Contributions of protein fractions to dough handling properties of wheat-rye translocation cultivars

Journal of Cereal Science 12 (1990) 113-122

Contributions of Protein Fractions to Dough Handling Properties of Wheat-Rye Translocation Cultivars A. S. DHALIWAL and F. MACRITCHIE

CSIRO Division of Plant Industry, Wheat Research Unit, P.O. Box 7, North Ryde, N.S. W. 2113, Australia Received 3 July 1989

Fractionation and reconstitution studies have been undertaken to assess the contribution of various flour components to dough stickiness and mixing properties, comparing rye translocation lines with their recurrent parents. For one line, approximately one third the stickiness originated in the water-soluble fraction and about two thirds in the gluten and, for another line, contributions from these two fractions each accounted for about half the stickiness. Reduction in the amount of water solubles below their natural level in the reconstituted flours improved Mixogram properties and decreased stickiness score. Similarly, dough stickiness was reduced by addition of flour protein fractions concentrated in glutenin proteins. When gluten was separated into two roughly equal fractions by differential solubility in dilute HCl, interchange experiments established that the difference in their effects on stickiness could be almost wholly attributed to the soluble portion. SE-HPLC revealed that the acid-soluble fraction from the translocation line SUN 89D had a lower proportion of glutenin than that of its parent Cook, whereas the HPLC profiles of the two acid-insoluble fractions were similar. SDS-PAGE analysis of protein fractions indicated that water-soluble and gluten acid-soluble fractions of translocation lines contained most of the rye secalins. The interchange experiments suggest that the weak and sticky dough properties of the translocation lines arise from a shift in the balance of the proportions of polymeric and monomeric proteins. This was supported by experiments in which this balance was varied by addition of specific protein fractions.

Introduction

The replacement of the IBs wheat chromosome by the IRs rye chromosome offers potential agronomic advantages and resistance to stem, leaf and stripe rustl . Wheatbreeding programmes to exploit IB/IR translocation cultivars for commercial use have been curtailed because of an inherent quality problem associated with reduced dough strength and dough stickiness. 2-6. Increased water absorption of the flours of the translocation wheats and higher content of soluble pentosans have been suggested to be involved in the development of sticky dough 2 • However, Dhaliwal et al. 6 and Martin and Stewart 4 did not find any definite correlation of pentosans with stickiness. The present study describes fractionation, reconstitution and interchange experiments aimed at identifying the components causing the weak and sticky dough properties of the translocation lines. Establishment of this may make it possible to put breeding strategies into place that 0733-5210/90/050113 + 10 $03.00/0

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could eliminate or reduce the defect while retaining the inherent advantages of these lines.

Experimental Two lB/IR translocation wheat cultivars (SUN 89D and M3345) and their recurrent" parents, Cook and Oxley, were used in the present study. The pedigrees of these translocation lines were described by Dhaliwal et at." All the lines were sown at the I. A. Watson Wheat Research Centre, Narrabri, N.S.W., Australia, during early June 1987 and harvested during early December 1987. Sunbird, another IBjlR CIMMYT line was included in the electrophoretic study. Grain was conditioned overnight to 15% moisture content and milled on a Quadrumat Senior Mill.

Preparation offractions The method used by MacRitchie 7 was employed for the preparation of various fractions except that flours were not defatted. Gluten was prepared by hand kneading doughs in distilled water at 10 dc. A standard procedure was adopted using seven washes, the first two with a ratio of 2: 1 waterjinitial flour weight, the second two with a ratio of I : I and the final three with a ratio of 1:2. Water washings were combined and then centrifuged to separate starch and solubles. The three fractions from each flour were freeze dried. Gliadins were initially obtained by extraction of glutens with 70 % ethanol. However, dough development times of reconstituted flours were considerably greater than those of the original flours as reported previouslyB.9. Therefore, dilute HCI was used for preparation of protein fractions 7. Gluten was extracted with a range of concentrations of dilute HCl (0'5 to 3 mM) Calibration curves of absorbance as a function of acid concentration were obtained, ensuring effects of turbidity were minimized by using dilutions to keep the absorbances between 0'1 and 0·2. Using these calibration curves, acid concentrations were chosen so as to extract approximately 50 % 'of the protein from each gluten to produce two fractions: gluten soluble and gluten insoluble. Gluten samples containing 30'0 g protein were stirred with 900 ml of dilute HCI (2'9 mM for Oxley and 2·2 mM for M3345, 2·0 roM for Cook and 2·3 mM for SUN 89D) in the Ultraturrax mixer for 2 min at the highest speed followed by centrifugation at 5000 g for 10 min. Supernatants and sediments were separated and brought to pH 5·8 by 0·1 MNaOH solution. Fractions were freeze dried, weighed, ground and protein (Kjeldahl) and moisture determinations made. For duplicate experiments, errors in weights of fractions were all less than 2·2 % of the means.

