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0733-5210/$ - se

doi:10.1016/j.jc

Abbreviations

dithiothreitol; F

polymer; HMW

low molecular w

sodium dodecy

tractable polym�Correspond

P.O. Box 8129,

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E-mail addr

Journal of Cereal Science 44 (2006) 127–136

www.elsevier.com/locate/yjcrs

HMW-GS affect the properties of glutenin particles in GMPand thus flour quality

C. Dona, G. Mannb, F. Bekesb, R.J. Hamera,c,d,�

aTNO Nutrition and Food Research, Zeist, NetherlandsbCSIRO Plant Industry, Canberra, ACT, Australia

cWageningen Centre for Food Sciences, Wageningen University, Wageningen, NetherlandsdCentre for Protein Technology TNO-WUR, P.O. Box 8129, 6700 EV Wageningen, The Netherlands

Received 12 August 2005; received in revised form 17 January 2006; accepted 21 February 2006

Abstract

Using a unique set of deletion lines, (Olympic�Gabo, varying in high molecular weight glutenin subunit (HMW-GS) composition,

but with the same genetic background) it was shown that the presence of glutenin particles in glutenin macropolymer (GMP) is directly

related to the presence of certain HMW-GS. In the absence of HMW-GS only a small amount of insoluble glutenin protein (GMP) could

be recovered from the flour that contained only LMW-GS. No particles were observed in this fraction. When one subunit (HMW-GS

Glu-Ax1) was present some particles could be observed, but when two or more HMW-GS were present particles could be clearly

identified. The amount of GMP increased with the increasing number of HMW-GS. All particles had the same LMW-GS composition

irrespective of HMW-GS-composition. Since the relative proportion of LMW-GS in GMP was dependent on the number of HMW-GS,

we postulate that LMW-GS become part of GMP through disulfide cross-linking with HMW-GS. GMP wet weight is correlated with the

average HMW density of the glutenin particle. These data were combined with previously published technological data from the same set

of wheats. Significant statistical relationships were observed between optimal mixing time and glutenin particle size and between thimble-

loaf height and GMP content. Taken together, these studies suggest that glutenin HMW-GS composition affects flour technological

properties through glutenin particle size.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: SDS; Unextractable glutenins; Glutenin macropolymer; Unextracted polymeric proteins; Glutenin network; Glutenin particles

1. Introduction

The very high molecular weight fraction of gluten isconsidered important in determining wheat flour quality.Lefebvre et al. (2000) showed that large gluten aggregatescontrol dough rheology. A clear correlation was shown

e front matter r 2006 Elsevier Ltd. All rights reserved.

s.2006.02.005

: CSLM, confocal scanning laser microscopy; DTT,

ITC, fluorescein isothiocyanate; GMP, glutenin macro-

-GS, high molecular weight glutenin subunits; LMW-GS,

eight glutenin subunits; PE, petroleum ether; SDS,

l sulphate; TFA, trifluoroacetic acid; %UPP, %unex-

eric protein

ing author. Centre for Protein Technology TNO-WUR,

6700 EV Wageningen, The Netherlands.

85383; fax: +31 317 485384.

ess: hamer@foodsciences.nl (R.J. Hamer).

between aggregate size and dough strength. Both Shewry etal. (1989) and MacRitchie (1999) have pointed out that: (1)HMW-GS composition affects the quantity of insolubleglutenin, (2) HMW-GS composition affects flour mixingtime, (3) insoluble glutenin fractions are polymericstructures containing HMW-GS and LMW-GS, and (4)that HMW-GS ‘5þ 10’ and ‘17þ 18’ play a key role in the‘polymeric’ size and the quantity of insoluble glutenins, andparallel dough and bread-making properties.Don et al. (2003a) showed that the SDS insoluble

glutenin fraction known as glutenin macropolymer (GMP)consists of glutenin particles. It was observed both withcommercial varieties (Don et al., 2003a) and with near-isogenic lines of the varieties Warigal and Lance (differingonly in HMW-GS, Don et al., 2005) that glutenin particlesextracted from flour of so-called 5þ 10 varieties are larger

ARTICLE IN PRESSC. Don et al. / Journal of Cereal Science 44 (2006) 127–136128

than particles extracted from flour of 2þ 12 varieties. Thisis in agreement with Popineau et al. (1994) who concludedthat both quantity and molecular size of the gluteninaggregates are largely determined by genetic factors.Furthermore, it has been proposed that glutenin particlesin GMP originate from protein particles in the wheatendosperm (Don et al., 2003a).

These studies clearly indicate a relationship betweenglutenin polymerisation and functionality controlled by itsHMW-GS composition. Here we further explore thepossible relation between HMW-GS composition on theone hand, and the quantity and properties of gluteninparticles, on the other.

