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Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale
Journal of Cereal Science 34 (2001) 19–27 doi:10.1006/jcrs.2000.0385, available online at http://www.idealibrary.com on
Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale H. Wieser and R. Kieffer Deutsche Forschungsanstalt fu¨r Lebensmittelchemie and Kurt-Hess-Institut fu¨r Mehl- und Eiweißforschung, Lichtenbergstraße 4, D-85748 Garching, Germany Received 13 May 1999
ABSTRACT Flour samples of 14 wheat cultivars previously characterised by rheological measurements and by baking tests on a micro-scale (Kieffer et al.: Journal of Cereal Science 27 (1998) 53–60) were analysed for the relative amounts of gluten protein types using a combined extraction/HPLC procedure. Regression analysis was used to find relations between wheat properties and protein quantities. The results indicated that the maximum resistance of dough and gluten and the gluten index were strongly dependent on the quantity of glutenin subunits (GS) in flour; additionally they were influenced by the ratio of gliadin to glutenin subunits. Within the family of glutenin proteins, the correlation coefficients for high-molecular-weight (HMW) and low-molecular-weight (LMW) GS were in a similar range, but twice the amount of LMW GS was necessary to get the same resistance as with HMW GS. Among HMW GS, the contribution of x-type GS was more important than those of y-type GS. The extensibility of dough and gluten was mainly dependent on the ratio of gliadin to total glutenin subunits, to HMW GS and LMW GS. Dough development time showed the highest correlation with total HMW GS and x-type HMW GS. Bread volume was influenced by the total amount of gluten protein more than by the amount of protein in different groups or of different types, probably because of the rather low range of flour protein content (8·7–12·0 %) within the set studied. Significant differences between gliadins and glutenins with respect to their effects on bread volume could not be detected. The correlation between bread volumes and the quantity of gluten proteins was higher, when dough was mixed to optimum. 2001 Academic Press
Keywords: wheat properties, gluten protein types, quantification, correlations.
INTRODUCTION Wheat quality is greatly influenced by genotype and growing conditions; with sound wheat especially gluten quantity and quality are important1. : CP=crude protein content; CV= coefficient of variation; Ext=extensibility; GI=gluten index; GS=glutenin subunits; MBT=micro-bakingtest; MRMT=micro-rapid-mix-test; ns=not significant; Rmax=maximum resistance; RP-HPLC= reversed-phase high-performance liquid chromatography. 0733–5210/01/040019+09 $35.00/0
Quantity is easy to determine, but is not sufficient to explain differences in flour or dough properties. Gluten quality is more difficult to define. Numerous studies tried to associate quality with the presence or absence of specific gluten protein fractions, but structural differences of allelic proteins in different wheat genotypes are small and, in many cases, restricted only to a few amino acid residues in their primary structures. The only strong evidence for a relevant structural difference has been revealed in HMW GS 5 with an additional cysteine residue at the beginning of domain B, which is missing in the allelic HMW GS 2001 Academic Press
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H. Wieser and R. Kieffer
22. The presence of HMW GS 5 is accompanied by a good breadmaking quality, whereas HMW GS 2 by a poor quality3; a different polymerisation behaviour via intermolecular disulphide bonds could be responsible for these quality differences4. In most cases, however, different amounts and proportions of gluten protein types essentially determine gluten quality. This has been demonstrated either by correlations between the amount of different gluten proteins and technological properties5–12 or by addition of protein fractions to base flour13–20. In most studies, however, only the influence of specific protein fractions has been described. Previously, the relationship between breadmaking performance and rheological dough and gluten properties of a German and an international wheat assortment were determined21. The aim of the present work was to quantify gluten proteins of these flours with a combined extraction/HPLC procedure22, and to correlate the technological properties with the quantities and ratios of all gluten protein types present in wheat flour. Moreover, the usefulness of the determination of technological properties and of gluten protein quantities, both on a micro-scale, should be demonstrated. EXPERIMENTAL Flours The flour of 13 international wheat cultivars grown at different locations and a commercial flour were taken from a previous study (assortment 2)21. All wheat was milled into flour using a ‘Bu¨hler Mahlautomat’. Flour was standardised at ash 0·55%.
Table I
Code for wheat samples and crude protein contents (%) of the flours
Wheata
No.
Bussard Canadian Western Red Spring Dutch commercial flour Dark Northern Spring Fresco Gambrinus Glenlea Hereward Kanzler Kraka Minaret Monopol Obelisk Soissons
1 2 3 4 5 6 7 8 9 10 11 12 13 14
a b
Code BUS CWR DCF DNS FRE GAM GLE HER KAN KRA MIN MON OBE SOI
CP (%)b 10·9 11·8 10·5 12·0 10·1 9·4 11·4 10·4 11·0 8·8 9·4 10·5 8·7 9·3
Corresponding to assortment 2 of previous studies21. N×5·7.