Measurement of dough stickiness In preliminary work, several approaches to measurement of dough stickiness were assessed, including the Digital Gram Gauge and the Universal Instron Tester. However, when a dough that exhibits stickiness is pulled by such instruments, the force measured is a composite of an adhesive (surface) force and a bulk force required to stretch the dough piece. The total force thus depends on the bulk rheological properties of the dough and it is usually not possible to separate the adhesive force. As a result, it was decided to assess stickiness by five subjective tests based on the tendency of the dough to adhere to different surfaces 10. The dough was first mixed to peak consistency on a 10 g Mixograph using a water absorption level of 60 % (13 % m.b.). Each dough was scored from o (no stickiness) to 4 (extreme stickiness) for each of the five tests, thus leading to a possible score of20 for a dough of maximum stickiness. The score of 0-5 represents not sticky; 6-10, marginally sticky; 11-15, sticky and 16-20, very sticky. The five tests and the scoring system were as follows. Test 1. Observe dough in bowl after mixing; 0, pulls out cleanly, no tendency to stick; 1, pulls out cleanly, a slight tendency to stick; 2, can be pulled out in one piece but requires time; 3, sticks to bowl, cannot be removed in one piece; 4, as for 3 but stickiness excessive.

PROTEINS AND DOUGH STICKINESS OF TRANSLOCATION LINES

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Test 2. Pick up dough piece and swap from one hand to other. 0, no stickiness; I, slightly sticky but comes off fingers; 2, sticky, leaves traces of dough on fingers; 3, sticky, leaves several points on fingers; 4, very sticky, covers fingers: Test 3. Squeeze dough between thumb and forefinger, then separate. 0, dough remains on one, leaves dent; 1, dough momentarily sticks to both but one releases; 2, remains stuck to both for approximately 3 cm; 3, remains stuck to both for approximately 10 cm and then comes free; 4, remains stuck to both, cannot be separated. Test 4. Place dough on bench, roll with clean Teflon rod. 0, rolls without sticking; I, rolls but visibly sticks transiently; 2, small amounts stick but pull free; 3, sticks as rolls, fragments remain stuck; 4, rod becomes embedded in centre of dough; Test 5. Place dough on paper towel, leave for 2 min, remove. 0, no sticking; I, trace stuck; 2, several points have stuck leaving debris; 3, thin covering where dough has stuck; 4, patch of dough diameter is covered thickly. In replicated blind trials, differences in stickiness score of the same samples were usually never greater than 2.

Electrophoretic characterization of proteins of different fractions Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed on proteins extracted from different fractions with a solution of 2% (w/v) SDS, 16% (v/v) N, Ndimethyl fonnamide and 62 mM Tris-HCI, pH 6·8. They were fractionated by discontinuous buffer system, SDS-PAGE on a 1·5 mm thick, 15 % T (T = acry1amide+ bisacrylamide) separating gel, as described by Skerritt et aZ,u for 2000 Vh. Gels were stained 20 h in 0·2 % (w/v) Coomassie blue R-250 in methanol, acetic acid and water (40: 10: 50) and destained 6 h in the same solvent minus dye.

SE-HPLC Total unreduced proteins for flour and freeze dried fractions were extracted with a sonic probe (A Branson B-12 model sonifier) in a solution of 2% (wIv) SDS in 50 mM Na phosphate buffer, pH 6.9 12 • The samples were extracted for 30 sec at a setting of 5. The sonicated samples were centrifuged at 15000 rev/min for 20 min. the clear supernatants were used for SE·HPLC. The supernatants were diluted with extracting buffer to give a final protein concentration of I mg/ml and filtered through 0-45 11m PVDF filters. An aliquot (20 Ill) of the sample solution was injected automatically for fractionation on a Protein Pak 300 column. The eluting solution was 0·1 % SDS, 0·05 MNa phosphate, pH 6,9, the flow rate 0·5 ml/min and the run time 40 min/sample. Detection was at 210 nm. The proportions of protein in each of the three major peaks were measured by integration of the areas. These three peaks in order of elution time have been shown to correspond closely to glutenins (with some high molecular weight albumins), gliadins and albumins/globulins 12 • Minimum absorbances between the peaks were used as cut-off points. From duplicate measurements, errors were less than 2 % of means.