Controlled expression of HMW-GS in wheat (Beasleyet al., 2002; Lafiandra et al., 1993) provides an opportunityto better understand how specific GS combine to form veryhigh molecular weight glutenin fractions, such as GMP. Aset of eight well-defined Olympic�Gabo lines withsystematic deletions (Beasley et al., 2002; Lawrence et al.,1988) was investigated to reveal possible links betweenglutenin HMW-GS composition and properties of gluteninparticles recovered in the GMP fraction. Further char-acterisation of these glutenin particles could also helpunravel the relationships between glutenin HMW-GScomposition, glutenin particle properties and wheat flourquality parameters. The latter have been previouslyreported for the same set of lines (Beasley et al., 2002;Uthayakumaran et al., 2002) enabling us to combine ourdata with their technological findings.

2. Experimental

2.1. Materials

A set of eight flour samples, prepared from near isogeniclines derived from the cross Olympic�Gabo was used. Theset was developed by Lawrence et al. (1988) and describedby Beasley et al. (2002) and Uthayakumaran et al. (2002)and was generated to have variations only in the Glu-A1, -B1 and -D1 loci. Table 1 shows the characteristics of theflour samples.

Table 1

The deletion lines used of Olympic�Gabo

Loci Olympic�Gabo A (1) B (17+18) D (5+10)

Triple null � � �

Double deletion + � �

Double deletion � + �

Double deletion � � +

Single deletion + + �

Single deletion + � +

Single deletion � + +

No deletion + + +

Presence or absence of alleles coding for different HMW glutenin subunits

are indicated with a+(present) or �(absent).

2.2. Isolation of GMP

Flour was defatted with petroleum ether (Don et al.,2003a,b). Defatted flour (1.4 g) was suspended in 1.5%SDS in water (28ml) and centrifuged at 80,000 g for 30minat 20 1C in a fixed angle rotor using a KontronUltracentrifuge (Kontron Instrument, Zfirich, Switzer-land). The supernatant was decanted and the gel-layercollected as GMP. Typically, the GMP gel consists ofHMW-GS and LMW-GS.

2.3. Rheology of GMP gels

GMP (1 g) was carefully taken from the top of the geland transferred into the measuring cell of a Bohlin VORrheometer (Bohlin, Sweden). The cell consisted of twoparallel plates ðd ¼ 30mmÞ with a gap of 1mm. Measure-ments were performed at 20 1C in a strain sweep modeat a frequency of 0.15Hz and at amplitudes ranging from1% to 100%. Data were expressed as G0 vs strain or delta(d) vs strain where G0 is the storage modulus. A higher G0

reflects a stiffer gel. The G0 is dependent on both thestrength of interactions between molecules in the gel andtheir number. The latter is related to protein concentration.Delta (d) is the ratio between storage and loss moduli (G00

over G0) and in this way less dependent on proteinconcentration. The d value is a measure of networkconnectivity and expressed in degrees. A d value lowerthan 451 indicates more connectivity and a more elastic gel.Typically, the strain sweep curve obtained covers both alinear and non-linear range. With GMP, typically G0 has aplateau in the linear range (up to a strain of ca. 0.1/amplitude of ca. 10%). At higher strains, G0 values decreasedue to breakdown of the gel structure. For this study,only G0 plateau values were taken. The same applies to thed value.

2.4. GMP dispersion

GMP dispersions were prepared by transferring ca. 1 g ofGMP gel to a tube containing 10ml 1.5% (w/v) SDSsolution. The gel was mixed with the solvent by brieflystirring with a spatula, the tube was then sealed and placedon a roller-bank at ambient temperature to homogenise thesample. High-energy input by mechanical stirring wasavoided. The dispersion produced was visually homoge-nous and opalescent, indicating that at least part of theprotein is not dissolved. Dispersions prepared in this waywere used for further characterisation.

2.5. Confocal scanning laser microscopy

GMP was isolated from flour of the eight wheat lines,followed by preparation of a GMP dispersion. Thedispersed GMP proteins were stained with fluoresceinisothiocyanate (FITC), a fluorescent probe for proteins,and observed using a Leica TCS NT confocal laser

ARTICLE IN PRESSC. Don et al. / Journal of Cereal Science 44 (2006) 127–136 129

scanning microscope. To allow resolution of the particlesGMP dispersions were examined at different proteinconcentrations (ranging between 0.1 and 5.0mg/ml).

2.6. Rheological characterisation of GMP dispersions

The viscosities of GMP dispersions were measured usingan Ubbelohde capillary viscometer with a capillarydiameter giving a flow through time of 300–400 s. Thedata were corrected for solvent viscosity and divided by theprotein concentration of the solution to give reducedviscosity values. From the plot of reduced viscosity vsconcentration the average voluminosity [Z] of the sus-pended particles was obtained as the intercept at zeroconcentration. Data were expressed as l/g.