(pH 7·5)+1% (w/v) dithioerythritol under nitrogen (2×1·0 mL)22. Aliquots of extracts were analysed by RP-HPLC on C8 silica gel, and quantification was achieved by using a detection wave-length of 210 nm22. A linear elution gradient (0 min 24% B, 50 min 56% B) was applied to separate gliadin and glutenin subunits. The coefficient of average variation for the total procedure (extraction plus HPLC) was between ±1·2 and ±3·5% (two determinations). Separation and quantification of single HMW GS was performed using special HPLC conditions (one determination)22. Statistic evaluations were performed with program Slide Write Plus (Advanced Graphics Software, Inc., Carlsbad, CA, U.S.A.). RESULTS AND DISCUSSION
Methods
Wheat properties
The preparation of dough and gluten, the determination of gluten index, and the extension and baking tests on a micro-scale were described previously21. Extension tests and micro-baking tests were performed 4–8 times (mean coefficient of variation CV=±5%), determination of Gl in duplicate (CV=±3%). For the quantification of gluten protein types, flour samples (100 mg) were stepwise extracted with 0·4 mol/L of NaCl+0·067 mol/L of HKNaPO4, pH 7·6 (2×1·0 mL), with 60% (v/v) aqueous ethanol (3×0·5 mL) and with 50% (v/v) 1propanol+2 mol/L urea+0·05 ml/L of Tris-HCl
For the present work, the flours of wheat assortment 2 (13 international wheat cultivars and a commercial flour) were taken from a previous study21 (Table I). They did not contain any wheat/rye translocation line. Seven of the wheats occurred also in the assortment previously investigated9, but growing locations and conditions were different. The flour samples were characterised by a relatively low crude protein contents (CP=8·7–12·0%). Technological properties of flours were studied by mixing, extension and baking tests on a micro-scale21. In detail, dough development time (DDT), maximum resistance to extension (Rmax) and extensibility (Ext)
Technological properties of wheat flours determined on a micro-scale
Table II
21
Quantitiesa and ratios of gluten protein types in flours AU
Wheat
BUS CWR DCF DNS FRE GAM GLE HER KAN KRA MIN MON OBE SOI ØCVc
Gluten protein
1460 1560 1369 1576 1337 1205 1525 1376 1465 1119 1212 1406 1073 1216 1·2
Gliadins (GLI)
Glutenins (GLU)b
Total 5 1, 2
980 54 74 1072 85 112 1018 61 82 1094 88 100 877 43 57 914 42 48 984 67 70 960 50 60 1000 55 65 749 73 60 839 41 67 938 47 60 757 36 62 773 36 61 2·3 3·5 3·0
468 417 518 429 399 464 460 490 523 376 413 458 394 351 2·9
384 458 357 477 378 360 387 360 357 240 318 373 265 325 2·2
Total HMW 480 488 351 482 460 291 541 416 465 370 373 468 316 443 2·9
152 158 113 152 134 81 179 120 134 99 102 145 93 144 2·6
Ratio
x
y
LMW
106 109 73 104 88 58 134 82 90 57 77 99 59 110 —d
46 49 40 48 46 23 45 38 44 42 25 46 34 34 —d
304 318 216 317 309 194 337 279 310 255 253 301 209 282 3·0
GLI/ GLI/ GLI/ LMW/ x/y GLU HMW LMW HMW 2·04 6·45 3·22 2·20 6·79 3·37 2·90 9·01 4·71 2·27 7·20 3·45 1·91 6·54 2·84 3·14 11·28 4·71 1·82 5·50 2·92 2·31 8·00 3·44 2·15 7·46 3·23 2·02 7·57 2·94 2·25 8·23 3·32 2·00 6·47 3·12 2·40 8·14 3·62 1·74 5·37 2·74
2·00 2·01 1·91 2·09 2·31 2·40 1·88 2·33 2·31 2·58 2·48 2·08 2·25 1·96
2·30 2·22 1·83 2·17 1·91 2·52 2·98 2·16 2·05 1·36 3·08 2·15 1·74 3·24
a
Absorbance units (AU) of HPLC corresponding to 1 mg flour. Values for glutenin-bound -gliadins (AU=12-25) are omitted. c Average coefficient of variation (in %, two determinations). d One determination. b
of dough and gluten, gluten index (GI) and bread volume derived from baking tests with constant (MRTM) and optimum mixing time (MBT) were determined. Quantity of gluten protein types Gliadin and glutenin subunits were extracted from flour according to a modified, micro-scale Osborne fractionation as described previously22. The quantification of total gliadins (GLI) and glutenin subunits (GLU) and of the different gluten protein types, 5-, 1, 2-, -,-gliadins and glutenin-bound (b) gliadins, HMW GS, LMW GS, was performed by RP-HPLC on C8 silica gel. The proportion of x-type HMW GS (nos. 1–7) and y-type HMW GS (nos. 8–12) according to total HMW GS was determined by modified HPLC conditions22. Table II summarises the relative amounts of protein groups and types expressed as HPLC absorbance units, which have been shown to be strongly correlated with protein amount22. Table II also includes the ratios of GLI, GLU and GLU types. In agreement with studies on the influence of nitrogen fertilisation23, the amount of total gluten proteins (GLI+GLU) was highly correlated (r=
0·99∗∗∗) with CP of flours (Table III). Among gluten protein fractions, GLI correlated better (r= 0·94∗∗∗) with CP than did GLU (r=0·76∗∗). The quantitative data of Table II demonstrate numerous flour specific differences, which could be caused by genotypes as well as by differences in growing conditions. For example, cultivar GAM was characterised by a very low amount of both HMW GS and LMW GS and, consequently, by a higher ratio of GLI to GLU and GLI to HMW GS and LMW GS. Cultivar SOI had a CP value and gluten protein content similar to GAM, but a higher amount of GLU and a lower amount of GLI. Thus the ratio of GLI to GLU was completely different. High values for both HMW GS and LMW GS were typical for cultivar GLE. This cultivar is known for over-producing HMW GS 79,11,24, which was confirmed by the highest amount of x-type GS, the lowest ratio of LMW to HMW GS and one of the highest ratio of x-type to ytype HMW GS (Table II). Altogether, the ratio of LMW to HMW GS were in a relatively small range (1·88 –2·58), whereas those of x-type to ytype HMW GS showed a broader range (1·36 –3·24). In the latter case, SOI had the highest ratio and cultivar KRA the lowest ratio.
