Characterization of Intercultivar Variation on the Linear Viscoelastic

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 29, 2018 | https://pubs.acs.org Publication Date: August 28, 2006 | doi: 10.1021/bk-2006-0935.ch008

Chapter 8

Characterization of Intercultivar Variation on the Linear Viscoelastic Network Properties of Wheat Gluten II: Effects of Temperature and L-Cysteine 1

1

2

Hongquan Liang , Chia C. Lee , Patricia Rayas-Duarte , and Steven J. Mulvaney 1

1

Department of Food Science, Cornell University, Ithaca, NY 14853

2

Food and Agricultural Products Center, Oklahoma State University, Stillwater, OK 74078

Both non-covalent and disulfide bonds are known to be important contributors to the unusual viscoelastic properties of wheat gluten, but it has been difficult to decouple their effects on such. In this work, the effects of added L-cysteine and an increase in temperature on the stress relaxation behavior of three glutens with different high molecular weight subunit composition were compared. An increase in temperature up to 40°C decreased the apparent network strength for all of the glutens, but the decrease in viscoelasticity varied between the glutens with different subunit composition. It was found that addition of 250 ppm L-cysteine at 25°C to these glutens eliminated most evidence of a network structure in their stress relaxation patterns, but the resulting extended power law stress relaxation curves did not superpose themselves at 25°C. These results suggest that use of the linear viscoelastic network strength of gluten shows promise as a new tool for assessing the intercultivar variations in gluten network structures.

© 2006 American Chemical Society

Fishman et al.; Advances in Biopolymers ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction Wet gluten is obtained from mixed wheatflourdough by washing out the water-soluble albumin proteins, the salt-soluble globulin proteins and starch. The cohesive gluten that is recovered after washing of the dough shows very interesting material properties. It can be stretched, and if not stretched too far, will return to its original shape, i.e., the deformation is recoverable like an elastic rubber band. Gluten obtained from doughs of different wheat cultivars are more or less resistant to extension, a property generally referred to as gluten strength. Dough and gluten can also be stretched until they break, the deformation at break representing the property referred to as extensibility. Wheat cultivars used for production of high loaf volume pan bread must give mixed doughs that have a certain balance between extensibility and elasticity to ensure both good dough handling properties and the ability to expand and hold C 0 gas during proofing and oven rise (/). In more general polymer science terms, soft cohesive materials like gluten and natural rubber that are both extensible and elastic are classified as viscoelastic materials. It is clear that the viscoelasticity of wheat gluten and dough is the result of specific gluten proteins forming an elastic network in the presence of water and some mechanical energy input. But, relating differences in the viscoelasticity of dough or gluten obtainedfromdifferent wheat cultivars to the cereal protein composition in grains orflouris complicated by the hierarchal structures that the cereal proteins adopt when hydrated. Shewry et al (2) remind us that the wheat gluten proteins are a highly complex mixture with at least 50 individual protein components being separated by two-dimensional isoelectric focusing/SDS-PAGE of reduced total fractions. Only some of these individual cereal proteins assemble themselves into an elastic network in gluten during mixing of wheat doughs. 2

Cereal chemistry and rheology: some background The exact biochemical basis for why differences in gluten protein composition inflour,e.g., presence or absence of certain wheat proteins or their proportions of the total gluten protein, causes differences in the viscoelastic properties of glutens and doughs is still not known for certain. However, wheat doughs preparedfromwheat lines that were devoid of a class of cereal proteins referred to as high molecular weight glutenin subunits (HMW-GS) were described as plastic, not elastic, and the gluten obtained from these doughs was