2.7. Coulter laser particle size analysis

Particle size distributions of diluted GMP dispersionswere determined by laser diffraction using a Coulter LS 130(Beckman Instruments, USA). In this apparatus laser lightis scattered by the suspended particles and the generateddiffraction pattern, a composite of the diffraction patternsfor all the particles, is measured. This diffraction pattern istransformed using Fraunhofer theory into a particle sizedistribution comprising 72 classes ranging from 0.3 to900 mm (for further information and a critical assessment ofthis method see Ley, 2005).

This theory assumes the presence of spherical particles,which is valid in this case. The particle distributionis plotted as volume percentage vs size. From thesedata a weighted average surface area of the particles(D3,2), and a weighted average volume (D4,3) can becalculated.

2.8. RP-HPLC

The HMW-LMW ratio of GMP from flour and doughsamples was assessed with reversed phase HPLC (RP-HPLC) using a Waters HPLC with a C8 bonded phasecolumn. GMP (1 g) was extracted twice with 1ml 50%(v/v) 1-propanol containing 0.02% (w/v) dithiothreitol(DTT) followed by extraction with 1ml of solvent A. Thetwo extracts were combined for HPLC analysis. Solvent Acontains 6M urea, 20% (v/v), acetonitrile, 0.1% (v/v) TFAand 0.02% (w/v) DTT. Solvent B contains 5.5M urea, 50%(v/v), 0.1% (v/v) and 0.02% (w/v) DTT. Loading was 10 mlof GMP extract. Column temperature was maintained at50 1C. The column was eluted at a flow rate of 1ml/min,with a multi-step gradient starting at 20% solvent B to80% solvent B. Typical elution times for the HMWglutenins were 30–45min and for LMW glutenins70–85min. HMW/LMW ratios were calculated from theirrespective areas under the RP-HPLC chromatogram. Themethod did not distinguish between LMW-GS and, forexample, g-gliadin.

3. Results

3.1. GMP protein from deletion lines Olympic�Gabo

GMP was extracted from flours from the set ofOlympic�Gabo lines with systematic deletions at theGlu-A, Glu-B and Glu-D loci (Table 1). The yields ofGMP and GMP-gel rheological parameters are shown inTable 2. Specific deletions have a demonstrable effect bothon the concentration of protein in GMP (Protein GMP-gel) and the total quantity of GMP and thus insolubleglutenin proteins (GMP protein) recovered. Fig. 1 showsclearly how the amount of GMP protein is related to thenumber and composition of HMW-GS present. This is inagreement with the results of He et al. (2005) who found aclear relationship between the total amount of HMWsubunits expressed and %UPP. These authors were notable to draw conclusions on effects of specific HMWsubunits but these differences become apparent with thedouble deletions used in this study. HMW-GS 17þ 18have a more pronounced effect on GMP protein quantitythan HMW-GS 5þ 10. On the other hand, the HMW-GS1 combined with HMW-GS 5þ 10 results in more GMPprotein than HMW-GS 1 combined with HMW-GS17þ 18.Since we expect that flour quality differences are both

related to GMP quantity and GMP structure, we studiedthe rheological parameters of the GMP gels.

3.2. GMP-gel rheology

There is a general consensus that HMW-GS play animportant role in determining the glutenin protein networkstructure. Lafiandra et al. (1993) showed the importance ofHMW-GS 5þ 10 wheat flour dough strength. We there-fore expected that differences in HMW-GS composition bereflected in differences in GMP rheological parameters.The GMP-gel stiffness (G0) is an indicator of glutenin

size (Don et al., 2003a,b,c) and was measured for seven ofthe Olympic�Gabo lines. The triple null variant (���)could not be measured rheologically because of the verylow quantity that was recovered from flour. Visually, thegel that was recovered was viscous and slimy. The GMP-gel structural parameters G0 and d for the other seven lineswere plotted (Fig. 2a and b) against the number of HMW-GS present. Fig. 2a shows that the value of G0 increaseswith the quantity of HMW-GS expressed. When the resultsare considered with respect to the number of deletions, itcan be seen that for double deletions, the (��+) curve hasthe highest G0. For single deletions the order for G0 isð� þ þÞ4ðþ � þÞ4ðþ þ �Þ. The G0 for the (�++) is thesame as the (+++) curve, considering the higher proteinconcentration of the (�++) (Table 2). This agrees withfindings of Popineau et al. (1994) that the presence ofHMW-GS ‘5þ 10’ increases gluten and dough elasticity.According to these authors this was not a result of proteincontent, but a result of HMW-GS. We also note that in the

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Table 2

Effect of HMWGS composition on the quantity and rheological characteristics of GMP

Sample GMP (g) Protein in GMP-gel (mg/g) GMP protein (mg) G0 (Pa) d (1)

��� 0.670.1 11.970.6 7.171.5 n.d. n.d.