Results and Discussion Interchange offlour fractions The fractions such as solubles, starch, acid soluble and insoluble gluten obtained according to the procedure used by MacRitchie 13 preserved the functional properties throughout the fractionation and reconstitution steps as judged on the basis of Mixograph parameters. The stickiness scores for reconstituted samples were higher for the two translocation lines and one parent than the original flours. The reason for this is not clear although some variability is expected for a subjective test of this nature.

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TABLE I. Composition of fractions (g dry weight) from a 100 g flour sample (dry weight)

Flour protein Gluten Total Protein Water-solubles Total Protein Starch Total Protein

Cook

SUN 89D

13-8

14·9

13-9

14'7

16·2 11·8

Hj·O 12'3

15·4 Jl·5

15-4 12·0

5·2

1-1

5·0 1·4

4·8 1·2

5·0 1'7

75·9 0·6

75'2 0'6

76·7 0·7

77·0 0'7

Oxley M3345

TABLE II. Dough stickiness scores for combinations of Cook (C)-SUN 89D (D) and Oxley (0)-M3345 (M) flour fractions Stickiness score Cook (C) SUN 89D (D) Cook-SUN 89D Gluten Solubles

Original flours Oxley (0) 3 11 M3345 (M) Reconstituted flours Oxley-M3345 Gluten Solubles

Starch

C C C C D C C D D D D C D C D D Composite scores Cook gluten SUN 89D gluten Cook solubles SUN 89D solubles Cook starch SUN 89D starch

C D D C C C D D

Stickiness score

6 6 11 II 16 11 13 18 34 58 36 56 44 48

0 0 0 0

M M M M

0

0

M M M 0

0

M

Oxley gluten M3345 gluten Oxley solubles M3345 solubles Oxley starch M3345 starch

5 16

Starch 0 M M 0 0 0 M M

5 7 13 11 19 15 17 20 36 71 44 63 50 57

However, the relative stickiness scores for each translocation line and parent were preserved and it is considered that conclusions drawn from interchange experiments are valid on this basis. The amounts of the three fractions obtained from each of the two translocation lines

PROTEINS AND DOUGH STICKINESS OF TRANSLOCATION LINES

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TABLE III. Quantities of freeze dried fractions obtained from 350 g flour samples of Cook and SUN 89D after gluten washing and fractional extraction of gluten

Cook

Weight (g) % of flour % protein

SUN 89D

Gluten

Starch and solubles

Gluten

Starch and solubles

54·4 17'7 71-6

251·9 82·2 2·1

52·4 17-0 72-7

255'9 83'0 2'7

Soluble Insoluble Weight (g) % compo % protein

19'8 40·3 89·8

29·3 59-6 59·9

Soluble Insoluble 19'9 40'3 91'4

29'4 59·7 63'0

and their recurrent parents are summarised in Table 1. The protein content was greater in the water-soluble fractions of SUN 89D and M3345 than in those of their recurrent parents. This increase was attributed to the presence of rye secatins, which are more water soluble than their wheat counterparts 6 • The dough stickiness scores of the reconstituted doughs are recorded in Table II. Interchange of fractions between the two IB/IR lines and their recurrent parents showed that both gluten and water solubles contributed to stickiness. In general, Mixograph parameters (not shown) paralleled the stickiness scores. The rate of breakdown after peak is regarded as a measure of dough strength and susceptibility to stickiness. The presence of glutens from the translocation lines caused the greatest decrease in width and height of the Mixogram trace. Peak development time was also reduced by gluten from SUN 89D, although not by M3345. Composite scores for each fraction were obtained by adding the total scores for each dough in which the given fraction was included (Table II). For the M3345-0xley pair, the contribution to stickiness of the gluten fraction was roughly double that of the water soluble fraction in agreement with previous results 10 , whereas for the SUN 89D-Cook pair, the effects of these two fractions were roughly equal. There did not appear to be a significant effect on stickiness following interchange of starch fractions between translocation lines and recurrent parents. Interchange of protein fractions

In further studies involving interchange of protein fractions, SUN 89D (lBjlR line) was compared with its recurrent parent Cook. Details of the gluten fractions prepared from each flour are recorded in Table III, and the stickiness measurements following reconstitution of the fractions are summarized in Table IV. On the basis of SDS-PAGE (Fig. 1) and SE-HPLC (Fig. 2), the soluble fraction contained mainly gliadins and the

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TABLE IV. Stickiness scores for combinations of Cook (C) and SUN 89D (D) fractions (GSgluten soluble, GI-gluten insoluble, R-solubles and starch) Combination OS C D D C

C D D C

GI C

D C D C C D D

R

C

D C C

D D C D

Stickiness 4 12 13

7 7 12 14 11

Composite scores for stickiness were: OS (Cook) 29 and OS (SUN 89D) 51; GI (Cook) 36 and GI (SUN 89D) 44; R (Cook) 38 and R (SUN 89D) 42.

