Vol. 74, No. 1, 1997 63 CARBOHYDRATES Sources of Variation for Starch Gelatinization, Pasting, and Gelation Properties in Wheat MING ZENG,1 CRAIG F. MORRIS,2,3 IAN L. BATEY,4 and COLIN W. WRIGLEY4 ABSTRACT Cereal Chem. 74(1):63���71 The starch of wheat (Triticum aestivum L.) flour affects food product quality due to the temperature-dependent interactions of starch with water during gelatinization, pasting, and gelation. The objective of this study was to determine the fundamental basis of variation in gelatiniza- tion, pasting, and gelation of prime starch derived from seven different wheat cultivars: Kanto 107, which is a partial waxy mutant line, and six near-isogenic lines (NILs) differing in hardness. Complete pasting curves with extended 16-min hold at 93��C were obtained using the Rapid Visco Analyser (RVA). Apparent amylose content ranged from 17.5 to 23.5% total amylose content ranged from 22.8 to 28.2%. Starches exhibited significant variation in onset of gelatinization. However, none of the parameters measured consistently correlated with onset or other RVA curve parameters that preceded peak paste viscosity. Peak paste viscosity varied from 190 to 323 RVA units (RVU). Higher peak, greater breakdown, lower final viscosity, negative setback, and less total setback were associated with lower apparent and total amylose contents. Each 1% reduction in apparent or total amylose content corresponded to an increase in peak viscosity of about 22 and 25 RVU, respectively, at 12% starch concentration. Of the seven U.S. cultivars, the lower amylose cultivars Penawawa and Klasic were missing the granule-bound starch synthase (GBSS ADPglucose starch glycosyl transferase, EC 2.4.4.21) protein associated with the Waxy gene locus on chromosome 4A (Wx-B1 locus). Kanto 107 was confirmed as missing both the 7A and 4A waxy proteins (Wx-A1 and Wx-B1 loci). The hardness NIL also were shown to be null at the 4A locus. Apparent and total amylose contents of prime starch generally corresponded well to the number of GBSS proteins although the hardness NIL tended to have somewhat higher amylose contents than did the other GBSS 4A nulls. We concluded that reduced quantity of starch amylose due to decreased GBSS profoundly affects starch gelatinization, pasting, and gelation properties. Starch is the primary component of wheat (Triticum aestivum L.) flour, and consequently, plays an important role as a determinant of food product quality. Most of the functional attributes of starch can be related to the temperature-dependent interactions of starch with water in the processes known as gelatinization, pasting, and gelation (retrogradation) (Dengate 1984, Atwell et al 1988). As starch is heated in the presence of water, granules swell and imbibe hydrogen bonds are disrupted with eventual irreversible loss of crystallite structure (gelatinization). Pasting generally refers to changes in viscosity just before, during, and after the sensu stricto event of gelatinization. Consequently, pasting also refers to changes in viscosity during gelation. As a starch-water system cools, starch polymer-water hydrogen bonds are replaced with polymer-polymer hydrogen bonds, and a gel network is formed. At a molecular level, this reassociation process is more aptly termed retrogradation. Although the literature is replete with research on many aspects of gelatinization, pasting, and gelation of starch, little work has been conducted on the basis of variation for these phenomena in wheat. In this report, the general phenom- ena of gelatinization, pasting, and gelation will be collectively referred to as pasting, especially in the context of viscosity, and unless special reference is made to the molecular or physical events associated with gelatinization and retrogradation. The study of the pasting of wheat starch dates back at least to the 1920s. Rask and Alsberg (1924) found distinct differences in peak paste viscosity among the total (prime plus tailing) defatted starches isolated from 11 commercial and known cultivar flours. They suggested that the changes in peak viscosity associated with changing starch concentration could best be explained by consid- ering the concentration of the amylopectin component of the starch, rather than the total starch concentration. Although this explanation was advanced as the basis for the observed concen- tration-dependent variation for a given starch, it was not extended to explain differences among starches derived from different wheats. Anker and Geddes (1944) reported that the differences in maximum paste viscosity of starches isolated from five commer- cial flours of diverse origin and market class were relatively small and of no significance. Hutchinson (1966) and Moss (1967) demonstrated that signifi- cant variation in pasting among wheat flours was independent of amylase activity, and therefore an inherent property of the flour. They further showed that Australian wheat in general (Hutchinson 1966), and certain Australian cultivars in particular (Moss 1967), had inherently higher paste