantial G0 compared toProlite 100. A gradual transformation of Prolite 100 doughfrom liquid-like to solid-like behavior indicated presence of

low molecular weight polymers of protein sub-fraction.Thermal properties of wheat proteins have been assessed atvarious moisture content and endothermic peaks werefound in wider temperature range. The Tg values for driedprotein isolates were almost alike. The dielectric propertiesof dough samples showed a substantial increase withtemperature in the range of protein denaturation anddiffered from each other. The penetration depth at lowerfrequency was significantly higher and decreased withtemperature for Prolite 100 whereas Prolite 200 exhibitedfrequency independent behavior at 2450MHz. Proteindenaturation appeared to play a significant role onrheological, calorimetric and dielectric properties of wheatisolates.

Acknowledgments

The authors acknowledge the financial support providedby the National Sciences and Engineering ResearchCouncil of Canada. The authors would like to thankMr. Yvan Gariepy and Mr. Anwer Ayad for theirassistance in dielectric instrument setup and quantificationof SDS–PAGE.

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