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Journal of Cereal Science 54 (2011) 181e186

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Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

Solvent effects on the molecular structures of crude gliadins as revealedby density and ultrasound velocity measurements

Zhuo Zhang, Martin G. Scanlon*

University of Manitoba, Department of Food Science, Winnipeg, Manitoba, Canada R3T 2N2

a r t i c l e i n f o

Article history:Received 10 July 2010Received in revised form14 February 2011Accepted 1 April 2011

Keywords:Crude gliadinsPartial specific adiabatic compressibilitycoefficientAqueous ethanolDilute acetic acidSpecific volume

* Corresponding author. Tel.: þ1 204 474 6480; faxE-mail address: scanlon@cc.umanitoba.ca (M.G. Sc

0733-5210/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.jcs.2011.04.006

a b s t r a c t

Gliadins were extracted from hard red spring wheat flour with 70% (v/v) aqueous ethanol and lyophi-lized. These crude gliadins were dissolved in 70% (v/v) aqueous ethanol or 4 mM acetic acid with orwithout ultrasonication. Precise measurements of the density and ultrasound velocity of the solutionswere made at 20 �C. For non-sonicated solutions, crude gliadins solubilized in ethanol had a slightlylarger partial specific volume (0.76 cm3 g�1), and a larger partial specific adiabatic compressibilitycoefficient (15� 10�11 Pa�1) compared to those solubilized in acid (0.739 cm3 g�1, 3.1� 10�11 Pa�1,respectively). Larger values are consistent with the existence of complexes formed by gliadins and lipidsin aqueous ethanol solutions. Utrasonication had no effect on these proteinelipid complexes based onmeasurements of density, but it did alter the compressibility of gliadins in dilute acid (making themalmost twice as compressible). Novel insights into gluten protein properties can be gained fromcompressibility measurements of solutions using ultrasonic resonators when coupled with measure-ments of protein specific volume.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Gliadins, the non-network proteins in dough (Gennadios andWeller, 1990), are consensually defined as the protein compo-nents of wheat flours or wheat glutens that are insoluble in wateror neutral salt solutions, but soluble in aqueous alcohols withoutreduction of disulphide bonds (Wieser, 1996). The functionality ofgliadins with respect to the mixing properties of dough and thebaking suitability of wheat flours has been a long-standing topic ofinvestigation for cereal chemists. MacRitchie (1987) extractedgliadin-rich fractions fromwheat flours using dilute HCl of high pHvalues (from about 5.0 to 5.8) and found that these fractionsdecreased dough strength and slightly depressed loaf volumes.Khatkar et al. (2002) extracted gliadins from flours using 70% (v/v)aqueous ethanol and chromatographically separated the gliadinsubgroups. From reconstitution experiments, they found that thegliadins tended to decrease the overall strength and stability of thedough, but both the total gliadins and the individual gliadinsubgroups substantially improved the loaf volume and the bread-making quality of the flours. Confounding results on the efficacy ofgliadins with respect to the baking suitability of flours might arisebecause of different extractionmethods, e.g., acidic versus alcoholicextraction. As a consequence, the behavior of gliadin-rich fractions

: þ1 204 474 7630.anlon).

All rights reserved.

in different solvents is valuable information for understanding theend-use functionalities of gliadins.

In recent years, there has been a substantial increase in the useof vibrating tube technology and ultrasonic resonators to ascertainfundamental thermodynamic parameters of proteins in solution(Chalikian et al., 1996; Sarvazyan, 1991; Wang et al., 2006). Thepartial specific adiabatic compressibility coefficient, which revealsthe pressure dependence of a protein’s molecular volume, is onesuch thermodynamic parameter that is experimentally obtainedfrom measurements of the density and ultrasonic velocity ofprotein solutions (Sarvazyan, 1991). This parameter is the subject ofsubstantial experimental investigation because of the insights itprovides on protein structure and conformation (Chalikian et al.,1995; Gekko, 2002), including how protein molecules interactwith solvent molecules in solution (Chalikian et al., 1995, 1996;Gekko and Yamagami, 1991). Despite the potential for probingprotein conformation and protein interactions with solvent mole-cules, measurements of the partial specific adiabatic compress-ibility coefficient of gliadins do not appear to have been made todate.