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H. Wieser and R. Kieffer
Table III
Correlation coefficients (r)a for the relationships between quantities of gluten protein types and wheat propertiesb CP
Dough DDT
Gluten proteins Gliadins total (GLI) Glutenin subunits total (GLU) HMW x y LMW GLI/GLU GLI/HMW GLI/x GLI/y GLI/LMW LMW/HMW x/y
Rmax
Gluten Ext
GI
Rmax
Bread Ext
MRMT
MBT
0·99
0·43
0·58
0·20
0·54
0·67
−0·34
0·71
0·88
0·94
0·18
0·26
0·47
0·21
0·37
−0·20
0·65
0·74
0·76 0·78 0·75 0·70 0·73 — — — — — — —
0·67 0·71 0·78 0·26 0·62 −0·56 −0·64 −0·74 −0·22 −0·49 −0·49 0·58
0·89 0·85 0·85 0·56 0·88 −0·80 −0·87 −0·92 −0·50 −0·73 −0·47 0·44
−0·27 −0·16 −0·09 −0·30 −0·35 0·74 0·61 0·47 0·63 0·77 −0·18 0·15
0·87 0·82 0·77 0·70 0·87 −0·86 −0·92 −0·87 −0·70 −0·79 −0·43 0·08
0·92 0·90 0·91 0·57 0·90 −0·75 −0·84 −0·91 −0·47 −0·68 −0·53 0·46
−0·72 −0·62 −0·58 −0·53 −0·76 0·84 0·81 0·77 0·59 0·80 0·17 −0·13
0·59 0·53 0·49 0·51 0·61 −0·23 −0·31 −0·37 −0·20 −0·21 −0·26 0·16
0·83 0·82 0·78 0·66 0·81 −0·40 −0·55 −0·62 −0·32 −0·33 −0·54 0·16
a
Level of significance: r=0·54–0·66, p=0·05 (∗); r=0·67–0·78, p=0·01 (∗∗); r>0·78, p=0·001 (∗∗∗). CP=crude protein content of flours; DDT=dough development time; Ext=extensibility; Rmax=maximum resistance to extension; GI=gluten index; MRMT=micro-rapid-mix-test; MBT=micro-baking test. b
Also within gliadin types typical differences can be observed. The North American wheat classes CWR and DNS had by far the highest amounts of 1,2- and -gliadins. The cultivars KAN and SOI were characterised by the highest and lowest values for -gliadins, respectively. Correlations between protein quantities and technological properties The correlation coefficients (r) calculated for the relations between protein quantities and ratios (Table II) and physical parameters21 are summarised in Table III. In accordance with previous studies8,9, the amount of GLI and gliadin subclasses are not correlated with any of the rheological dough and gluten properties, except when their ratio to glutenins was considered. DDT showed moderate correlations (r=0·62∗– 0·71∗∗) with GLU, HMW GS and LMW GS. xType HMW GS were related with DDT much more (r=0·78∗∗) than y-type HMW GS (0·26ns). The importance of x-type HMW GS was most pronounced for the cultivar GLE, which was characterised by the highest amount of x-type HMW GS (Table II) and an extremely long DDT (16 min). Distinctly strong dough mixing properties of GLE were already described by Sapirstein and
Fu11. The coefficients for the ratios of GLI to GLU and for the ratios of GLI and GLU types were negative and in a lower range (−0·22ns to −0·74∗∗). In contrast to the incorporation studies of Uthayakumaran et al.20, the ratio of HMW GS to LMW GS was not correlated with DDT (r= −0·49ns). Rmax of both dough and gluten was highly correlated with the quantity of GLU, HMW GS and LMW GS (r=0·85∗∗∗–0·92∗∗∗); x-type HMW GS (r=0·85∗∗∗ for dough and 0·91∗∗∗ for gluten) influenced Rmax much more than y-type HMWs (r=0·56∗ and 0·57∗, respectively). Quantities of GLI and gliadin types were not related with Rmax (r=0·08ns–0·55∗), but the negative correlation of the ratio of GLI to GLU was in a similar range as the positive correlation obtained with glutenin quantity. The decrease in dough strength caused by the addition of gliadins was also demonstrated previously13,15,16. These results very well reflected the different functionality of both gluten fractions: glutenin subunits form high Mr aggregates, and a higher quantity in flour favours dough and gluten strength. Monomeric gliadins act as a ‘solvent’ for glutenins; a high ratio of GLI to GLU leads to a more viscous material. The different roles of glutenin and gliadin for the rheological properties are supported by previous experiments, which
Technological properties of wheat flours determined on a micro-scale
23
(b)
(a) 430
430
14 362
362
294
294
Rmax (mN)
Rmax (mN)
5
226
226
158
158
90 150
196
242 288 LMW (AU)
334
380
90 53
11
6 78
104 129 HMW (AU)
155
180
(c) 430
Rmax (mN)
362
294
226 10
158 13
90 5.00
6.30
7.60 8.90 GLI/HMW
10.20
11.50
Figure 1 Correlations between Rmax of dough and (a) amount of LMW GS (r=0·88∗∗∗), (b) amount of HMW GS (r= 0·85∗∗∗) and (c) ratio of GLI to HMW GS (r=−0·87∗∗∗).
24
H. Wieser and R. Kieffer
(a)
(b)
732
732
614
614 Rmax (mN)
850
Rmax (mN)
850
496
496
378
378
260 40
60
80
100
120
140
x (AU)
260 12
22
32
41
51
61
y (AU)
Figure 2 Correlations between Rmax of gluten and amount of (a) x-type HMW GS (r=0·91∗∗∗) and (b) y-type HMW GS (r=0·57∗∗).
demonstrated that Rmax of gluten was significantly increased by the addition of reoxidised HMW GS and decreased by the addition of gliadins13,15. Differences between the coefficients for Rmax of dough and gluten are small (Table III). This indicates that dough strength is predominantly determined by gluten proteins. In agreement with studies of Gupta et al.6, the relation of HMW GS and LMW GS to Rmax of dough and gluten were similar (Table III); this can be explained by the ability of both protein types to form aggregates by means of intermolecular disulphide bonds2. However, the regression analysis showed that twice the amount of LMW GS was necessary to get the same Rmax as with HMW GS [Fig. 1(a,b)]. Comparing the regression lines for the relations of Rmax with the quantity of HMW GS and the ratio of GLI to HMW GS [Fig. 1(b, c)], cultivars FRE (no. 5), GAM (6), MIN (11) and SOI (14) showed the greatest deviation from the regression line for Rmax and HMW GS [Fig. 1(b)]. This could be interpreted by the fact that the ratio
of GLI to HMW GS was either relatively low (FRE, SOI) or high (GAM, MIN). Cultivars KRA (10) and OBE (13) were outliers in the relation of Rmax to the ratio GLI to HMW GS [Fig. 1(c)]. They were characterised by a very low quantity of HMW GS (Table II). In consequence, a reliable predication of Rmax of dough and gluten appears to be possible, if the amounts of HMW GS as well as the ratio of GLI to HMW GS are considered. However, it should be mentioned that correlations between HMW GS and Rmax are lowered, when the set contains wheat/rye translocation lines25. In this case the correlation with LMW GS is higher. With respect to the influence of x-type and ytype HMW GS on Rmax, previous results7,9 were confirmed: x-type HMW GS had a strong effect (r=0·85∗∗∗ for dough and 0·91∗∗∗ for gluten) and y-type HMW GS a weak effect (r=0·56∗, 0·57∗; Fig. 2). Differences between x- and ytype HMW GS were also demonstrated by the incorporation studies of Veraverbeke et al.18,19 Dough or gluten Ext was mainly influenced by
Technological properties of wheat flours determined on a micro-scale
(a)
25
(b)
18
18
15
15 Ext (cm)
20
Ext (cm)
20
13
13
10
10
8 3.60
5.32
7.04
8.76
10.48
12.20
GLI/HMW
Figure 3 0·77∗∗).
