Name: analysis and specification of starch granule size distributions
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Description: Watch this Scientific Journal Video about Analysis and Specification of Starch Granule Size Distributions at JoVE.com

This work is partly supported by the Cooperative Agriculture Research Center, and Integrated Food Security Research Center of the College of Agriculture and Human Sciences, Prairie View A&#38;M University. We thank Hua Tian for his technical support.

1. Preparation of starch samples
<ol>
	<li>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 grains13,29, and maize kernels30, etc.).</li>
	<li>Thoroughly wash starch samples with acetone or toluene 3-4x to minimize granule aggregates and dry them...

<ol>
	<li>Shannon, J. C., Garwood, D. L., Boyer, C. D., BeMiller, J., Whistler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Starch:Chemistry and Technology Food Science and Technology. , 23-82 (2009).</li><li>Singh, N., Singh, J., Kaur, L., Singh Sodhi, N., Singh Gill, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological,+thermal+and+rheological+properties+of+starches+from+different+botanical+sources.">Morphological, thermal and rheological properties of starches from different botanical sources.</a> Food Chemistry. 81 (2), 219-231 (2003).</li><li>Lindeboom, N., Chang, P. R., Tyler, R. 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L., Kasemsuwan, T., Leas, S., Zobel, H., Robyt, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthology+of+starch+granule+morphology+by+scanning+electron+microscopy.">Anthology of starch granule morphology by scanning electron microscopy.</a> Starch-St&#228;rke. 46 (4), 121-129 (1994).</li><li>Matsushima, R., Nakamura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Starch: Metabolism and Structure. , 425-441 (2015).</li><li>Wang, S. -. q., Wanf, L. -. l., Fan, W. -. h., Cao, H., Cao, B. -. s. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+analysis+of+common+edible+starch+granules+by+scanning+electron+microscopy.">Morphological analysis of common edible starch granules by scanning electron microscopy.</a> Food Science. 32 (15), 74-79 (2011).</li><li>Baldwin, P. M., Adler, J., Davies, M. C., Melia, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Holes+in+starch+granules:+confocal,+SEM+and+light+microscopy+studies+of+starch+granule+structure.">Holes in starch granules: confocal, SEM and light microscopy studies of starch granule structure.</a> Starch-St&#228;rke. 46 (9), 341-346 (1994).</li><li>Chakraborty, I., Pallen, S., Shetty, Y., Roy, N., Mazumder, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+microscopy+techniques+for+revealing+molecular+structure+of+starch+granules.">Advanced microscopy techniques for revealing molecular structure of starch granules.</a> Biophysical Reviews. 12 (1), 105-122 (2020).</li><li>Bechtel, D. B., Wilson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloplast+formation+and+starch+granule+development+in+hard+red+winter+wheat.">Amyloplast formation and starch granule development in hard red winter wheat.</a> Cereal Chemistry. 80 (2), 175-183 (2003).</li><li>Evers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+wheat+starch.+III.+Granule+development+in+the+endosperm.">Scanning electron microscopy of wheat starch. III. Granule development in the endosperm.</a> Starch-St&#228;rke. 23 (5), 157-162 (1971).</li><li>Wang, Y. J., White, P., Pollak, L., Jane, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+starch+structures+of+17+maize+endosperm+mutant+genotypes+with+Oh43+inbred+line+background.">Characterization of starch structures of 17 maize endosperm mutant genotypes with Oh43 inbred line background.</a> Cereal Chemistry. 70, 171-179 (1993).</li><li>Peng, M., Gao, M., Abdel-Aal, E. S. M., Hucl, P., Chibbar, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+and+characterization+of+A-and+B-type+starch+granules+in+wheat+endosperm.">Separation and characterization of A-and B-type starch granules in wheat endosperm.</a> Cereal Chemistry. 76, 375-379 (1999).</li><li>Wilson, J. D., Bechtel, D. B., Todd, T. C., Seib, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+wheat+starch+granule+size+distribution+using+image+analysis+and+laser+diffraction+technology.">Measurement of wheat starch granule size distribution using image analysis and laser diffraction technology.</a> Cereal Chemistry. 83 (3), 259-268 (2006).</li><li>Liu, Q., Weber, E., Currie, V., Yada, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+properties+of+starches+during+potato+growth.">Physicochemical properties of starches during potato growth.</a> Carbohydrate Polymers. 51 (2), 213-221 (2003).</li><li>Chmelik, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+size+characterization+of+barley+starch+granules+determined+by+electron+and+optical+microscopy,+low+angle+laser+light+scattering+and+gravitational+field-flow+fractionation.">Comparison of size characterization of barley starch granules determined by electron and optical microscopy, low angle laser light scattering and gravitational field-flow fractionation.</a> Journal of the Institute of Brewing. 107 (1), 11-17 (2001).