L LMW glutenin

'B

"'-oUodin{ 10 ~

...-Mtolln IR Q,

fJ.

Y{lB. ~

Ollodins

Albumins

and

globulins

FIGURE 1. SDS/PAGE patterns of unreduced proteins from flours and different fractions of the cultivar Cook and two IB/IR lines, Sun 89D and Sunbird. Left to right in each set of three tracks are Cook, Sun 89D and Sunbird; a, band c, flour; d, e and f, gluten; g, hand i, gluten soluble fractions; j, k and I, gluten insoluble fractions; ro, nand 0, water soluble fractions.

insoluble mainly glutenins. This was shown by the greater intensities of bands corresponding to r:J., and ~-gliadins in the soluble fractions and the greater streaking due to glutenins in the insoluble fractions of the gels (Fig. 1). Similarly, the SE-HPLC profiles (Fig. 2) showed relatively larger second peaks (gliadins) for the soluble fractions and larger first peaks (glutenins) for the insoluble fractions. The proportions are quantified in Table V. Fig. 1 also illustrates the absence of IB w-gliadins in the flours and fractions of the translocation lines. 1R w-secalins were seen to be present in the flours, concentrated in the water solubles and present in the gluten solubles fractions but not

PROTEINS AND DOUGH STICKINESS OF TRANSLOCATION LINES

119

~ c I-...I--..I.'-L_--L--===.......... _LL.._-L-=:o::!:==l

N
5

20

5 Elution time (min)

FIGURE 2. SE-HPLC patterns for protein from the parent flours and different fractions of the cultivar Cook (left) and its lBjlR derivative, Sun 89D (right). Top row, flours; 2nd row, gluten soluble fractions; 3rd row, gluten insoluble fractions; bottom row, water soluble fractions. TABLE V. Distribution of protein in different regions of SE-HPLC chromatograms for flours and fractions of Cook and SUN 89D SUN 89D

Cook

Flour Gluten soluble Gluten insoluble Water soluble

Peak I

Peak 2

Peak 3

Peak 1

Peak 2

Peak 3

34·8 29·9 53·5

46·3 52-8 25·0 46·4

18·9 17-4 21'5 53·7

25·9 16·3 54·5

54'5 62·2 25·5 54·0

19·7 21·5 19'9 46'0

The values presented are the area beneath each peak expressed as a proportion (%) of the total area beneath the chromatogram.

120

A. S. DHALIWAL AND F.

MAC RITCHIE

FIGURE 3. Mixograms showing the effects of reduction in the water soluble fraction of flours. Traces on the left are for the normal flours. Traces on right are for reconstituted flours with a reduction in their water solubles content of 25 %. Stickiness scores (S) are also recorded. in the gluten insolubles fractions of these lines. It is apparent from the composite scores in Table IV that the difference in stickiness between SUN 89D and Cook was caused mainly by a difference in gluten-soluble fractions. This can be related to the protein composition of the fractions, which is best illustrated in Table V. Whereas the insoluble gluten fractions were similar in composition, the soluble fraction of SUN 89D had a much lower ratio of polymeric (glutenin) proteins to monomeric (gJiadins, albumins and globulins). The changes in the balance of polymeric to monOmeric proteins therefore appears to account for the different effects of the soluble fractions. The effects on Mixograms and stickiness of varying the amounts of these fractions (solubles, gluten soluble and insoluble) were studied further. The Mixogram properties improved and stickiness score decreased following 25 % decrease of water solubles in the reconstituted doughs of both translocation lines and their parents (Fig. 3). The effect was more pronounced in SUN 89D than M3345. There was an increase in the stickiness scores of Cook and Oxley with an increase in their water solubles by 15 %. These results are summarized in Table VI.

PROTEINS AND DOUGH STICKINESS OF TRANSLOCATION LINES

121

TABLE VI. Effects on dough stickiness of variations in the amounts of water solubles in flours of translocation lines and recurrent parents

Per cent of water solubles fraction

Variety Cook SUN 89Da Oxley M3345 a

a

115 100 75 100 75 115 100 75 100 75

Per cent protein in reconstituted flour

Stickiness score

12·1 11·9 11-7 12,8 12'5 12'0 11·9 11·7 12·7 12·3

9 5 0 18 6 9 6 6 19 11

(Original flour) (Original flour) (Original flour) (Original flour)

Doughs too sticky to measure at 115 % level of water solubles.