Therefore, the objective of this study was to determine thepartial specific adiabatic compressibility coefficients of ethanol-extracted proteins (known as crude gliadins or gliadin-rich frac-tions) from hard red spring wheat flour resolubilized in either 70%(v/v) aqueous ethanol or 4 mM acetic acid. Deductions were madeon the behavior and properties of gliadin molecules in these

Nomenclature

bS Adiabatic compressibilitycoefficientofa solution,Pa�1

bSo Adiabatic compressibilitycoefficientof a solvent, Pa�1

boS Partial specific adiabatic compressibility coefficient

of a solute (protein), Pa�1

Fo Apparent volumetric fraction of a solvent, -r Density of a solution, kgm�3

ro Density of a solvent, kgm�3

KS Apparent specific adiabatic compressibility,cm3 g�1 Pa�1

P Pressure, Pau Ultrasonic velocity through a solution, m s�1

uo Ultrasonic velocity through a solvent, m s�1

v Volume of a liquid, m3

v Apparent specific volume, cm3 g�1

vo Partial specific volume, cm3 g�1

Z. Zhang, M.G. Scanlon / Journal of Cereal Science 54 (2011) 181e186182

solvents, particularlywith respect to interaction of gliadinswith co-extracted molecules such as lipids (Békés et al., 1983a, 1983b, 1992;Cornell et al., 2002; McCann et al., 2009).

2. Experimental

2.1. Flour samples

No. 1 Canada Western Red Spring 13.5 (CWRS) wheat flour(13.6� 0.1% protein, N� 5.7) that had been milled as a straightgrade flour at the Canadian International Grains Institute (CIGI,Winnipeg, Canada) pilot mill was used for this study.

2.2. Extraction of crude gliadins

Crude gliadins were extracted with 70% (v/v) ethanol directlyfrom flours. Wheat flour samples (25 g) were mixed with 70% (v/v)ethanol (100 mL) at room temperature (23�1 �C) for 30 min andthen centrifuged at 480g for 10 min (Centrifuge RC5C, SorvallInstruments, USA). The supernatant was collected and solvent wasevaporated by continuous blowing of air over the extract surface.The residue was then lyophilized in a freeze drier (Pilot lyophilizer,VirTis, USA). Five extractions were performed to prepare sufficientfreeze-dried material for all experiments. The freeze-dried prod-ucts from the five extractions were blended and ground intoa powder that was sealed and stored at 4 �C. The predominantmaterial in this powder is considered to be gliadins (Robertsonet al., 2007).

2.3. Preparation of crude gliadin solutions

Two solvents, either 70% (v/v) ethanol or 4 mM acetic acid,were used to dissolve the crude gliadins and to make up theserial dilutions for ultrasound and density measurements. Crudegliadin samples (375.0� 0.1 mg) were dissolved in solvent(30 mL) by vortexing with additional brief vortexing every 3 minat room temperature (23�1 �C) for a total of 10 min. Thesuspension was then centrifuged at 17,200g for 10 min (Centri-fuge RC5C, Sorvall Instruments, USA), and the supernatant ofcrude gliadin solution (z30 mL) was poured into a covered testtube and sealed. A portion (10 mL) of the solution was retained asthe most concentrated solution and another 10 mL was used forserial dilutions (1/2, 1/4, 1/8 and 1/16). The rest of the solutionwas used for micro-Kjeldahl nitrogen determination (Kjeldahl

system 1002 distilling unit, FOSS Tecator AB, Sweden) (AmericanAssociation of Cereal Chemists, 2000) and for determination oftotal dissolved solids. Thus, the concentrations of protein in eachsolution could be directly related to measurements performed onother portions of the same solution. Three replicate series ofsolutions were prepared from the lyophilized crude gliadins foreach treatment.