8 1.90
2.60
3.30
4.00
4.70
5.40
GLI/LMW
Correlations between Ext of gluten and the ratio of GLI to (a) HMW GS (r=0·61∗) and (b) to LMW GS (r=
the ratio of GLI to GLU (r=0·74∗∗ and 0·84∗∗∗, respectively) and the ratios of single types, e.g. GLI/LMW GS (r=0·77∗∗, 0·80∗∗∗). The quantity of glutenin subunits showed a negative effect, but only on gluten Ext (GLU: r=−0·72∗∗; LMW: −0·76∗∗). In almost all cases, coefficients were higher for gluten than for dough, but lower than for Rmax, probably because of the lower accuracy of Ext measurements. Again, the influence of HMW GS was approximately twice as large as that of LMW GS, when the results were based on the same quantity of protein (Fig. 3). For the determination of GI (ICC standard no. 15526), wet gluten is pressed through a special sieve by centrifugal forces. The portion of gluten retained by the sieve is used as an indicator for gluten quality. The correlation coefficients (Table III) showed that GI was strongly influenced by GLU and subunit types of GLU (r=0·70∗∗– 0·87∗∗∗) and, negatively, by the ratios of GLI to GLU and of GLI to GLU subunit types (r= −0·70∗∗ to −0·92∗∗∗) whereas the amount of
gliadin was not important. Significant differences between glutenin subunits could not be detected. Finally, loaf volume data from two different baking tests were correlated with protein quantity (Table III). The main difference between the tests was a constant mixing time (2 min) for MRMT and a variable mixing time (identical with DDT) for MBT21. The correlation coefficients showed that the influence of protein quantity on loaf volume and on the rheological properties was quite different. The highest coefficients (r=0·71∗∗ for MRMT, r=0·88∗∗∗ for MBT) were found for the amounts of total gluten proteins, which were strongly correlated with CP (r=0·99∗∗∗). Because the range of CP of the assortment was very low (Table I), it can be concluded that the amount of total gluten proteins was the most important factor for explaining variation in loaf volume, and the importance of protein groups or types was secondary. Thus, the coefficients for GLI, GLU and GLU subunit types were lower. Loaf volumes were not statistically related to the ratios of different
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H. Wieser and R. Kieffer
(a)
(b) 67
56
60
51
54 Vol (mL)
Vol (mL)
62
45
47
40
41
34 1040
1192
1344
1496
1648
1800
Gluten (AU)
34 1040
1158
1276
1394
1512
1630
Gluten (AU)
Figure 4 Correlations between amounts of total gluten proteins and loaf volume of (a) MRMT (r=0·71∗∗) and (b) MBT (r=0·88∗∗∗).
proteins. The coefficients for MBT were generally higher than for MRMT; this could mean that the cultivar specific effects of gluten proteins were more pronounced, when mixing time was optimised for each flour. To understand the relations between breadmaking quality and quantity of single protein types, flours with higher CP (>13%) have to be investigated. CONCLUSIONS Previous studies of thirty wheat samples5,7–9 and the present studies of 14 cultivars showed that maximum resistance of dough and gluten is strongly determined by both the amounts of glutenin subunits and by the ratio of gliadin to glutenin subunits. The correlations of LMW and HMW GS with rheological properties are similar, but if equal amounts of protein are considered, the effect of HMW GS on maximum resistance is twofold. The contributions of x-type HMW GS to these properties are much higher than those of y-
type HMW GS. The extensibility of dough and gluten is mainly influenced by the ratios of gliadin to glutenin subunits. In agreement with literature the contents of glutenin subunits explained the variation of dough and gluten properties better than the contents of gliadins. Loaf volume of flour with relatively low protein contents, as used in the present study, is more influenced by the amount of total gluten proteins and less by the amount of single protein types. The present studies demonstrated that the combination of micro-scale methods for the determination of technological properties and gluten protein quantities is useful for the evaluation of wheat flour quality. Acknowledgements This work was supported by the FEI (Forschungskreis der Erna¨hrungsindustrie e.V., Bonn), the AiF and the Ministry of Economics, Project No. 10228N). We thank U. Schu¨tzler for excellent technical assistance and S. Bijewitz for statistical analysis.
Technological properties of wheat flours determined on a micro-scale
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26.