125 described as having the consistency of chewed gum (3). Thus, it seems certain that the HMW-GS are necessary in order to observe the property of recoverable deformation in wheat gluten and dough, but determining how exactly they interact with each other and the other cereal proteins to form an elastic network structure in gluten remains a challenge. One major problem that arises in any attempt to relate HMW-GS composition to the viscoelasticity of hydrated cereal proteins is the fact that determination of the individual HMW-GS composition of a wheat cultivar requires reduction of disulfide bonds to liberate the individual subunits from the native crosslinked glutenin structures that are naturally present in wheat flour. Another problem is that only a portion of the glutenin proteins are soluble with the presently used methodology, even after reduction of disulfide bonds and alkylation, and limited information is known about the remaining insoluble proteins. Thus, explaining viscoelasticity of gluten in the hydrated state requires a hypothetical "reassembling" of all of the subunits back into crosslinked clusters, and ultimately into a connected glutenin network structure. Ewart (4) provides a review of the major hypotheses for glutenin network structures that have resulted primarily from extraction and fractionation studies of the wheat proteins fromflour.Kasarda (5) reviews how molecular modeling has led to a better understanding of how the structures of individual glutenin subunits, in particular the number and locations of cysteine residues, might determine the properties of glutenin polymers. It is important to keep in mind here though that the term "glutenin polymers" in the context of cereal science refers to structures that result presumably mainly from the disulfide crosslinking of certain subunits into larger molecular weight clusters that exist in wheatflour,and not individual linear molecules as would usually be the case in synthetic polymer science. This is an important, and sometimes overlooked, conceptual difference when it comes to interpreting experimental viscoelastic data in terms of underlying network structures. This point will be emphasized further in the results and discussion section of this paper.

The HMW-GS and wheat quality Despite some of the obvious difficulties in relating HMW-GS composition to viscoelasticity of gluten or dough, the basis of elucidating structure-function relationships in wheat has narrowed over the last 20 years or so to focus mainly on the HMW-GS (2). A numbering system (nomenclature) for cataloging the HMW-GS of wheat cultivars based on their electrophoretic patterns was



126 published by Payne and Lawrence (5) and remains in worldwide use today. In a widely cited study, Payne et al. (7) determined the HMW-GS in grain for about 185 varieties of wheat, and observed differences in the both the number (3 to 5) of HMW-GS and their mobility in SDS-PAGE analysis. In a nutshell, the HMWGS are coded by three chromosomes (1A, IB, and ID), and allelic variation is observed at each locus. A majorfindingof the early work with HMW-GS was that the ID allelic subunit pair designated as "5+10" was strongly correlated with improved bread loaf volume relative to its allelic alternative ID subunit pair 2+12 (8, 9). But, there were a large number of 2+12 cultivars in the sample set. Of the 92% of the 185 cultivars that had either the 5+10 or the 2+12 ID coded pair of subunits, 55% were 2+12. On the other hand, analysis of the HMW-GS subunits in 70 Canadian grown wheats showed that essentially all of the hard wheats had the "5+10" subunits (70), while 43 out of 44 hard red spring wheats grown in North Dakota also contained the 5+10 ID coded pair of subunits {11). Apparently, most of the wheat breeders in North America had been selecting for stronger gluten when the end use was yeasted bread, unknowing with a result of a gene pool in which 5+10 is common. Likewise, breeders had been selecting for weaker gluten when the end-use was baked products such as cookies, cakes and crackers, unknowing resulting in gene pools with an abundance of 2+12 cultivars. But, there are a number of exceptions found in which relatively strong gluten wheat cultivars also contain 2+12 subunits. Given the above, it is not too surprising that tremendous efforts have been made to determine whether correlations between the intercultivar variations in the composition of the HMW-GS alone are sufficient for prediction of wheat quality in breeding programs. Primard et al (72) tested this exact hypothesis with 286 experimental wheat lines, and followed up with an additonal study involving 100 hard winter wheat lines of diverse origin (73). They found no significant differences in mean quality parameters (grain protein, dough mix time and dough tolerance to overmixing) in lines that contained the alternative 1A coded subunits 1 or 2*, or the IB coded alternatives 7+8 versus 7+9, or 7+8 versus 13+16, and also observed a wide range of mixograph characteristics for a population of wheat lines all carrying 5+10 ID subunits. In a later study it was concluded that allelic variations did not always result in significant quality differences, nor did allelic variation result in changes in the complete spectrum of quality variables (73). Khan et al (77) noted that interactions between gliadin and glutenin protein fractions seemed to be the important factor in influencing breadmaking quality parameters. Thus, it appears that knowing the HMW-GS subunit composition is only part of the "complete package" of information needed by breeders and cereal chemists for prediction of the functional traits in early generation experimental wheat lines. In the next section we describe how linear viscoelastic rheological


127 measurements on gluten can be used to obtain a direct measure of the network strength in gluten, which can be recovered from mixed doughs easily, and still retains the unique extensibility and elasticity of hydrated wheat proteins. The latter two being important functional aspects of total wheat quality.