viscosity. In that same era, Medcalf and Gilles (1965) found that four durum wheat starches generally had elevated amylose levels and reduced pasting peaks compared to 15 hard wheat starches. Loney and coworkers (Loney and Meredith 1974, Loney et al 1975) further substantiated that genetic factors (i.e., differences among cultivars) were a significant source of variation for peak paste viscosity of prime starch. The Australian cultivars Gamenya, Raven, Falcon, and Gamut were significantly higher pasting than four New Zealand wheat cultivars, irrespective of where they were grown. Loney et al (1975) appear to be the first to extend the observation for rice (Halick and Kelly 1959) that lower amylose, and therefore higher amylopectin, is associated with higher peak viscosity in wheat. Falcon and Gamut were ���1.5���3% or 2���5% lower in amylose than the New Zealand cultivars, depending on method of analysis (Loney et al 1975). A number of subsequent reports have supported the view that genetic variation in both peak paste viscosity and amylose exist in wheat, and that, generally, lower amylose corresponds to higher 1Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164-6394. Washington State University Scientific Paper 9610-15. 2USDA-ARS Western Wheat Quality Laboratory, E-202 Food Science & Human Nutrition Facility East, Washington State University, Pullman, WA 99164-6394. Mention of trademark or proprietary products does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. 3 Corresponding author. E-mail: morrisc@wsunix.it.wsu.edu, Fax: 509/335-8573, Phone 509/335-4062 4Grain Quality Research Laboratory, C.S.I.R.O. Division of Plant Industry, P.O. Box 7, North Ryde, NSW, 2113 Australia. Publication no. C-1997-0110-03R. This article is in the public domain and not copyrightable. It may be freely re- printed with customary crediting of the source. American Association of Cereal Chemists, Inc., 1997.

CEREAL CHEMISTRY 64 peak paste viscosity. Total starch of Australian Standard White (ASW) wheat from Western Australia had 2.0% less amylose and higher peak viscosity when compared to U.S. Western White wheat (Oda et al 1980). However, Norin 61, a Japanese domestic cultivar, exhibited an amylose content similar to that of Western White, but a peak viscosity more similar to the ASW starch. Peak paste viscosity was significantly correlated with apparent amylose (iodine blue value) among starches obtained from several individ- ual Australian cultivars (r = ���0.73 and ���0.67) (Moss 1980 and Moss and Miskelly 1984, respectively). Gamenya and Halberd cultivars were higher pasting and had apparent amylose contents of 19.2 and 22.6%, respectively, while the cultivars Oxley and Egret were lower pasting and had apparent amylose contents of 28.0 and 32.0%, respectively (Moss 1980). The amylose content of prime starch of commercial Western Australia ASW was ���3% lower than commercial U.S. Western White wheat, and three Japanese domestic cultivars (Chihoku, Norin 61, and Horoshiri) (Endo et al 1989). Chihoku exhibited the highest peak paste vis- cosity followed by the ASW starch. Kuroda et al (1989, cited in Yamamori et al 1992) reported variation in flour amylose content of 21���30% among Japanese cultivars. The two lowest amylose lines, Kanto 107 and Kanto 79, had flour amylose contents of ���21���23%. In a survey of 66 wheat cultivars dating back to 1860, McCormick et al (1994) found that most high-pasting cultivars were derivatives of the cultivar Currawa, released in 1912, while most semi-dwarf material developed from the CIMMYT intro- ductions, WW80 and WW15, had low peak viscosity. King et al (1994) found that among three U.S. cultivars, the prime starch derived from Klasic was markedly higher in peak paste viscosity than that of McKay and Madsen. In addition to peak paste viscosity, other gelatinization, pasting, and gelation properties are known to vary among different wheats. Anker and Geddes (1944) found that the soft wheat starch sample included in their study had a lower transition temperature (onset of pasting) and a lower temperature of maximum viscosity when compared to the other four samples. Oda et al (1980) found that starches with lower amylose content tended to reach peak viscos- ity at a lower temperature (and in less time) and also had greater breakdown (amylograph paste stability, or D-value). Moss and Miskelly (1984) also found that the two cultivars with lower starch amylose, Gamenya and Halberd, had greater breakdown. Endo et al (1989) reported that the ASW and Chihoku starches, which exhibited high peak viscosity, exhibited lower gelatinization temperature and lower breakdown. However, from the results presented (Endo et al 1989) it is clear that these two starches exhibited greater breakdown when compared to the Norin 61, Western White, and Horoshiri samples. Batey et al (1993) reported that the prime starch of Klasic was markedly higher pasting, and exhibited much greater breakdown and final viscosi- ties than did McKay both are U.S. cultivars. Konik et al (1992) found that starch