2.4. Ultrasonication treatment

Ultrasonication (using high intensity ultrasound) has beenshown to improve the extractability of wheat flour proteins (Singhet al., 1990). Dissolution aided by ultrasonication was performed asin Section 2.3 except that an ultrasonication treatment was appliedjust before the final vortexing. Ultrasonication treatments of60 W� 1.5 minwere applied to the solutionwith a VC 60 ultrasonicprocessor (Sonics & Materials, Inc., Newton, CT, USA). Although60W was the power that the instrument applied at the probe(diameter 13 mm), the actual power transferred from probe tosolutions was 12.5 W. During ultrasonication, centrifuge tubes fil-led with the solution being extractedwere inserted in cold water sothat the increase in temperature of the solution (either in 70%ethanol or 4 mM acetic acid) was maintained below 7 �C. Thus, thetemperature rise was considered to have little effect on solventevaporation.

2.5. Density and ultrasound velocity measurements

The ultrasound velocities of each solvent and each serial dilutionof the crude gliadins were determined with a ResoScan System(TF Instrument Inc., Germany) at 20� 0.02 �C. Determination ofdensities on the same solution and solvent samples were per-formed with a DMA 5000 density meter (Anton Paar, Austria) at20� 0.006 �C. Determinations of ultrasonic velocity in the solutionand solution density were performed on two subsamples takenfrom each replicate solution.

2.6. Calculations of volume and compressibility parameters

The partial specific volume (vo) is calculated from density (r)results using the zero extrapolation method (Gekko et al., 2004):

voh limc/0

v ¼ limc/0

1� Fo

c(1)

where v is the apparent specific volume of the solute; c is theconcentration of the solute; Fo is the volume fraction of the solventin the solution and is calculated as Fo ¼ ðr� cÞ=ro from thedensities of the solution (r) and the solvent (ro).

The adiabatic compressibility coefficient, bS, of a liquid (solutionor solvent), defined as the pressure (P) dependence of the relativevolume of a liquid, is calculated from the Laplace equation based ondensity (r) and ultrasound velocity (u) measurements (Sarvazyan,1991):

bSh� 1v��vv

vP

�S¼ 1

ru2

where v represents the volume of the liquid and the subscript S

indicates the entropy is constant (i.e., measurements are performedunder adiabatic conditions).

The apparent specific adiabatic compressibility (KS) of thesoluble crude gliadins, indicating the pressure dependence ofmolecular volumes of soluble crude gliadins at each concentration,is calculated as (Sarvazyan, 1991):

0.000

0.001

0.002

0.003

0.004

0.005

0 2 4 6 8 10 12 14

Crude Gliadin Concentration / g dm-3

Spec

ific

Den

sity

0.000

0.001

0.002

0.003

0 2 4 6 8 10 12 14Crude Gliadin Concentration / g dm-3

Spec

ific

Velo

city

A

B

Fig. 1. Specific density (A) and specific ultrasonic velocity (B) of serial dilutions ofcrude gliadins in 70% (v/v) ethanol (-) and in 4 mM acetic acid (:); gliadins extractedwithout ultrasonication.

0.60

0.65

0.70

0.75

0.80

0 2 4 6 8 10 12 14

Crude Gliadin Concentration / g dm-3

Appa

rent

Spe

cific

Vol

ume

/ cm

3 g

-1

Fig. 2. Apparent specific volume, v, of crude gliadins in 70% (v/v) ethanol (-) and in4 mM acetic acid (:); gliadins extracted without ultrasonication.

Z. Zhang, M.G. Scanlon / Journal of Cereal Science 54 (2011) 181e186 183

KSh��vv

vP

�S¼ bSo �

bS=bSo � Fo

c(2)

The partial specific adiabatic compressibility coefficient (boS), which

is a measure of the pressure dependence of the relative volume ofideally isolatedmolecules of crude gliadins in solution, is calculatedby performing zero extrapolation on the results of the plot ofapparent specific adiabatic compressibility versus concentration(Gekko et al., 2004):

boS ¼ 1

volimc/0

KS ¼�bSovo

�� lim

c/0

bS=bSo � Fo

c(3)

Limit extrapolations of Eqs. (1) and (3) were performed usingOrigin 7.5 (www.originlab.com) employing a weighted linear fitwith the weighting determined from the standard deviations of thedensity or velocity measurement for each serial dilution.