+�� 0.870.1 11.470.6 9.171.6 2.170.2 25.071.2

�+� 2.670.2 11.570.5 29.973.6 2.470.2 23.470.8

��+ 1.770.2 13.670.6 23.172.9 9.470.5 16.871.2

+�+ 2.870.2 12.570.6 35.074.1 21.071.1 15.870.6

++� 2.270.2 13.670.7 29.973.9 7.170.4 17.770.5

�++ 3.070.2 15.770.8 47.175.7 59.472.2 12.170.5

+++ 3.570.3 14.770.8 50.476.7 54.272.1 12.870.8

GMP was extracted from 1.4 g flour from each of the deletion lines and the control. The table gives the quantity of the wet GMP gel (in g), its protein

content (in mgprotein/g wet gel) and the total amount of GMP protein (in mg) per 1.4 g flour.

0

10

20

30

40

50

60

0 1 2 3 4 5Number of HMW-GS

GM

P p

rote

in(m

g)

/1.

4 g

flo

ur

(g)

(---) (+--)

(-+-)

(--+)

(+-+)

(++-)

(-++)(+++)

Fig. 1. Effect of the number of HMW-GS expressed on the quantity of

GMP. Flour sample from each line was extracted and GMP was isolated

as described in Materials and Methods. The total amount of protein in the

gel (expressed as mg protein/1.4 g flour) is plotted against the number of

HMW-GS. Results from lines expressing HMWGS 5þ 10 are indicated

with the filled symbols; (+,�) refer to the deletions made in the respective

wheat lines. See Table 1 for an explanation.

0

10

20

30

40

50

60

0 1 2 3 4 5Number of HMW-GS

Gel

sti

ffn

esG

' (P

a)

10

15

20

25

0 1 2 3 4 5Number of HMW-GS

Del

ta (

°)

(a)

(b)

Fig. 2. The effect of the number of HMW-GS expressed on the

rheological properties of the GMP gel. GMP gel extracted from flour of

the respective wheat lines was characterized using a Bohlin VOR

rheometer (see Section 2.3). Symbols are as in Fig. 1. (a) Effect on gel

stiffness. From the resulting strain sweep curves the G0 plateau value was

taken. This value is plotted against the number of HMW-GS expressed.

(b) Effect on small deformation elasticity (Delta). Here from the resulting

strain sweep curves Delta is calculated (G00/G’). This value is plotted

against the number of HMW-GS expressed.

C. Don et al. / Journal of Cereal Science 44 (2006) 127–136130

four lines with 2 and 3 HMW-GS, the GMP-gels contain-ing HMW-GS ‘5þ 10’ have higher G0 values than thosecontaining HMW-GS ‘17þ 18’. Also, with more HMW-GS, the phase angle d is lower, indicating a higher gelelasticity. As a structural parameter, the phase angle doesnot depend on protein concentration, as does the gelmodulus G0. Therefore, a lower d can only indicate that theaverage cross-link density in the GMP-gel increases with ahigher number of HMW-GS. For this set of lines, the GMPrheology confirms the relationship between HMW-GScomposition and glutenin structural properties.

3.3. RP-HPLC analyses for GMP from the

Olympic�Gabo lines

We have previously reported that GMP consists ofHMW and LMW glutenins (Don et al., 2005). Nowanalyses of the GS composition of both flour and GMPusing RP-HPLC reveal how GMP composition is affectedby systematic deletions of HMW-GS. Fig. 3 shows HPLCprofiles for the respective wheat lines. The triple nullsample, shows little HMW-GS and the GMP consistsessentially of LMW-GS. Apparently, LMW-GS can alsoform aggregates that are insoluble in SDS. Veraverbeke

et al. (2000a,b) reported that LMW-GS can polymerise toform larger structures although it is not clear whether theirLMW-GS polymers were also insoluble or partially solublein SDS.All other lines show a GS composition that matches the

GS composition of the flour and for all the lines the LMWglutenin pattern is the same. Apparently, the compositionof LMW-GS present in GMP is not affected by thedifferences in HMW-GS composition.The calculated HMW/LMW ratios indicate whether the

presence of HMW-GS affect the quantity of LMW-GSrecovered in GMP. The expression of HMW-GS 5þ 10 is

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Fig. 3. HMW and LMW-GS composition of GMP from deletion lines as

characterized by reversed Phase HPLC. GMP was isolated from flour of

each line and subjected to RP-HPLC analysis as described in Section 2.8.

The positions of the various HMW-GS are indicated as well as the overall

position of LMW-GS. (+++) refers to the allelic compositions as

described in Table 1. From the traces a HMW/LMW ratio is calculated.

The respective H/L values are included.

0

5

10

15

20

25

30

0 1 2 3 4 5Number of HMW-GS

Glu

ten

in p

rote

in (

mg

)

Fig. 4. The effect of number of HMW-GS expressed on the quantity of

HMW and LMW protein recovered in GMP. The absolute amount of

HMW-GS protein and LMW-GS protein was calculated from the H/L

ratios obtained using RP-HPLC and the protein content of GMP. These

values are plotted against the number of HMW-GS expressed. Open

squares: LMW; open circles: HMW; filled symbols refer to lines with

HMW-GS 5þ 10.