Table VII. Effects on dough stickiness of addition of ghlten protein fractions to flours at a level of 1 g/lOO g of flour. OS is gluten soluble fraction and OJ is gluten insoluble fraction. Stickiness score Cook flour + GI (SUN 89D) + OS (SUN 89D) SUN 89D flour + GI (SUN 89D) + OS (SUN 89D) +GI (Cook) +OS (Cook) SUN 89D flour + Cook fn 1 + Cook fn 3 + Cook fn 4 + Cook fn 7 +Cook fn 10

5 3 6 10 7 13 7 11 10 16 13 4 5 4

Per cent glutenin in flour 34·8

25·9

25·9

Per cent glutenin in fraction 54·5 16·3 54·5 16·3 53·5 29'9 35·7 36'9 52·1 66·5 72-5

Additions of protein fractions

The effects on dough stickiness of varying the protein composition by addition of gluten protein fractions was also studied and the results are summarized in Table VII. Two sets of fractions were used and added to flours at a level of I g of protein/ I00 g of flour. The first set comprised the gluten soluble and insoluble fractions described above. Their

122

A. S. DHALIWAL AND F. MAcRITCHIE

glutenin contents were taken from the SE-HPLC data of Table V. The effects of these fractions were as expected from the interchange experiments of Table IV. The gluten solubles fraction of SUN 89D caused the largest increase in stickiness score, while the gluten insolubles fractions of Cook and Sun 89D decreased stickiness score by the same amounts. The second set comprized fractions from Cook, which were prepared by fractional extraction of gluten with dilute HCI as described previously7. The proportions of glutenin in these fractions were determined by densitometry of SDS-PAGE patterns under reducing and non-reducing conditions 14 • In this case, early extracted fractions, having relatively low proportions of glutenin, increased the stickiness score above that of the control, whereas later extracted fractions of high glutenin content decreased stickiness markedly.

Conclusions Dough stickiness may arise from a number of different causes. However, the particular stickiness associated with rye translocation lines appears to arise from a shift in balance of the protein composition. This is caused by two factors. First, there is a decrease in the strength-contributing glutenin as a result of the loss of the LMW glutenin subunits encoded by the IB chromosome. This is confirmed by the HPLC profiles, the lower proportions of glutenin in both the original SUN 89D flour and its gluten-soluble fraction are consistent with the observed reduced dough strength and increased stickiness compared with corresponding samples of Cook. Second, there is a gain of monomeric secalins introduced by the IR chromosome short arm. This shows up in the increased stickiness of the water soluble fractions from the translocation lines.

References 1. Mettin, D., Bluthner, W. D. and Schlegal, G. in 'Prec. Int. Wheat Genet. Symp. 4th', Mo Agric Stn, Columbia MO. (1973). 2. Zeller, F. J., Gunzel, G., Fischbeck, G., Gerstenkoen, P. and Weipert, D. Getreide Mehl Brot. 36, (1982) 141-143. 3. Moonen, J. H. E. and Zeven, A. C. Euphytica 33 (1984) 3-8. 4. Martin, D. J. and Stewart, B. G. Euphytica 35 (1986) 225-232 5. Dhaliwal, A. S., Mares, D. J., and Marshall, D. R. Cereal Chern. 64 (1987) 72-76. 6. Dhaliwal, A. S., Mares, D. J., Marshall, D. R. and Skerritt, J. H. Cereal Chern. 65 (1988) 143-149. 7. MacRitchie, F. J. Cereal Sci. 6 (1987) 259-268. 8. Hoseney, R. C., Finney, K. F., Pomeranz, Y. and Shogren, M. D. Cereal Chern. 46 (1969) 606-613. 9. MacRitchie, F. and Gras, P. W. Cereal Chern. 50 (1973) 292-302. 10. MacRitchie, F., Skerritt, J. H., Wrigley, C. W., Campbell, W. P. and Dhaliwal, A. S. in Proc. Royal Australian Chemical Institute 36th Cereal Chemistry Conference, Adelaide (1986) pp 78-82. 11. Skerritt, J. H., Smith, R., Wrigley, C. W. and Underwood, P. A. J. Cereal Sci. 2 (1984) 215-224. 12. Singh, N. K., Donovan, G. R., Batey, I. L. and MacRitchie, F. Cereal Chern. 67 (1990) 150. 13. MacRitchie, F. J. Cereal Sci. 3 (1985) 221-230. 14. MacRitchie, F., Kasarda, D. D. and Kuzmicky, D. D. (unpublished results).