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Eigenschaften von rekonstituierten Weizenklebern mit variierenden Anteilen an Prolaminfraktionen verschiedener Getreidearten. Zeitschrift fu¨r Lebensmittel-Untersuchung und -Forschung 186 (1988) 16–21. Bekes, F., Gras, P.W., Gupta, R.B., Hickman, D.R. and Tatham, A.S. Effect of a high Mr glutenin subunit (1B×20) on the dough mixing properties of wheat flour. Journal of Cereal Science 19 (1994) 3–7. Schropp, P. and Wieser, H. Effects of HMW subunits of glutenin on the rheological properties of wheat gluten. Cereal Chemistry 73 (1996) 410–413. Fido, R.J., Bekes, F., Gras, P.W. and Tatham, A.S. Effects of -, -, - and -gliadins on the dough mixing properties of wheat flour. Journal of Cereal Science 26 (1997) 271–277. Uthayakumaran, S., Gras, P.W., Stoddard, F.L. and Bekes, F. Effect of varying protein content and gluteninto-gliadin ratio on the functional properties of wheat dough. Cereal Chemistry 76 (1999) 389–394. Veraverbeke, W.S., Larroque, O.R., Bekes, F. and Delcour, J.A. In vitro polymerization of wheat glutenin subunits with inorganic oxidizing agents. I. Comparison of single-step and stepwise oxidations of high molecular weight glutenin subunits. Cereal Chemistry 77 (2000) 582– 588. Veraverbeke, W.S., Larroque, O.S., Bekes, F. and Delcour, J.A. In vitro polymerization of wheat glutenin subunits with inorganic oxidizing agents. II. Stepwise oxidation of low molecular weight glutenin subunits and a mixture of high and low molecular weight glutenin subunits. Cereal Chemistry 77 (2000) 589–594. Uthayakumaran, S., Stoddard, F.L., Gras, P.W. and Bekes, F. Effects of incorporated glutenins on functional properties of wheat dough. Cereal Chemistry 77 (2000) 737–743. Kieffer, R., Wieser, H., Henderson, M.H. and Graveland, A. Correlations of the breadmaking performance of wheat flour with rheological measurements on a micro-scale. Journal of Cereal Science 27 (1998) 53–60. Wieser, H., Antes, S. and Seilmeier, W. Quantitative determination of gluten protein types in wheat flour by reversed-phase high-performance liquid chromatography. Cereal Chemistry 75 (1998) 644–650. Wieser, H. and Seilmeier, W. The influence of nitrogen fertilisation on quantities and proportion of different protein types in wheat flour. Journal of the Science of Food and Agriculture 76 (1998) 49–50. Lukow, O.M., Forsyth, S.A. and Payne, P.I. Over-production of HMW glutenin subunits coded on chromosome 1B in common wheat. Triticum aestivum. Journal of Genetics and Breeding 46 (1992) 187–192. Wieser, H., Kieffer, R. and Lelley, T. The influence of 1B/1R chromosome translocation on gluten protein composition and technological properties of bread wheat. Journal of the Science of Food and Agriculture 80 (2000) 1640– 1647. Standard Methods of the ICC (International Association for Cereal Sciences and Technology), Scha¨fer, Detmold.
Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale H. Wieser and R. Kieffer Deutsche Forschungsanstalt fu¨r Lebensmittelchemie and Kurt-Hess-Institut fu¨r Mehl- und Eiweißforschung, Lichtenbergstraße 4, D-85748 Garching, Germany Received 13 May 1999
ABSTRACT Flour samples of 14 wheat cultivars previously characterised by rheological measurements and by baking tests on a micro-scale (Kieffer et al.: Journal of Cereal Science 27 (1998) 53–60) were analysed for the relative amounts of gluten protein types using a combined extraction/HPLC procedure. Regression analysis was used to find relations between wheat properties and protein quantities. The results indicated that the maximum resistance of dough and gluten and the gluten index were strongly dependent on the quantity of glutenin subunits (GS) in flour; additionally they were influenced by the ratio of gliadin to glutenin subunits. Within the family of glutenin proteins, the correlation coefficients for high-molecular-weight (HMW) and low-molecular-weight (LMW) GS were in a similar range, but twice the amount of LMW GS was necessary to get the same resistance as with HMW GS. Among HMW GS, the contribution of x-type GS was more important than those of y-type GS. The extensibility of dough and gluten was mainly dependent on the ratio of gliadin to total glutenin subunits, to HMW GS and LMW GS. Dough development time showed the highest correlation with total HMW GS and x-type HMW GS. Bread volume was influenced by the total amount of gluten protein more than by the amount of protein in different groups or of different types, probably because of the rather low range of flour protein content (8·7–12·0 %) within the set studied. Significant differences between gliadins and glutenins with respect to their effects on bread volume could not be detected. The correlation between bread volumes and the quantity of gluten proteins was higher, when dough was mixed to optimum. 2001 Academic Press
Keywords: wheat properties, gluten protein types, quantification, correlations.
INTRODUCTION Wheat quality is greatly influenced by genotype and growing conditions; with sound wheat especially gluten quantity and quality are important1. : CP=crude protein content; CV= coefficient of variation; Ext=extensibility; GI=gluten index; GS=glutenin subunits; MBT=micro-bakingtest; MRMT=micro-rapid-mix-test; ns=not significant; Rmax=maximum resistance; RP-HPLC= reversed-phase high-performance liquid chromatography. 0733–5210/01/040019+09 $35.00/0
Quantity is easy to determine, but is not sufficient to explain differences in flour or dough properties. Gluten quality is more difficult to define. Numerous studies tried to associate quality with the presence or absence of specific gluten protein fractions, but structural differences of allelic proteins in different wheat genotypes are small and, in many cases, restricted only to a few amino acid residues in their primary structures. The only strong evidence for a relevant structural difference has been revealed in HMW GS 5 with an additional cysteine residue at the beginning of domain B, which is missing in the allelic HMW GS 2001 Academic Press
20
H. Wieser and R. Kieffer
22. The presence of HMW GS 5 is accompanied by a good breadmaking quality, whereas HMW GS 2 by a poor quality3; a different polymerisation behaviour via intermolecular disulphide bonds could be responsible for these quality differences4. In most cases, however, different amounts and proportions of gluten protein types essentially determine gluten quality. This has been demonstrated either by correlations between the amount of different gluten proteins and technological properties5–12 or by addition of protein fractions to base flour13–20. In most studies, however, only the influence of specific protein fractions has been described. Previously, the relationship between breadmaking performance and rheological dough and gluten properties of a German and an international wheat assortment were determined21. The aim of the present work was to quantify gluten proteins of these flours with a combined extraction/HPLC procedure22, and to correlate the technological properties with the quantities and ratios of all gluten protein types present in wheat flour. Moreover, the usefulness of the determination of technological properties and of gluten protein quantities, both on a micro-scale, should be demonstrated. EXPERIMENTAL Flours The flour of 13 international wheat cultivars grown at different locations and a commercial flour were taken from a previous study (assortment 2)21. All wheat was milled into flour using a ‘Bu¨hler Mahlautomat’. Flour was standardised at ash 0·55%.
Table I
Code for wheat samples and crude protein contents (%) of the flours
Wheata
No.
Bussard Canadian Western Red Spring Dutch commercial flour Dark Northern Spring Fresco Gambrinus Glenlea Hereward Kanzler Kraka Minaret Monopol Obelisk Soissons
1 2 3 4 5 6 7 8 9 10 11 12 13 14
a b
Code BUS CWR DCF DNS FRE GAM GLE HER KAN KRA MIN MON OBE SOI
CP (%)b 10·9 11·8 10·5 12·0 10·1 9·4 11·4 10·4 11·0 8·8 9·4 10·5 8·7 9·3
Corresponding to assortment 2 of previous studies21. N×5·7.