C. Don et al. / Journal of Cereal Science 44 (2006) 127–136 131

important for the HMW/LMW ratio, because the (+�+)and (�++) samples have a higher HMW/LMW than the(++�) curve. As expected, the (+++) trace has themost HMW glutenins in the GMP fraction. By combiningdata from Table 2 with the HMW/LMW ratios, thequantity of HMW-GS and LMW-GS (mg) in GMP can becalculated allowing assessment of the extent to whichHMW-GS affects the presence of LMW-GS in GMP. Theresults are shown in Fig. 4. With the triple null line, RP-HPLC only reveals LMW-GS in the GMP. In the sevenlines in which HMW-GS are present, the absolute amountof LMW-GS in the GMP is raised above the triple null line.This is probably due to the formation of cross-linksbetween HMW-GS and LMW-GS as demonstrated byKeck et al. (1995) and Kasarda (1999).

If we assume HMW-GS to have an average molecularmass of 2.5 times the mass of LMW-GS, their molar ratios

can be calculated. From Fig. 4 it is clear that more LMW-GS becomes part of GMP when only 2 HMW-GS areexpressed than when 5 HMW-GS are expressed. This canonly be explained if HMW-GS can also form HMW-GS-HMW-GS cross-links, thereby limiting the possibilities forHMW-GS-LMW-GS cross-linking. Also, in the course ofHMW-GS polymerisation, the number of possibilities forlinking LMW-GS and HMW-GS becomes smaller. Thiscould explain the higher HMW/LMW ratio found in GMPcompared with the HMW/LMW ratio found for therespective flours (Beasley et al., 2002).

3.4. CSLM observations of glutenin particles

The specific HMW-GS in this unique set of deletion linesaffect both GMP quantity, rheology and composition.GMP consists of glutenin particles, which are heteroge-neous in size and which average size differed betweenvarieties (Don et al., 2003a,b). To further analyse the effectof glutenin composition on the glutenin particle, GMPdispersions were examined for particulate structures.Fig. 5a–h shows images obtained with CSLM. No gluteninparticles were found in the triple null line (Fig. 5a)although the process of GMP isolation would concentrateany particles present. Some protein was stainedand possibly represents poorly aggregated LMW-GS,but there was no evidence of particles in the absence ofHMW-GS.Dispersed GMP of the (+��) line displayed some

particulate structures (Fig. 5b). Lines (�+�) and (��+)(Fig. 5c and d, respectively) showed clearer gluteninparticle structures than (+��) (Fig. 5b). The gluteninparticle structures are abundant in lines (�++), (+++),(++�) and (+�+) (Fig. 5e–h, respectively). HMW-GScomposition is clearly important for the formation ofstable glutenin particles.

ARTICLE IN PRESS

Fig. 5. CSLM micrographs of GMP isolated from wheat lines with systematic expressions/deletions of HMW-GS. GMP was isolated from flour obtained

from each deletion line and prepared for CSLM analysis as described in Sections 2.4 and 2.5. The graphs show an image of 120� 100mm, representative

for each sample. (a–h) respective images, (+,�) refer to the deletions made in the respective wheat lines. See Table 1 for an explanation.

C. Don et al. / Journal of Cereal Science 44 (2006) 127–136132

3.5. Glutenin particle size

The average glutenin particle sizes for the different linesare summarised in Table 3. The samples containing HMW-GS 5þ 10 have average particle voluminosity [Z] valuesbetween 0.24 and 0.28 l/g, whereas samples lacking this pairof HMW-GS have lower [Z] values of ca. 0.14 l/g. Thepresence of HMW-GS 5þ 10 is necessary but not sufficientto give a large average particle size; apparently HMW-GS17þ 18 are also required. Fig. 6 shows the size distribu-tions, normalised for peak height. Since the amount ofGMP with the triple null sample (���) was very low, thiswas relatively more contaminated with starch. Small starchgranules are visible at ca. 5.5 mm. Furthermore, nostructures 450 mm were present, indicating the absence ofglutenin particles. In contrast, GMP from the (��+) linecontained particles 4100m. The (�++) and (+++)lines have more large particles in the 10–100m range thanthe (��+) line. This is consistent with their higher [Z]values. These results are also consistent with the higher G0

values for their gels (Fig. 2a).There was a clear contrast between particles from the

(��+) line with 4100 mm sized particles and other doubledeletion lines in which particles are mainly o100 mm. Thesize distribution found with the (��+) line indicated an

important effect of HMW-GS 5þ 10 on the size of theglutenin particle as discussed by Lafiandra et al. (1993) andPopineau et al. (1994). It has to be noted however, thatthese authors interpret a higher amount of unextractablepolymeric protein as an indicator of a larger size of thepolymeric aggregate. This need not necessarily be the case.In our study, the correlation between yield of GMP andGMP particle size is not strong ðR2 ¼ 0:58Þ. Thus the yieldof GMP is controlled by both the amount of gluteninpresent and the size of the glutenin particle.The size of the glutenin particle seems to the related GS

composition: a correlation, R2 ¼ 0:76, was observedbetween the HMW/LMW ratio of GMP (Fig. 3) and theaverage voluminosity of the particles. Such a relation wasalready observed previously by Don et al. (2003a,b), wherea viscometric method was used to measure the average sizeof the glutenin particles. Interestingly, the two verydifferent approaches correlated well: R2 of averagevoluminosity [Z] vs D3,2 or D4,3 are 0.96 and 0.93,respectively (Table 3).

