Source: UNIV OF IDAHO submitted to
STARCH GRANULE STRUCTURE AND REACTIVITY CHARACTERIZATION USING HIGH PERFORMANCE SIZE EXCLUSION CHROMATOGRAPHY WITH FLUORESCENCE DETECTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0203842
Grant No.
2005-35503-16218
Project No.
IDA00502-CG
Proposal No.
2005-01320
Multistate No.
(N/A)
Program Code
71.1
Project Start Date
Sep 1, 2005
Project End Date
Aug 31, 2006
Grant Year
2005
Project Director
Huber, K. C.
Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803
Performing Department
FOOD SCIENCE & TOXICOLOGY
Non Technical Summary
Starch, which represents the second greatest biomass on the planet, is utilized in many food and industrial applications because of its abundance, relative low cost, and properties. It exists in the form of semi-crystalline, water insoluble granules comprised of two polymers: amylose and amylopectin. End-use applications of starch depend on its physiochemical properties, which are determined by starch granule composition and molecular structure. A comprehensive understanding of starch structure and reactivity is key to maximizing starch utilization and application in food and industrial settings. The acquisition of an HPSEC system with fluorescence analysis capability will provide increased insight and interpretative power into the molecular structural elements of starch granules and starch based materials. This instrument will extend the research capability by providing a novel capability presently not available. Further, it is anticipated to stimulate multidisciplinary collaborations and cross-sharing of ideas between researchers of multiple colleges (Agricultural and Life Sciences; Natural Resources; Engineering; Science). The instrument will also enhance the ability to generate both preliminary and full-scale data necessary for the procurement of significant research funding, which will facilitate the expansion, competitiveness, and long-term stability of the associated research programs.
Animal Health Component
(N/A)
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5021310200040%
5021543200060%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1543 - Soft white wheat; 1310 - Potato;

Field Of Science
2000 - Chemistry;
Goals / Objectives
To acquire a Waters Breeze HPSEC System coupled with a Waters 2475 Fluorescence detector (Waters Corp., Milford, MA). This equipment will greatly enhance ability to characterize and understand starch granule structure and reactivity in relation to functional properties of starch within food and non-food systems, and will extend the research capability in support of both current and future research projects.
Project Methods
Starch molecules, which consist of both linear (amylose) and branched (amylopectin) polymers of alpha-D-glucose, are packaged together and assembled in the form of semi-crystalline aggregates, called granules. Starch granules, which are insoluble in room temperature water, are stabilized by regions of complex molecular order, and require heating sufficient to disrupt the native granular order to achieve starch solubility and functionality. Methods such as x-ray diffraction, enzyme labeling, chemical gelatinization, and atomic force microscopy have all been used to gain a better understanding of starch granule architecture. Recently, the use of fluorescent dyes in combination with confocal scanning laser microscopy (CSLM) has proven to be a very effective method for visual elucidation of starch granule structural features. It has also provided significant visual insight as to how granules react with chemical reagents to generate chemically modified starches. While the use of fluorescence in combination with CSLM provides an overall view of the starch granule and its gross anatomical structures through optical sectioning, it lacks the ability to provide specific details of starch granule architecture at the molecular level. High performance size exclusion chromatography (HPSEC) has been used extensively for molecular characterization of amylose and amylopectin polymers. Physical characteristics of amylose and amylopectin such as molecular weight, radius of gyration, branching prevalence, and branch chain length distribution are all routinely evaluated when the resolving power of HPSEC is coupled with light scattering, viscometric, pulsed amperometric, and/or refractive index detectors. While HPSEC has proved useful for investigating the molecular characteristics of amylose and amylopectin, it sheds little light on the original location and spatial arrangement of amylose and amylopectin within the native starch granule. Nevertheless, by combining the use of fluorescence imaging with the capabilities of HPSEC, the gap between granule microstructure and starch molecular attributes can be more effectively bridged.

Progress 09/01/05 to 08/31/06

Outputs
The Waters Breeze HPLC system along with the Waters 2475 fluorescence detector was installed and set up for operation in November 2005. An existing Waters 2410 refractive index detector was also coupled to the system as pledged in the original proposal, after which the system was set up for size exclusion analysis (SEC) of starch polymers. Over the past year, a novel microwave-assisted starch dissolution method was pioneered in our laboratory that has greatly streamlined sample preparation (takes only 30 minutes compared to 12-24 hours for traditional methods) for starch characterization via SEC analysis (work funded by USDA-NRICGP Proposal No: 2003-01730). While the method has been useful for starch analysis via low-pressure chromatography systems (requires 2 days for a single run), it has not been applicable to more rapid analysis using HPSEC systems due to interference from co-eluting peaks present in the initial dissolution medium. However, this limitation has been recently overcome, paving the way for both rapid starch dissolution and structural analysis via HPSEC (entire process can be accomplished in 2-3 hours). Thus, the Waters Breeze HPLC system is now poised to play a meaningful role in our starch characterization efforts. In the original proposal, three primary research projects were described to justify the need for the instrument (1. Analysis of Starch Granule Surface Structure and Composition; 2. Development of Potato-based Ingredients with Moderated Glycemic Response; 3. Investigation of Wheat Starch Composition and Reactivity). Work on all three of the projects has now commenced. Project 1 involves site-directed derivatization of starch granules with a fluorescent probe and subsequent analysis of molecular patterns of fluorescence using the newly acquired Waters HPLC system to bridge the knowledge gap between starch granular and molecular structures. It has been verified that the new HPLC system is capable of detecting molecular patterns of fluorescence, which capability provides the foundation for a USDA-NRICGP grant that will be submitted for FY 2007. For Project 2, a similar approach using a fluorescent probe will be used to characterize and optimize development of potato-based resistant starch products with moderated glycemic response. The HPLC system is critical to characterizing starch molecular structures that are resistant to digestion and result in a reduced glycemic response. Lastly, Project 3, which is currently funded by USDA-NRICGP (Proposal Number 2003-01730), seeks to understand structural differences between A- and B-type starch granule populations of wheat as they relate to chemical modification processes and end-use functionality. The HPLC system is being used to investigate differences in molecular structure and reactivity for the two granule populations to aid development of modified starch products from wheat. A total of three to four refereed publications is expected to result from current projects utilizing the new HPLC system.

Impacts
Starch is an important polymeric material common to a wide range of agricultural commodities, and represents the second greatest component biomass on the planet. Its annual production continues to grow each year, and an understanding of starch structure and behavior is key to effective utilization in new and emerging markets. In short, the acquired Breeze HPLC system provides a significant capability and tool needed to understand molecular aspects of starch polymers. It has already enhanced the competitiveness of our research laboratory through direct contribution to both existing and planned USDA-NRICGP projects and grants. Thus, the HPLC system has extended the research potential and capability of my laboratory, has facilitated collaborations and funding opportunities for development of future projects, and will continue to enhance the potential for meaningful scientific contribution related to US agricultural interests.

Publications

  • No publications reported this period