</li><li>Moon, M. H., Giddings, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+separation+and+measurement+of+particle+size+distribution+of+starch+granules+by+sedimentation/steric+field-flow+fractionation.">Rapid separation and measurement of particle size distribution of starch granules by sedimentation/steric field-flow fractionation.</a> Journal of Food Science. 58 (5), 1166-1171 (1993).</li><li>Wriedt, T., Wolfram, H., Wriedt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mie Theory: Basics and Applications. , 53-71 (2012).</li><li>Schuerman, D. W., Wang, R. T., Gustafson, B. &. #. 1. 9. 7. ;. S., Schaefer, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+studies+of+light+scattering.+1:+Particle+shape.">Systematic studies of light scattering. 1: Particle shape.</a> Applied Optics. 20 (23), 4039-4050 (1981).</li><li>Goering, K. J., Fritts, D. H., Eslick, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+starch+granule+size+and+distribution+in+29+barley+varieties.">A study of starch granule size and distribution in 29 barley varieties.</a> Starch-St&#228;rke. 25 (9), 297-302 (1973).</li><li>Chen, Z., Schols, H. A., Voragen, A. G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch+granule+size+strongly+determines+starch+noodle+processing+and+noodle+quality.">Starch granule size strongly determines starch noodle processing and noodle quality.</a> Journal of Food Sciences. 68 (5), 1584-1589 (2003).</li><li>Dai, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch+granule+size+distribution+in+grains+at+different+positions+on+the+spike+of+wheat+(Triticum+aestivum+L.).">Starch granule size distribution in grains at different positions on the spike of wheat (Triticum aestivum L.).</a> Starch-Starke. 61 (10), 582-589 (2009).</li><li>Edwards, M. A., Osborne, B. G., Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+endosperm+starch+granule+size+distribution+on+milling+yield+in+hard+wheat.">Effect of endosperm starch granule size distribution on milling yield in hard wheat.</a> Journal of Cereal Science. 48 (1), 180-192 (2008).</li><li>Karlsson, R., Olered, R., Eliasson, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+starch+granule+size+distribution+and+starch+gelatinization+properties+during+development+and+maturation+of+wheat,+barley+and+rye.">Changes in starch granule size distribution and starch gelatinization properties during development and maturation of wheat, barley and rye.</a> Starch - Starke. 35 (10), 335-340 (1983).</li><li>Li, W. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+starch+granule+size+distribution+between+hard+and+soft+wheat+cultivars+in+Eastern+China.">Comparison of starch granule size distribution between hard and soft wheat cultivars in Eastern China.</a> Agricultural Sciences China. 7 (8), 907-914 (2008).</li><li>Park, S. H., Wilson, J. D., Seabourn, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch+granule+size+distribution+of+hard+red+winter+and+hard+red+spring+wheat:+Its+effects+on+mixing+and+breadmaking+quality.">Starch granule size distribution of hard red winter and hard red spring wheat: Its effects on mixing and breadmaking quality.</a> Journal of Cereal Science. 49 (1), 98-105 (2009).</li><li>Limpert, E., Stahel, W. A., Abbt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Log-normal+distributions+across+the+sciences:+keys+and+clues.">Log-normal distributions across the sciences: keys and clues.</a> Bioscience. 51 (5), 341-352 (2001).</li><li>Gao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-preserving+lognormal+volume-size+distributions+of+starch+granules+in+developing+sweetpotatoes+and+modulation+of+their+scale+parameters+by+a+starch+synthase+II+(SSII).">Self-preserving lognormal volume-size distributions of starch granules in developing sweetpotatoes and modulation of their scale parameters by a starch synthase II (SSII).</a> Acta Physiologiae Plantarum. 38 (11), 259 (2016).</li><li>Wattebled, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STA11,+a+Chlamydomonas+reinhardtii+locus+required+for+normal+starch+granule+biogenesis,+encodes+disproportionating+enzyme.+Further+evidence+for+a+function+of+alpha-1,4+glucanotransferases+during+starch+granule+biosynthesis+in+green+algae.">STA11, a Chlamydomonas reinhardtii locus required for normal starch granule biogenesis, encodes disproportionating enzyme. Further evidence for a function of alpha-1,4 glucanotransferases during starch granule biosynthesis in green algae.</a> Plant Physiology. 132 (1), 137-145 (2003).</li><li>Ji, Y., Seetharaman, K., White, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+a+Small-Scale+Corn-Starch+Extraction+Method+for+Use+in+the+Laboratory.">Optimizing a Small-Scale Corn-Starch Extraction Method for Use in the Laboratory.</a> Cereal Chemistry. 81 (1), 55-58 (2004).</li><li>Halloy, S., Whigham, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lognormal+as+universal+descriptor+of+unconstrained+complex+systems:+a+unifying+theory+for+complexity.">The lognormal as universal descriptor of unconstrained complex systems: a unifying theory for complexity.</a> Proceedings of the 7th Asia-Pacific Complex Systems Conference. , 309-320 (2004).</li><li>Furusawa, C., Suzuki, T., Kashiwagi, A., Yomo, T., Kaneko, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquity+of+log-normal+distributions+in+intra-cellular+reaction+dynamics.">Ubiquity of log-normal distributions in intra-cellular reaction dynamics.</a> Biophysics (Nagoya-shi). 1, 25-31 (2005).</li></ol>

