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United States Department of Agriculture

Agricultural Research Service

Title: A method for the molecular characterization of rice starch using an aqueous HPSEC-MALLS-RI system

Authors
item Chen, Ming-Hsuan
item Bergman, Christine - UNLV

Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: January 1, 2006
Publication Date: February 15, 2006
Citation: Chen, M.H., Bergman, C.J. 2006. A method for the molecular characterization of rice starch using an aqueous HPSEC-MALLS-RI system. In: 31st Proceedings of the Rice Technical Working Group Meeting Proceedings. 2006 CDROM.

Technical Abstract: Rice starch composes approximately 90% of milled rice. It is made up of two major glucose polymers, amylose and amylopectin. The amylose content of rice starch ranges from 0 to 30% w/w. Cooked rice texture and rice starch functional properties are reported to be primarily impacted by amylose content. However, evidence is building that variation in other aspects of rice kernels are also important determinants of rice cooking and processing quality. Some of these attributes include: water soluble versus insoluble amylose content and debranched amylopectin chain length (CL). Examples of other starch characteristics that might influence rice functional properties that have received limited attention are molecular mass and mass distributions and branching parameters such as root mean-square radius (Rg) and connectivity between branches. In this report, we present the development and evaluation of a method for studying various aspects of these rice starch characteristics. Eight rice cultivars used for this study included two high, intermediate, and low amylose-, and glutinous-types. Protein and lipid fractions were extracted using NaOH and 85% methanol, respectively. Starch was gelatinized with dimethylsulfoxide and digested with isoamylase. The isoamylolysates were resolved and the molecular structure of amylose was characterized using high-performance size exclusion chromatography and multi-angle laser light scattering and refractive index detectors (HPSEC-MALLS-RI). The columns used were one guard column and two analytical size-exclusion columns connected in tandem, which were packed with polystyrene divinylbenzene cross-linked copolymer, and were maintained at 70 ºC. The mobile phase was 50 mM NaNO3 and 0.02% NaN3. The debranched starch was precipitated with the addition of butanol, and the supernatant amylopectin chain structure was characterized using HPSEC-MALLS-RI. Three different mass fractions of amylose were observed and their distributions differed among the samples analyzed. The specific parameters determined included: weight-average molecular mass (Mw) (or weight-average degree of polymerization, DPw), number-average molecular mass (Mn) (or number-average degree of polymerization, DPn), polydispersity, and Rg. Most of the molecular parameters for the amylose fraction and its subfractions were reproducible (coefficients of variation < 5%). The range of Mw and Mn of amylose was 5.1 – 6.9 x 105 and 1.4 – 1.8 x 105, respectively, for six cultivars varying in amylose content. Small linear chains of DPn < 3.07 x 103 (Mn = 3.66 x 105) accounted for >90% of the molar population. Considering the molar-distribution, the high amylose-type and the low amylose-type primarily differed quantitatively, relative to the intermediate amylose-type, Dellmont, in the smaller-DPn range of the linear chains. Two debranched amylopectin subfractions, the high- (AmpF1) and the low- (AmpF2) hydrodynamic-volume fractions were observed. Good repeatability was obtained for the Mw determination of AmpF1 and AmpF2 (CV < 2%) and for the mass ratio of AmpF2/F1 (CV <6%). The molar-distributions of the amylopectin weight-average chain length (CLw) of L201 and Bengal demonstrated that L201, an intermediate gelatinization-temperature cultivar, has a higher molar proportion between CLw 15 to 25 and less between 9 to12 relative to Bengal, a low gelatinization-temperature cultivar. These results are comparable to those reported using high-performance anion-exchange chromatography with pulsed-amperometric detection in that the higher gelatinization-temperature cultivars are rich in chains of 12<DP<24 and lower in chains of DP<11. However, no artificial normalization of mass response to CL is required in the present method. The method described above enables the measurement of starch characteristics that are known to impact rice functionality as well as others that are hypothesized to, including: weight- and molar-based distributions of DP, and Mw and Mn of amylose and amylopectin fine structure. With this method, large sample sets can be analyzed within a relatively short time frame with good repeatability, thus making it suitable for use in studies directed at understanding rice starch functionality and the genetics controlling these traits. This method should also be applicable for starch structural characterizations of diverse botanical sources.

Last Modified: 8/1/2014
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