4. Discussion

Graveland et al. (1985) proposed that HMW gluteninpolymers are a key to GMP formation. In this study we

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Table 3

Particle voluminosity data of glutenin particles determined with visco-

metry and Coulter laser granulometry

Sample [Z] (L/g) D3,2 (mm) D4,3 (mm)

��� 0.10 4.3 8.3

+�+ 0.24 5.8 13.7

�+� 0.11 4.5 8.5

++� 0.15 5.0 10.9

��+ 0.16 5.1 10.2

+�� 0.13 4.9 9.3

+++ 0.28 7.2 25.6

�++ 0.27 6.6 21.2

For each line, GMP was isolated, dispersed in SDS and its glutenin

particles analyzed as described in Section 2.7. [Z] is taken as a measure of

the average voluminosity of the particles, D(3,2) is a measure of the

weighted average surface area and D(4,3) is a measure of the weighted

average volume of the particles.

Size (µm)

0.1 1 10 100 1000

0.1 1 10 100 1000

0.1 1 10 100 1000

0.1 1 10 100 1000

0.1 1 10 100 1000

0.1 1 10 100 1000

0.1 1 10 100 1000

0.1 1 10 100 1000

0

1

No

rmal

ized

for

pea

k

---

+--

-+-

--+

++-

-+++

+-+

+++

Fig. 6. Effect of HMW-GS composition on the size distribution patterns

of glutenin particles extracted from flour. GMP was isolated, dispersed

and analysed using granulometry as described in Section 2. Representative

volume distribution graphs (%vol vs size (mm)) are shown for each of the

deletion lines studied. (+,�) refer to the deletions made in the respective

wheat lines. See Table 1 for an explanation. The dotted line at 100mmposition is added to allow comparisons of the different volume

distributions.

C. Don et al. / Journal of Cereal Science 44 (2006) 127–136 133

investigated to what extent HMW-GS are indeed requiredto form larger aggregates. Although not conclusive,evidence from CSLM and laser light scattering is presentedindicating that the triple null line, lacking HMW-GS, doesnot appear to contain glutenin particles that are sufficientlystable in SDS. The low amount of GMP harvested with thisline contained only LMW-GS. Veraverbeke et al. (2000a,b)have already shown that LMW-GS can form polymericaggregates. Our findings confirm this. We propose thatdepending on the concentration and provided that HMW-GS are absent, LMW-GS aggregates are soluble in SDS.We consider such aggregates to be the result of the physicalaggregation of oligomers. By definition, physical networksdissolve upon dilution. This feature was previouslyobserved with GMP from the wheat variety Caprimus(HMW-GS 6þ 8, 2þ 12) and the double deletion lineGalahad-7 (HMW-GS 7) (Don et al., 2003a).

The observations reported in this study, as well as inearlier studies, indicate that cross-linking between HMW-GS and LMW-GS is required to form glutenin particlesthat remain insoluble in SDS. This could also explain whyin this study (Fig. 1) and those of others (Beasley et al.,2002; Uthayakumaran et al., 2002) the quantity of GMP or%UPP is reported to be more sensitive to the number ortype of HMW-GS than to flour protein content. This is inagreement with the established differences in the ability ofHMW-GS to form cross-links with other HMW-GS orLMW-GS (Shewry et al., 2003). In the presence of HMW-GS, particles are formed that consist predominantly ofHMW-GS and LMW-GS. We did not specifically analysefor the presence of gamma gliadins, that have a free SH-group allowing their cross-linking to glutenin structures,but RP-HPLC analysis of GMP shows mainly HMW andLMW-GS. Fig. 3 demonstrates that the content of LMW-GS in GMP increases when the number of HMW-GSis increased. This clearly indicates that the presence ofLMW-GS in glutenin particles is related to the presence ofHMW-GS.

Fig. 3 provides another interesting observation. RP-HPLC analysis of GMP shows a large variation in HMW-GS, as expected. However, the LMW-GS composition ofGMP is constant. We have provided arguments that this isnot due to a mechanism in which LMW-GS polymeriseand become part of GMP independent of HMW-GS. Morelikely, oligomers of LMW-GS cross-link to HMW-GS andthus become part of GMP. The presence of such LMW-GSoligomers was postulated by Graveland et al. (1985)and has also been demonstrated using partial reductionstudies (Hamer et al., 1995). Our results are in agreementwith the Graveland model in which glutenin ‘polymers’consist of a backbone of HMW-GS linked by disulphidebonds to which oligomers of LMW-GS are attached

ARTICLE IN PRESSC. Don et al. / Journal of Cereal Science 44 (2006) 127–136134

(Graveland et al., 1985; Keck et al., 1995; Shewry et al.,1989; Weegels et al., 1996).