3. Results

3.1. Protein content of soluble crude gliadins in different solutions

The freeze-dried powder material contained 74�1% proteins,and so it is designated as “crude gliadins”. Attempts to enhance theextraction of proteins relative to other components by using a cold-extraction technique for gliadins (Arrhenius, 1937) were noteffective, since protein contents were actually lower at 72�1%.

Somenon-gliadinproteins (mainly LMW-glutenins) (Békés et al.,1992) andnon-proteinmaterials (mainly lipids) (Cornell et al., 2002;Ponteetal.,1967)dissolveinthetwosolvents.Theproteincontentsofthe solubilized crude gliadinsweredetermined tobe 75%� 1% in the70% (v/v) ethanol solutions and 79%� 1% in the 4 mM acetic acidsolutions;ultrasonicationtreatmentslightlyimprovedtheamountofmaterial dissolved in the solvents but it had no effect on improveddissolutionofproteinineithersolvent.

3.2. Specific density and specific ultrasonic velocity

Specific density and specific ultrasonic velocity were deter-mined in the same way that specific viscosity is determined, i.e.,dividing the difference between the values in solution and insolvent by the value in the solvent. Both parameters are plotted asa function of solution concentration in Fig.1. It can be seen that bothtechniques have outstanding precision when measuring the prop-erties of gliadins in acidic aqueous solutions, and good precision ofmeasurement in the ethanol solutions. Quantifying this statement:for the aqueous solutions, average coefficients of variation based onsubsample measurements were 0.0007% for density (i.e., 7 partsper million) and 0.0013% for ultrasonic velocity. These figures are inline with repeatabilities reported on aqueous solutions by otherresearchers using quartz resonance instruments (Chalikian et al.,1996; Gekko, 2002; Kharakoz, 1991; Sarvazyan, 1991). Precisiondeteriorates by a factor of approximately two for replicates, andvariability in protein solubility in the individual replicates is likelyresponsible. Repeatabilitiy, although still good, was poorer foranalyses of the ethanol solutions, with average coefficients ofvariation based on measurements on subsamples being 0.02% fordensity and 0.029% for ultrasonic velocity.

3.3. Specific volume

The apparent specific volume of crude gliadins in solution, v,calculated from densities and solute concentrations according toEq. (1), is shown over a narrow range of specific volume in Fig. 2.

Uncertainty increases for the most dilute solutions, particularly forthe gliadins in ethanol. Similar results were obtained for ultra-sonicated samples (not shown), so ultrasonication has no effect onthe apparent specific volume of soluble crude gliadins in eitherethanol or acetic acid; results are reported in Table 1. Values inTable 1 are not dissimilar to the partial specific volumes acquiredwithout vibrating tube technology that ranged from 0.712 to0.736 cm3 g�1 for gliadins in 8 mMHCle8 mMKCl aqueous solution(Krejci and Svedberg, 1935) and 0.724 cm3 g�1 for gliadins in 62%aqueous ethanol (Foster and French, 1945).

Table 1Partial specific volume, vo , and partial specific adiabatic compressibility coefficient,boS , of crude gliadins extracted by two techniques in different solvents. Errors

represent standard deviations (n¼ 3).

Solvents Ultrasonication vo (cm3 g�1) boS (�10�11 Pa�1)

4 mM acetic acid Not applied 0.739� 0.005 3.1� 0.660 W� 1.5 min 0.735� 0.003 5.9� 1.1

70% (v/v) ethanol Not applied 0.76� 0.03 15� 960W� 1.5 min 0. 69� 0.03 Not reported

Z. Zhang, M.G. Scanlon / Journal of Cereal Science 54 (2011) 181e186184

3.4. Compressibility parameters

The values of apparent specific adiabatic compressibility, KS, ofcrude gliadins in solution are calculated according to Eq. (2), andvalues for serial dilutions of the non-ultrasonicated extractions areplotted against solute concentration in Fig. 3.

Using linear extrapolations of the KS values, the partial specificadiabatic compressibility coefficient, b

oS , is calculated using Eq. (3).