peak paste viscosity, holding strength (minimum viscosity), breakdown, final viscosity, setback, and pasting tem- perature varied among 42 cultivars and breeding lines of Austra- lian wheat. All pasting parameters were significantly correlated with each other (r 0.50, P 0.001). In a later study, Konik et al (1994) reported similar results and all starch pasting parameters were again significantly correlated to one another, except peak viscosity. Beyond these simple correlations and observations between amylose content and pasting properties of starch, no additional insight into the fundamental basis of variation for gelatinization, pasting and gelation properties was obtained. Although ��ebeFiD (1989) found no significant relationship between amylose content of prime starch and the peak amylograph viscosity of flours, they did refer to unidentified starch physicochemical properties as a primary and fundamental cause for differences in amylograph peak viscosity. Endo et al (1989) indicated that sodium dodecyl sulfate (SDS) extracts of total starch of ASW, Chihoku, and Norin 61 possessed a substantially greater amount of a late-eluting high- performance liquid chromatography (HPLC) peak compared to the lowest pasting type, Horoshiri. A significant advance came when Yamamori et al (1992) correlated the relative quantity of granule- bound starch synthase (GBSS) with the amylose content of 31 Japanese cultivars as reported by Kuroda et al (1989). GBSS is the enzyme associated with the synthesis of amylose and with what is known as the waxy gene locus (Tsai 1974). Yamamori et al (1992) found that the amylose content of flour sifted from whole grain wheat meal ranged from 21.6 to ���30%, and corresponded rela- tively well with the amount of waxy protein detected as a single band in gel electrophoresis. Kanto 107 and Kanto 79 were reported as having both the lowest observed amylose content and the lowest quantity of GBSS. In a subsequent report, Nakamura et al (1993) characterized the GBSS proteins of these two Kanto lines and found that two of the three GBSS proteins were not detected. As such, they were termed ���partial waxy mutants.��� Beyond the research described above, we are aware of only two reports that relate the genetics of GBSS with starch amylose con- tent or pasting properties. Miura and Tanii (1994) found that the amylose content of prime starch varied among five wheats from 21.3% (Kanto 107) to a high of 24.5% (Cheyenne). Significant differences between cultivars across years were noted. However, the peak viscosity of flour tended not to be correlated with amylose content of starch. Cheyenne differed significantly from all other cultivars in having a higher flour peak viscosity, yet a high starch amylose content. Although the cultivars were characterized eletrophoretically and classified into three groups based on the pattern of GBSS, no assignment to flour pasting properties was advanced. Starch amylose content, however, generally followed an inverse relation- ship with GBSS levels. Sprouting occurred in some samples and these authors, as well as others (��ebeFiD 1989), have pointed out that sprouting or the presence of amylases may preclude the drawing of appropriate conclusions. Zhao et al (1995) found that those progeny possessing the GBSS protein of the Waxy locus on chromosome 4A as a group exhibited significantly higher starch amylose content compared to those that lacked the protein (partial waxy 4A-nulls). The objective of this study was to determine some of the fun- damental bases of variation in gelatinization, pasting, and gelation of prime starch derived from different wheat cultivars, especially the associations among presence of GBSS isozymes, starch amy- lose content and starch pasting properties. MATERIALS AND METHODS Samples Two sets and two individual grain samples were used for a total of 14 different wheats and derived prime starches (Table I). Sam- ple Set 1 was comprised of six U.S. wheat cultivars: Lewjain, Stephens, Hill 81, and Madsen (soft white winter), and Klasic and Penawawa (hard and soft white spring cultivars, respectively). Set 1 was grown at the Spillman Agronomy Farm, Pullman, WA, in 1993. Penawawa and Klasic were known a priori to exhibit inher- ently higher flour paste viscosity compared to most other cultivars in the Pacific Northwest (data not shown). To these six samples were added a sample of Butte 86 hard red spring wheat variety, grown at Spillman Agronomy Farm in 1991, and Kanto 107, a soft red spring wheat produced in Japan. Kanto 107 possesses the null allele for two of the three Waxy loci which code for GBSS (Nakamura et al 1993). Based on results from Set 1 and Butte 86, six samples were selected from a set of near- isogenic lines (NIL) that differ in grain hardness and added to the study (designated Set 2, Table I). These NILs were produced by crossing the Australian cultivars Heron (soft) and Falcon (hard), and making six back crosses using Falcon as the recurrent parent (Symes 1969). They have the Australian Winter Cereals Collec-