Our study also provides information on the size of theglutenin particles formed, as demonstrated from CSLM,viscometric and granulometric techniques. CSLM (Fig. 5)clearly demonstrates a polydisperse collection of particles.The size distributions in Fig. 6 confirm this. We considerthese sizes to reflect the status of the particles in a swollenstate. In this respect, they are both related to the originalsize of the particles when they are formed in the wheatkernel and their ability to swell. The latter is again relatedto the degree of cross-linking (or cross-link density) andhence the composition of the glutenin particles. This couldhelp explain the observed correlation between HMW/LMW ratio and aggregate size (Table 3). In this respect thepresence of HMW-GS 5þ 10 leads to a lowering of thephase angle, delta, of GMP (Fig. 2b). The lower deltaindicates a higher cross-link density of the GMP-gel.Southan and MacRitchie (1999) pointed out that theHMW/LMW ratio affects the size of glutenins, but againwe note that these authors refer to the quantity of gluteninpolymers extracted after sonication (%UPP). Interestingly,our observations on particles are supported by a number ofstudies on solubilised aggregates (e.g. via ultrasonication).For example, Ueno et al. (2002) have shown using free flowfractionation that acetic acid insoluble fractions containglutenin aggregates with a larger Stokes diameter than theacetic acid soluble glutenins.

In addition to the number of HMW-GS, also thepresence of specific HMW-GS can play a role in determin-ing the quantity and size of glutenin networks. Southanand MacRitchie (1999) report that the introduction ofHMW-GS ‘5þ 10’ and HMW-GS ‘17þ 18’ is stronglyassociated with an increase of insoluble glutenin size(%UPP). The replacement of HMW-GS ‘2þ 12’ withHMW-GS ‘5þ 10’ is reported by many authors to increasedough strength (for a review, see Shewry et al., 2003).According to Lefebvre et al. (2000) this is related to anincrease in the size of glutenin aggregates. Veraverbeke etal. (2000a,b) discussed the role of certain x-type HMW-GSin the formation of larger glutenin aggregates. Morerecently, Don et al. (2005) reported that under non-stressedgrowing conditions, GMP isolated from the Warigal andLance lines with HMW-GS ‘5þ 10’ contained largerglutenin particles than Warigal and Lance lines whichwere genetically identical but expressed HMW-GS ‘2þ 12’rather than ‘5þ 10’.

We conclude that, in addition to quantity, the presenceof (specific) HMW-GS helps control the HMW/LMWratio which in turn controls the apparent size of theglutenin particles isolated from wheat flour. Nevertheless,LMW-GS may also have a specific effect on gluteninaggregation (DuPont and Altenbach, 2003; D’Ovidio andMasci, 2004; Veraverbeke et al., 2000a,b), but an investiga-tion of this was outside the scope of this study. Thedifferences here are related to HMW-GS only. Our results,combined with evidence from previous reports, strengthen

our view that HMW-GS control the physical properties ofthe glutenin particles. We therefore postulate that:

�

HMW-GS are required for glutenin particle formation. � The size of the glutenin particles is affected by the

HMW/LMW-GS ratio.

� HMW-GS ‘5þ 10’ shifts the size distribution pattern

towards larger sizes.

� HMW-GS affect the internal structure of the glutenin

particles (Fig. 2b).

Since the glutenin network also plays an important roletechnologically, we expect to find correlations betweenglutenin particle properties and technological properties.Beasley et al. (2002) and Uthayakumaran et al. (2002)analysed the technological properties of the same set ofmaterials as studied here. This allowed us to test ourhypothesis.

4.1. Parallels between GMP, %UPP and dough

Uthayakumaran et al. (2002) showed that doughrheological strength correlates with %UPP. Don et al.(2003a,b) have demonstrated a clear relationship betweenGMP and dough rheological properties. Here we show thatthe amount of GMP protein parallels %UPP (Fig. 7a). The%UPP is a measure of the glutenins insoluble inpropanolþ SDS. Since %UPP and GMP are clearlycorrelated ðR2 ¼ 0:91Þ and since both relate to insolubleglutenin protein, we suggest that the UPP fraction alsorepresents glutenin particles. The quantity of insolubleglutenin particles or aggregates has been shown to correlatewith flour end-use quality. Mecham et al. (1965) havepreviously suggested that large gluten aggregates, insolublein acetic acid, strongly affect gluten dough properties.Beasley et al. (2002) determined dough development