(pH 7·5)+1% (w/v) dithioerythritol under nitrogen (2×1·0 mL)22. Aliquots of extracts were analysed by RP-HPLC on C8 silica gel, and quantification was achieved by using a detection wave-length of 210 nm22. A linear elution gradient (0 min 24% B, 50 min 56% B) was applied to separate gliadin and glutenin subunits. The coefficient of average variation for the total procedure (extraction plus HPLC) was between ±1·2 and ±3·5% (two determinations). Separation and quantification of single HMW GS was performed using special HPLC conditions (one determination)22. Statistic evaluations were performed with program Slide Write Plus (Advanced Graphics Software, Inc., Carlsbad, CA, U.S.A.). RESULTS AND DISCUSSION
Methods
Wheat properties
The preparation of dough and gluten, the determination of gluten index, and the extension and baking tests on a micro-scale were described previously21. Extension tests and micro-baking tests were performed 4–8 times (mean coefficient of variation CV=±5%), determination of Gl in duplicate (CV=±3%). For the quantification of gluten protein types, flour samples (100 mg) were stepwise extracted with 0·4 mol/L of NaCl+0·067 mol/L of HKNaPO4, pH 7·6 (2×1·0 mL), with 60% (v/v) aqueous ethanol (3×0·5 mL) and with 50% (v/v) 1propanol+2 mol/L urea+0·05 ml/L of Tris-HCl
For the present work, the flours of wheat assortment 2 (13 international wheat cultivars and a commercial flour) were taken from a previous study21 (Table I). They did not contain any wheat/rye translocation line. Seven of the wheats occurred also in the assortment previously investigated9, but growing locations and conditions were different. The flour samples were characterised by a relatively low crude protein contents (CP=8·7–12·0%). Technological properties of flours were studied by mixing, extension and baking tests on a micro-scale21. In detail, dough development time (DDT), maximum resistance to extension (Rmax) and extensibility (Ext)
Technological properties of wheat flours determined on a micro-scale
Table II
21
Quantitiesa and ratios of gluten protein types in flours AU
Wheat
BUS CWR DCF DNS FRE GAM GLE HER KAN KRA MIN MON OBE SOI ØCVc
Gluten protein
1460 1560 1369 1576 1337 1205 1525 1376 1465 1119 1212 1406 1073 1216 1·2
Gliadins (GLI)
Glutenins (GLU)b
Total 5 1, 2
980 54 74 1072 85 112 1018 61 82 1094 88 100 877 43 57 914 42 48 984 67 70 960 50 60 1000 55 65 749 73 60 839 41 67 938 47 60 757 36 62 773 36 61 2·3 3·5 3·0
468 417 518 429 399 464 460 490 523 376 413 458 394 351 2·9
384 458 357 477 378 360 387 360 357 240 318 373 265 325 2·2
Total HMW 480 488 351 482 460 291 541 416 465 370 373 468 316 443 2·9
152 158 113 152 134 81 179 120 134 99 102 145 93 144 2·6
Ratio
x
y
LMW
106 109 73 104 88 58 134 82 90 57 77 99 59 110 —d
46 49 40 48 46 23 45 38 44 42 25 46 34 34 —d
304 318 216 317 309 194 337 279 310 255 253 301 209 282 3·0
GLI/ GLI/ GLI/ LMW/ x/y GLU HMW LMW HMW 2·04 6·45 3·22 2·20 6·79 3·37 2·90 9·01 4·71 2·27 7·20 3·45 1·91 6·54 2·84 3·14 11·28 4·71 1·82 5·50 2·92 2·31 8·00 3·44 2·15 7·46 3·23 2·02 7·57 2·94 2·25 8·23 3·32 2·00 6·47 3·12 2·40 8·14 3·62 1·74 5·37 2·74
2·00 2·01 1·91 2·09 2·31 2·40 1·88 2·33 2·31 2·58 2·48 2·08 2·25 1·96
2·30 2·22 1·83 2·17 1·91 2·52 2·98 2·16 2·05 1·36 3·08 2·15 1·74 3·24
a
Absorbance units (AU) of HPLC corresponding to 1 mg flour. Values for glutenin-bound -gliadins (AU=12-25) are omitted. c Average coefficient of variation (in %, two determinations). d One determination. b
of dough and gluten, gluten index (GI) and bread volume derived from baking tests with constant (MRTM) and optimum mixing time (MBT) were determined. Quantity of gluten protein types Gliadin and glutenin subunits were extracted from flour according to a modified, micro-scale Osborne fractionation as described previously22. The quantification of total gliadins (GLI) and glutenin subunits (GLU) and of the different gluten protein types, 5-, 1, 2-, -,-gliadins and glutenin-bound (b) gliadins, HMW GS, LMW GS, was performed by RP-HPLC on C8 silica gel. The proportion of x-type HMW GS (nos. 1–7) and y-type HMW GS (nos. 8–12) according to total HMW GS was determined by modified HPLC conditions22. Table II summarises the relative amounts of protein groups and types expressed as HPLC absorbance units, which have been shown to be strongly correlated with protein amount22. Table II also includes the ratios of GLI, GLU and GLU types. In agreement with studies on the influence of nitrogen fertilisation23, the amount of total gluten proteins (GLI+GLU) was highly correlated (r=
0·99∗∗∗) with CP of flours (Table III). Among gluten protein fractions, GLI correlated better (r= 0·94∗∗∗) with CP than did GLU (r=0·76∗∗). The quantitative data of Table II demonstrate numerous flour specific differences, which could be caused by genotypes as well as by differences in growing conditions. For example, cultivar GAM was characterised by a very low amount of both HMW GS and LMW GS and, consequently, by a higher ratio of GLI to GLU and GLI to HMW GS and LMW GS. Cultivar SOI had a CP value and gluten protein content similar to GAM, but a higher amount of GLU and a lower amount of GLI. Thus the ratio of GLI to GLU was completely different. High values for both HMW GS and LMW GS were typical for cultivar GLE. This cultivar is known for over-producing HMW GS 79,11,24, which was confirmed by the highest amount of x-type GS, the lowest ratio of LMW to HMW GS and one of the highest ratio of x-type to ytype HMW GS (Table II). Altogether, the ratio of LMW to HMW GS were in a relatively small range (1·88 –2·58), whereas those of x-type to ytype HMW GS showed a broader range (1·36 –3·24). In the latter case, SOI had the highest ratio and cultivar KRA the lowest ratio.