Linear viscoelastic network strength of gluten as an index of wheat quality It is clear that many different combinations of chemical and/or physical dough properties and/or loaf volume measurement have been traditionally used as indices of wheat quality. Overall, mixed results have been obtained when attempting to correlate these measures of wheat quality with various wheat protein fractions or subunit composition. However, none of these methods that have been traditionally used as indices of wheat quality actually provide a direct non-destructive measure of the intercultivar variation in the "strength" of the elastic network in whole gluten. Thus, determination of the native network strength of whole gluten itself has been largely left out of the wheat quality loop, although the amount of wet gluten recovered from wheat flour has been used. Based on the above discussion, we think that development of an experimental rheological technique that allows for determination of the native network strength in gluten would be an important addition to the tools used for assessment of intercultivar variations in wheat quality. Linear viscoelastic measurements are very sensitive to small relative differences in network connectivity, which has been demonstrated by their ability to track very well changes in the extent of curing in chemical crosslinking reactions (14, 15). Characterizing the network strength of gluten in the fundamental (universal) units of polymer science will also allow for direct comparisons with a wide range of synthetic polymer networks with varied, but well characterized types of network structures and degrees of crosslinking. This should help us to better understand the basic nature of the interactions (entanglements, disulfide crosslinks, and/or non-covalent interactions) involved in the elastic gluten network structure, which in turn can be related to the chemistry of the cereal proteins. This concept will be tested here by determining the viscoelastic properties of glutens from "5+10" wheat cultivars differing only in their IB HMW-GS compositions. The effects of temperature and addition of a reducing agent on the viscoelastic properties will also be determined. The former to test the hypothesis that differences in viscoelastic properties between these cultivars may diminish as the temperature is increased, and the latter to verify which portion of the viscoelastic spectrum corresponds to the disulfide crosslinked network in whole gluten.


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Materials and Methods


Two single cultivar hard white spring wheat (HWS) flours, Golden 86 and Argent, were obtainedfromCook Natural Products (Oakland, CA.). A Canadian Western Extra Strong (CWES) red wheat cultivar, Glenlea, was used as an example of a very strong wheat for comparison. L-cysteine was obtained from Sigma Chemical Co. (St. Louis, MO).

Table 1. Protein content offloursand the HMW-GS (1A, IB, ID) subunits for the three cultivars. '

~—•—Cultivars

Properties Flour Protein (%) HMW-GS subunits 1A IB ID

Argent "—

Golden 86

Glenlea

. 13.5

13.17

12.9

2* 7+9 5+10

2* 7+8 5+10

2* 7+8 5+10

Thus, all three cultivars contain the lull complement of five HMW-GS, and differ in only one HMW-GS subunit, lBy9 for Argent versus lBy8 for Glenlea and Golden 86. Wet gluten was obtainedfromeachflouraccording to AACC method 38-10 (AACC) with some modification as described in Lee and Mulvaney (16). Gluten samples (60% water content on total weight basis) were prepared for rheological studies as described previously (16). Briefly, the gluten powder was rehydrated and then pressed between two aluminum plates with a 2.5 mm gap for 1 hour to form a gluten sheet of uniform thickness. Shear stress relaxation experiments (1% strain) were done with a Bohlin VOR-M rheometer (Bohlin Instruments, Cranbury, NJ), using parallel plate geometry as described previously (16). The shear relaxation modulus (G(t)) was obtained for 10,000s for control glutens at 15, 25, 35, and 40 °C, but only at 15, 25 and 35 °C for L-cysteine containing samples due to limited quantities offlours.Minitab version 12.0 (Minitab Inc, State College, PA, USA) was used for statistical analysis of rheological parameters. Differences in results were considered statistically significant at P

Characterization of Intercultivar Variation on the Linear Viscoelastic

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