times (DDT) for the same set of lines used in this studyusing a Z-blade mixer. The amount of GMP extractedcorrelated to some extent with these DDT valuesðR2 ¼ 0:67Þ. However, the glutenin particle sizes of thelines correlated strongly with DDT (R2 ¼ 0:95, Fig. 7b), inagreement with Don et al. (2003a,b,c). In this way, HMW-GS composition is correlated to a technological parameter,dough development time, through the effect on gluteninparticle size. Again, we cannot comment on the possiblerole of LMW-GS here, since this is outside the scope of thispaper.Beasley et al. (2002) also studied the effect of HMW-GS

on loaf volume. Miniature (thimble) loaves were preparedwith flour from the set of Olympic�Gabo lines. We foundthat for the same set of lines, GMP wet weight showed thebest correlation with loaf height (R2 ¼ 0:92 (Fig. 7c)). Aftermixing and during dough rest, disrupted glutenin particlesre-assemble (Don et al., 2005; Weegels et al., 1996). As aconsequence, the dough becomes more elastic withincreasing dough rest time (Don et al., 2005). Differencesin the amount and properties of the glutenin network after

ARTICLE IN PRESS

R2 = 0.95

0

50

100

150

200

250

0.05 0.10 0.15 0.20 0.25 0.30particle voluminosity [η], L/g

DD

T (

Z-b

lad

e), s

0

10

20

30

40

50

60

0 10 20 30 40 50 60GMP protein quantity (mg GMP

protein / 1.4g flour)

% U

PP

(+++)

(-++)(+-+)

(++-)(-+-)

(--+)(+--)

(---)

(A)

(B)

R2 = 0.92

25

30

35

0 1 2 3 4GMP wet weight (g)

Lo

af H

eig

ht

(mm

)

(C)

Fig. 7. Comparison of biochemical and technological characteristics of

the Olymbic�Gabo deletion lines. (A) %UPP data reported for the same

set of Olympic�Gabo deletion lines (Uthayakumaran et al., 2002), are

plotted against GMP values reported in this study ðR2 ¼ 0:92Þ. (B) Beasleyet al. (2002) reported dough development times (DDT) for each of the

deletion lines. These values are plotted here against the particle

voluminosity value [Z] as reported in this study. (C) Thimble-loaf heights

(Beasley et al. 2002) also correlated with GMP wet weight values reported

in this study. (+,�) refer to the deletions made in the respective wheat

lines. See Table 1 for an explanation.

R2 = 0.91

0

1

2

3

4

0 1 2 3 4 5HMW (mg) / D3,2 (µm)

GM

P w

et w

eig

ht

(g)

Fig. 8. Relation between the amount of HMWGS expressed and size of

the glutenin particle (D3,2) and GMP wet weight. The ratio of the amount

of HMWGS expressed (mg) vs the weighted average surface area (D3,2

(mm)) is plotted against the wet weight of GMP (g) recovered from 1.4 g of

flour.

C. Don et al. / Journal of Cereal Science 44 (2006) 127–136 135

resting will be reflected in differences in loaf height. Thenumber and strength of interactions determine the proper-ties of the network. Both the size and internal structure ofglutenin particles could play a role here. In Fig. 8 GMP wetweight is plotted against the ratio of the amount of HMW-GS (in mg) and the weighted average surface area, D3,2.This ratio represents the average HMW-GS density of the

glutenin particles and was found to correlate with the wetweight of GMP ðR2 ¼ 0:91Þ (with %UPP ðR2 ¼ 0:90Þ).Again, it is not known how this relationship is affectedwhen the composition of LMW-GS is also varied.Although not as straightforward as with DDT, this againlinks a technological property (loaf volume) to theproperties of glutenin particles, this time through anintermediate parameter, the quantity of GMP.In summary, the formation of glutenin particles, stable

in SDS-solution, requires the presence of HMW-GS.Within the set of samples used in this study, HMW-GSfrom either Glu-1B 17, 18 or Glu-1D 5, 10 proved to beessential. The amount and presence of specific HMW-GSalso affected glutenin particle properties, such as cross-linkdensity and size. Our results are in agreement with a modelin which HMW-GS form the ‘‘backbone’’ structures towhich polymers of LMW-GS become attached. Theanalysis of our glutenin particle results together with thetechnological data of Beasley et al. (2002) suggests thatHMW-GS affect flour technological quality throughglutenin particles. Further information on the internalglutenin particle structure will improve our understandingof glutenin quantity/glutenin particle structure relation-ships in dough mixing and bread-making. Moreover, thecombination of glutenin particle analysis and the use ofnear-isogenic wheat lines opens new possibilities forunderstanding factors determining glutenin structure,aggregate size and their relation with flour end-use quality.

Acknowledgements

The assistance of J. Klok in performing the CSLMexperiments and Anne v/d Pijpekamp for her assistancewith the RP-HPLC analyses is gratefully acknowledged.We also like to thank J. Plijter and T. van Vliet for theircritical comments on the manuscript.

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