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H. Wieser and R. Kieffer
Table III
Correlation coefficients (r)a for the relationships between quantities of gluten protein types and wheat propertiesb CP
Dough DDT
Gluten proteins Gliadins total (GLI) Glutenin subunits total (GLU) HMW x y LMW GLI/GLU GLI/HMW GLI/x GLI/y GLI/LMW LMW/HMW x/y
Rmax
Gluten Ext
GI
Rmax
Bread Ext
MRMT
MBT
0·99
0·43
0·58
0·20
0·54
0·67
−0·34
0·71
0·88
0·94
0·18
0·26
0·47
0·21
0·37
−0·20
0·65
0·74
0·76 0·78 0·75 0·70 0·73 — — — — — — —
0·67 0·71 0·78 0·26 0·62 −0·56 −0·64 −0·74 −0·22 −0·49 −0·49 0·58
0·89 0·85 0·85 0·56 0·88 −0·80 −0·87 −0·92 −0·50 −0·73 −0·47 0·44
−0·27 −0·16 −0·09 −0·30 −0·35 0·74 0·61 0·47 0·63 0·77 −0·18 0·15
0·87 0·82 0·77 0·70 0·87 −0·86 −0·92 −0·87 −0·70 −0·79 −0·43 0·08
0·92 0·90 0·91 0·57 0·90 −0·75 −0·84 −0·91 −0·47 −0·68 −0·53 0·46
−0·72 −0·62 −0·58 −0·53 −0·76 0·84 0·81 0·77 0·59 0·80 0·17 −0·13
0·59 0·53 0·49 0·51 0·61 −0·23 −0·31 −0·37 −0·20 −0·21 −0·26 0·16
0·83 0·82 0·78 0·66 0·81 −0·40 −0·55 −0·62 −0·32 −0·33 −0·54 0·16
a
Level of significance: r=0·54–0·66, p=0·05 (∗); r=0·67–0·78, p=0·01 (∗∗); r>0·78, p=0·001 (∗∗∗). CP=crude protein content of flours; DDT=dough development time; Ext=extensibility; Rmax=maximum resistance to extension; GI=gluten index; MRMT=micro-rapid-mix-test; MBT=micro-baking test. b
Also within gliadin types typical differences can be observed. The North American wheat classes CWR and DNS had by far the highest amounts of 1,2- and -gliadins. The cultivars KAN and SOI were characterised by the highest and lowest values for -gliadins, respectively. Correlations between protein quantities and technological properties The correlation coefficients (r) calculated for the relations between protein quantities and ratios (Table II) and physical parameters21 are summarised in Table III. In accordance with previous studies8,9, the amount of GLI and gliadin subclasses are not correlated with any of the rheological dough and gluten properties, except when their ratio to glutenins was considered. DDT showed moderate correlations (r=0·62∗– 0·71∗∗) with GLU, HMW GS and LMW GS. xType HMW GS were related with DDT much more (r=0·78∗∗) than y-type HMW GS (0·26ns). The importance of x-type HMW GS was most pronounced for the cultivar GLE, which was characterised by the highest amount of x-type HMW GS (Table II) and an extremely long DDT (16 min). Distinctly strong dough mixing properties of GLE were already described by Sapirstein and
Fu11. The coefficients for the ratios of GLI to GLU and for the ratios of GLI and GLU types were negative and in a lower range (−0·22ns to −0·74∗∗). In contrast to the incorporation studies of Uthayakumaran et al.20, the ratio of HMW GS to LMW GS was not correlated with DDT (r= −0·49ns). Rmax of both dough and gluten was highly correlated with the quantity of GLU, HMW GS and LMW GS (r=0·85∗∗∗–0·92∗∗∗); x-type HMW GS (r=0·85∗∗∗ for dough and 0·91∗∗∗ for gluten) influenced Rmax much more than y-type HMWs (r=0·56∗ and 0·57∗, respectively). Quantities of GLI and gliadin types were not related with Rmax (r=0·08ns–0·55∗), but the negative correlation of the ratio of GLI to GLU was in a similar range as the positive correlation obtained with glutenin quantity. The decrease in dough strength caused by the addition of gliadins was also demonstrated previously13,15,16. These results very well reflected the different functionality of both gluten fractions: glutenin subunits form high Mr aggregates, and a higher quantity in flour favours dough and gluten strength. Monomeric gliadins act as a ‘solvent’ for glutenins; a high ratio of GLI to GLU leads to a more viscous material. The different roles of glutenin and gliadin for the rheological properties are supported by previous experiments, which
Technological properties of wheat flours determined on a micro-scale
23
(b)
(a) 430
430
14 362
362
294
294
Rmax (mN)
Rmax (mN)
5
226
226
158
158
90 150
196
242 288 LMW (AU)
334
380
90 53
11
6 78
104 129 HMW (AU)
155
180
(c) 430
Rmax (mN)
362
294
226 10
158 13
90 5.00
6.30
7.60 8.90 GLI/HMW
10.20
11.50
Figure 1 Correlations between Rmax of dough and (a) amount of LMW GS (r=0·88∗∗∗), (b) amount of HMW GS (r= 0·85∗∗∗) and (c) ratio of GLI to HMW GS (r=−0·87∗∗∗).