The partial specific adiabatic compressibility coefficients, boS , of

crude gliadins solubilized in different solvents and with differentextraction conditions are shown in Table 1. Due to a large standarddeviation for the value of b

oS of crude gliadins solubilized by soni-

cation in ethanol, a result has not been reported.

4. Discussion

4.1. Effects of solvent

From Figs. 2 and 3, it is clear that except in the most diluteethanol solutions, the apparent specific volume, v, and the apparentspecific adiabatic compressibility, KS, of crude gliadins in aqueousethanol are independent of solute concentration. Because gliadinssolubilized in alcohol (70% ethanol) have larger values for bothparameters, their molecules occupy more space and are morecompressible. Békés et al. (1983a, 1983b, 1992) reported thatproteins (LMW-glutenins and gliadins) aggregate with lipids inethanol, while it was reported that polar lipids, especially phos-pholipids, associate with gliadins through hydrogen bonds andhydrophobic interactions, and that these lipidegliadin complexesare absent in dilute acid (McCann et al., 2009; Pomeranz andChung, 1978). The specific volume of lipids (0.85e1.1 cm3 g�1) issubstantially greater than that of proteins, which varies from 0.58to 0.76 cm3 g�1 (Durchschlag, 1989). However, it is difficult todeduce structural information from composition alone because

-5

-3

-1

1

3

5

7

9

11

13

15

0 2 4 6 8 10 12 14

Crude Gliadin Concentration / g dm-3

Appa

rent

Spe

cific

Adi

abat

ic C

ompr

essi

bilit

y / 1

0-1

1 cm

3 g-1

Pa-1

Fig. 3. Apparent specific adiabatic compressibility,KS , of crude gliadins in 70% (v/v)ethanol (-) and in 4 mM acetic acid (:); gliadins extracted without ultrasonication.

effects on the specific volume from the neighboring layer of solventmolecules are neglected, and solvent ordering effects are unlikelyto be the same for the protein and for the proteinelipid complex.Additional insights into the structure of a solute in its solvated form,such as proteins, can be acquired from compressibility measure-ments (Gekko, 2002; Sarvazyan, 1991).

Crude gliadins have smaller values of partial specific adiabaticcompressibility coefficient, b

oS in 4 mM acetic acid (Table 1). Two

factors associated with solvent exclusion and solvent-solute inter-actions are likely responsible for differences in gliadin compress-ibility. The weak bonding in the proteinelipid complexes that hasbeen observed in 70% ethanol (Békés et al., 1983a, 1983b, 1992;McCann et al., 2009) is likely to produce more solvent-inaccessible void space than protein or lipid molecules alone.Greater void space is known to increase the intrinsic compressibilityof the dissolved molecules (Andrews et al., 2002; Chalikian et al.,1994; Gekko and Hasegawa, 1986; Gekko and Yamagami, 1991),and thus, lipideprotein complexation in aqueous ethanol mayaccount for much of the difference in the b

oS of gliadins in different

solvents that is observed inTable 1. In addition, proteinmolecules in4 mM acetic acid have more net charges compared to in aqueousethanol (Damodaran, 1996). The increased ordering of watermolecules around the charged groups causes the hydratedmoleculeto be less compressible (Chalikian et al., 1996; Kharakoz, 1991). Incontrast, crude gliadins in 70% (v/v) ethanol interact mainly withethanol molecules through hydrophobic interactions (Damodaran,1996; McCann et al., 2009), where the clathrate structuring isopen and the hydratedmolecule is substantially more compressible(Chalikian et al., 1994, 1996; Kharakoz, 1991). Both factors willreduce molecular compressibility, thus accounting for the smallvalues of b

oS for gliadins in 4 mM acetic acid compared to in ethanol.

Although uncertainty increases at the lower concentrations inaqueous ethanol, both v and KS of crude gliadins are smaller asconcentration decreases. One plausible explanation is the dissoci-ation of proteinelipid complexes brought about by dilution. As thelipids dissociate from the proteins, inter-molecular solvent-inac-cessible void space within the proteinelipid complexes disappearsand the value of v decreases. Similarly, the loss of solvent-inaccessible void space decreases the intrinsic compressibilityand increases the solvent-accessible surface of solute molecules,both of which decrease the apparent specific adiabatic compress-ibility. A number of food proteins (Sakurai et al., 2001) and proteinsof biological significance (Kövér et al., 2008) are known to self-associate at physiological concentrations but dissociate at lowprotein concentration. Given the weak molecular forces betweengliadins and lipids in aqueous ethanol (McCann et al., 2009), it isnot unlikely that the dissociation constant for complexation is lowenough to be affected by dilution.

