Analysis and Specification of Starch Granule Size Distributions

Summary

Presented here is a procedure for reproducible and statistically valid determinations of starch granule size distributions, and for specifying the determined granule lognormal size distributions using a two-parameter multiplicative form. It is applicable to all granule sizing analyses of gram-scale starch samples for plant and food science research.  

Abstract

Starch from all plant sources are made up of granules in a range of sizes and shapes having different occurrence frequencies, i.e., exhibiting a size and a shape distribution. Starch granule size data determined using several types of particle sizing techniques are often problematic due to poor reproducibility or lack of statistical significance resulting from some insurmountable systematic errors, including sensitivity to granule shapes and limits of granule-sample sizes. We outlined a procedure for reproducible and statistically valid determinations of starch granule size distributions using the electrical sensing zone technique, and for specifying the determined granule lognormal size distributions using an adopted two-parameter multiplicative form with improved accuracy and comparability. It is applicable to all granule sizing analyses of gram-scale starch samples, and, therefore, could facilitate studies on how starch granule sizes are molded by the starch biosynthesis apparatus and mechanisms; and how they impact properties and functionality of starches for food and industrial uses. Representative results are presented from replicate analyses of granule size distributions of sweetpotato starch samples using the outlined procedure. We further discussed several key technical aspects of the procedure, especially, the multiplicative specification of granule lognormal size distributions and some technical means for overcoming frequent aperture blockage by granule aggregates.

Introduction

Starch granules are the physical structure in which two main reserve homoglucan polymers in plant photosynthesis and storage tissues, the linear or sparsely branched amylose and the highly branched amylopectin, are orderly packed along with some minor components, including lipids and proteins. Starch granules from various plant species exhibit many three-dimensional (3D) shapes (reviewed in ref.^1,2), including spheres, ellipsoids, polyhedrons, platelets, cubes, cuboids, and irregular tubules. Even those from the same tissue or different tissues of the same plant species could have a set of shapes with varying ....

Protocol

1. Preparation of starch samples

1. Prepare two (or three) gram-scale replicate starch samples from starch-accumulating tissues of various plant species following the established procedures (e.g., potatoes¹⁵, sweetpotatoes²⁸, wheat grains^13,29, and maize kernels³⁰, etc.).
2. Thoroughly wash starch samples with acetone or toluene 3-4x to minimize granule aggregates and dry them completely.
   NOTE: Use extraction procedures that yield more than 1 g of starch per preparation. One or two 0.5-g aliquots from each of the three....

Results

To validate the procedure, and demonstrate reproducibility of the determined granule size distribution, we performed replicate sizing analyses of sweetpotato starch samples. We prepared replicate (S1 and S2) starch samples from field-grown sweetpotatoes of a breeding line SC1149-19 at a similar developmental age using a previously described procedure²⁸. From each starch extract, two 0.5 g aliquots (a and b) were sampled, suspended in 5 mL of methanol and sonicated with several pulses of low-energy.......

Discussion

The outlined procedure has resolved some critical issues in several existing methods for starch granule size analyses, including inappropriate 1D or 2D sizing of 3D granules, distortion of sizing measurements due to none-uniform granule shapes, poor reproducibility and dubious statistical validity due to limited granule-sample sizes, inaccurate or improper specification (especially the use of the average size) of granule sizes in the presence of both granule shape and none-normal size distributions. It uses the ESZ techn.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Analytical beaker	Beckman Coulter Life Sciences	A35595	Smart-Technology (ST) beaker
Aperture tube, 100 µm	Beckman Coulter Life Sciences	A36394	For the MS4E
Disposable transfer pipettor,	Fisher Scientific (Fishersci.com)	13-711-9AM	Other disposable transfer pipettors with similar orifice can also be used.
Fisherbrand Conical Polypropylene Centrifuge Tubes, 50 ml	Fisher Scientific (Fishersci.com)	05-539-13	Any other similar types of tubes can be used.
Glass beakers, 150 to 250 ml	Fisher Scientific (Fishersci.com)	02-540K	These beakers are used to contain methanol for washing the aperture tube and stirrer between runs.
LiCl	Fisher Chemical	L121-100
Methanol	Fisher Chemical	A412-500	Buy in bulk as the analysis uses a large quantity of methanol.
Mettler Toledo ML-T Precision Balances	Mettler Toledo	30243412	Any other precision balance with a readability 0.01 g to 1 mg will work.
Multisizer 4e Coulter Counter	Beckman Coulter Life Sciences	B23005	The old model, Multisizer 3 can also be used with slight adjustment of parameters. The 4e model comes with a 100 μm aperture tube. Other aperture tubes of different diameter can be purchased separately from the company.
Ultrasonic processor UP50H	Hielscher Ultrasound Technology	UP50H	Other laboratory sonicator having a low-power (<50 Watt) output can be also used. Both MS1 and MS2 sonotrodes for the particular sonicator can be used to disperse starch granules in 5 ml methanol. Always use the lowest setting first, 20% amplitude and 0.1 or 0.2 cycle, and raise the setting if aggregates persist in suspension.