24
H. Wieser and R. Kieffer
(a)
(b)
732
732
614
614 Rmax (mN)
850
Rmax (mN)
850
496
496
378
378
260 40
60
80
100
120
140
x (AU)
260 12
22
32
41
51
61
y (AU)
Figure 2 Correlations between Rmax of gluten and amount of (a) x-type HMW GS (r=0·91∗∗∗) and (b) y-type HMW GS (r=0·57∗∗).
demonstrated that Rmax of gluten was significantly increased by the addition of reoxidised HMW GS and decreased by the addition of gliadins13,15. Differences between the coefficients for Rmax of dough and gluten are small (Table III). This indicates that dough strength is predominantly determined by gluten proteins. In agreement with studies of Gupta et al.6, the relation of HMW GS and LMW GS to Rmax of dough and gluten were similar (Table III); this can be explained by the ability of both protein types to form aggregates by means of intermolecular disulphide bonds2. However, the regression analysis showed that twice the amount of LMW GS was necessary to get the same Rmax as with HMW GS [Fig. 1(a,b)]. Comparing the regression lines for the relations of Rmax with the quantity of HMW GS and the ratio of GLI to HMW GS [Fig. 1(b, c)], cultivars FRE (no. 5), GAM (6), MIN (11) and SOI (14) showed the greatest deviation from the regression line for Rmax and HMW GS [Fig. 1(b)]. This could be interpreted by the fact that the ratio
of GLI to HMW GS was either relatively low (FRE, SOI) or high (GAM, MIN). Cultivars KRA (10) and OBE (13) were outliers in the relation of Rmax to the ratio GLI to HMW GS [Fig. 1(c)]. They were characterised by a very low quantity of HMW GS (Table II). In consequence, a reliable predication of Rmax of dough and gluten appears to be possible, if the amounts of HMW GS as well as the ratio of GLI to HMW GS are considered. However, it should be mentioned that correlations between HMW GS and Rmax are lowered, when the set contains wheat/rye translocation lines25. In this case the correlation with LMW GS is higher. With respect to the influence of x-type and ytype HMW GS on Rmax, previous results7,9 were confirmed: x-type HMW GS had a strong effect (r=0·85∗∗∗ for dough and 0·91∗∗∗ for gluten) and y-type HMW GS a weak effect (r=0·56∗, 0·57∗; Fig. 2). Differences between x- and ytype HMW GS were also demonstrated by the incorporation studies of Veraverbeke et al.18,19 Dough or gluten Ext was mainly influenced by
Technological properties of wheat flours determined on a micro-scale
(a)
25
(b)
18
18
15
15 Ext (cm)
20
Ext (cm)
20
13
13
10
10
8 3.60
5.32
7.04
8.76
10.48
12.20
GLI/HMW
Figure 3 0·77∗∗).
8 1.90
2.60
3.30
4.00
4.70
5.40
GLI/LMW
Correlations between Ext of gluten and the ratio of GLI to (a) HMW GS (r=0·61∗) and (b) to LMW GS (r=
the ratio of GLI to GLU (r=0·74∗∗ and 0·84∗∗∗, respectively) and the ratios of single types, e.g. GLI/LMW GS (r=0·77∗∗, 0·80∗∗∗). The quantity of glutenin subunits showed a negative effect, but only on gluten Ext (GLU: r=−0·72∗∗; LMW: −0·76∗∗). In almost all cases, coefficients were higher for gluten than for dough, but lower than for Rmax, probably because of the lower accuracy of Ext measurements. Again, the influence of HMW GS was approximately twice as large as that of LMW GS, when the results were based on the same quantity of protein (Fig. 3). For the determination of GI (ICC standard no. 15526), wet gluten is pressed through a special sieve by centrifugal forces. The portion of gluten retained by the sieve is used as an indicator for gluten quality. The correlation coefficients (Table III) showed that GI was strongly influenced by GLU and subunit types of GLU (r=0·70∗∗– 0·87∗∗∗) and, negatively, by the ratios of GLI to GLU and of GLI to GLU subunit types (r= −0·70∗∗ to −0·92∗∗∗) whereas the amount of
gliadin was not important. Significant differences between glutenin subunits could not be detected. Finally, loaf volume data from two different baking tests were correlated with protein quantity (Table III). The main difference between the tests was a constant mixing time (2 min) for MRMT and a variable mixing time (identical with DDT) for MBT21. The correlation coefficients showed that the influence of protein quantity on loaf volume and on the rheological properties was quite different. The highest coefficients (r=0·71∗∗ for MRMT, r=0·88∗∗∗ for MBT) were found for the amounts of total gluten proteins, which were strongly correlated with CP (r=0·99∗∗∗). Because the range of CP of the assortment was very low (Table I), it can be concluded that the amount of total gluten proteins was the most important factor for explaining variation in loaf volume, and the importance of protein groups or types was secondary. Thus, the coefficients for GLI, GLU and GLU subunit types were lower. Loaf volumes were not statistically related to the ratios of different
26
H. Wieser and R. Kieffer
(a)
(b) 67
56
60
51
54 Vol (mL)
Vol (mL)
62
45
47
40
41
34 1040
1192
1344
1496
1648
1800
Gluten (AU)
34 1040
1158
1276
1394
1512
1630
Gluten (AU)
Figure 4 Correlations between amounts of total gluten proteins and loaf volume of (a) MRMT (r=0·71∗∗) and (b) MBT (r=0·88∗∗∗).
proteins. The coefficients for MBT were generally higher than for MRMT; this could mean that the cultivar specific effects of gluten proteins were more pronounced, when mixing time was optimised for each flour. To understand the relations between breadmaking quality and quantity of single protein types, flours with higher CP (>13%) have to be investigated. CONCLUSIONS Previous studies of thirty wheat samples5,7–9 and the present studies of 14 cultivars showed that maximum resistance of dough and gluten is strongly determined by both the amounts of glutenin subunits and by the ratio of gliadin to glutenin subunits. The correlations of LMW and HMW GS with rheological properties are similar, but if equal amounts of protein are considered, the effect of HMW GS on maximum resistance is twofold. The contributions of x-type HMW GS to these properties are much higher than those of y-
type HMW GS. The extensibility of dough and gluten is mainly influenced by the ratios of gliadin to glutenin subunits. In agreement with literature the contents of glutenin subunits explained the variation of dough and gluten properties better than the contents of gliadins. Loaf volume of flour with relatively low protein contents, as used in the present study, is more influenced by the amount of total gluten proteins and less by the amount of single protein types. The present studies demonstrated that the combination of micro-scale methods for the determination of technological properties and gluten protein quantities is useful for the evaluation of wheat flour quality. Acknowledgements This work was supported by the FEI (Forschungskreis der Erna¨hrungsindustrie e.V., Bonn), the AiF and the Ministry of Economics, Project No. 10228N). We thank U. Schu¨tzler for excellent technical assistance and S. Bijewitz for statistical analysis.
Technological properties of wheat flours determined on a micro-scale
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15. 16.
17.
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