4.2. Effects of ultrasonication

From Table 1, the partial specific volumes, vo, of crude gliadins in4 mM acetic acid (without and with ultrasonication) are notdifferent from each other. In contrast, the partial specific adiabaticcompressibility coefficient, b

oS , of ultrasonicated gliadins in acetic

acid is substantially larger than that of gliadins extracted withoutultrasonication (Fig. 4, Table 1). The values of apparent specificadiabatic compressibility, KS, are larger at all concentrations andthe difference increases as concentration decreases due to theprogressive increase in KS of ultrasonicated gliadins upon dilution.This progressive increase calls into question whether a linearextrapolation from the whole of the concentration range is valid,and a tangent method at lower concentration might furnish theappropriate value for the partial specific adiabatic compressibilitycoefficient of the gliadins solubilized by sonication. In such a case,

0.0

2.0

4.0

6.0

8.0

10.0

121086420

Crude Gliadin Concentration / g dm-3

Appa

rent

Spe

cific

Adi

abat

ic C

ompr

essi

bilit

y / 1

0-11 c

m3 g

-1 Pa-1

Fig. 4. Apparent specific adiabatic compressibility, KS , of crude gliadins in 4 mM aceticacid with extraction performed with (6) or without (:) ultrasonication.

Z. Zhang, M.G. Scanlon / Journal of Cereal Science 54 (2011) 181e186 185

the value would be substantially larger than the 5.9�10�11 Pa�1

reported in Table 1.It has been shown that high hydrostatic pressure distorts the

tertiary structure of g46 gliadin dissolved in aqueous acid, and thatthis distortion is associated with enhanced exposure of hydro-phobic surfaces (Lullien-Pellerin et al., 2001). It is therefore notinconceivable that the numerous high intensity condensations andrarefactions that occur during 90 s of ultrasonication will changethe tertiary structure of the gliadins and so alter their measuredproperties (Güzey et al., 2006). Changes in ultrasonic velocityassociated with a thermally-induced transition from native topartially unfolded conformation have been reported for a-lactal-bumin (Wang et al., 2006), while velocity changes in two cyto-chrome protein solutions arose from the pH-induced transition ofthe proteins from native (N) to compact intermediate (CI) state(Nölting and Sligar, 1993). According to Chalikian et al. (1995), thepartial specific adiabatic compressibility increases as the extent oftransition from N to CI state increases. Therefore, compressibilitymeasurements indicate that there are pressure-induced changes ingliadin structure brought about by ultrasonication (althoughspectroscopic studies would be needed to confirm this). Smallercompressibilities with increasing concentration of these loosely-structured gliadins is a consequence of an overall decrease in thefree energy of the solution; the structured water around exposedhydrophobic surfaces relaxes as the flexible ultrasonicated gliadinsrearrange to lessen the molecule’s cavity size, a phenomenonobserved for other proteins (Gekko and Hasegawa, 1986). Alterna-tively, the transition from structured to bulk water that permitsa lowering of the free energy of the solution may arise from self-association of the gliadins, an unresolved issue in cereal science(Ang et al., 2010; Liang et al., 2008).

5. Conclusion

Thermodynamic parameter measurements of crude gliadinsindicate that the specific volume and the compressibility of crudegliadins are affected by the solvent. The use of ultrasonicationduring gliadin solubilization in dilute acid sensitizes gliadins so thatthey aremore compressible and their compressibility changes upondilution.

Acknowledgments

The authors appreciate the financial assistance received fromthe Natural Sciences and Engineering Research Council of Canada,

and we also thank the Canadian Malting Barley Technical Center(CMBTC, Winnipeg, Manitoba) for use of their density meter.

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