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...

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 occurrence frequencies. In other words, starch granules from a plant species may have a characteristic statistical shape distribution, rather than a specific shape. The non-uniform and non-spherical granule shapes make it difficult to properly measure and define starch granule sizes. Additionally, starch granules from the same tissues of a plant species are of a range of sizes with different proportions, i.e., exhibiting a characteristic size distribution. This size distribution further complicates the analysis and description of starch granule sizes.
Starch granule sizes have been analyzed using several categories of particle sizing techniques (reviewed in ref.3), including microscopy, sedimentation/steric field-flow fractionation (Sd/StFFF), laser diffraction and electrical sensing zone (ESZ). However, these techniques are not equally suited for the determination of starch granule sizes in the presence of a granule shape and a size distribution. Microscopy, including light, confocal and scanning electron microscopy, is excellent for the studies of morphology4,5,6,7, structure8,9 and development10,11 of starch granules, but hardly suited for defining their size distributions due to some inherent shortcomings. Direct measurements of microscopic granule images or software-assisted image analysis of optical microscopy data (IAOM), which have been used for the determination of granule sizes of starches from several species, including maize12, wheat13,14, potato15 and barley16, can measure only 1D (usually maximal length) or 2D (surface area) sizes of very limited numbers (tens to a few thousands) of starch granule images. The small granule sampling sizes that are inherently constrained by the techniques could rarely be statistically representative, considering the enormous granule numbers per unit weight of starch (~120 x 106 per gram, assuming all 10 &#181;m spheres at 1.5 g/cm&#179; density), and, therefore, could lead to the poor reproducibility of the results. The Sd/StFFF technique may have high speed and resolution, and narrow size fractions of starch granules17, but has been rarely used probably because its accuracy could be severely affected by damages, different shapes, and density of starch granules. The laser diffraction technique is the most widely used, and has been applied for starch granule size analyses for all major crop species3,14,16. Although the technique has many advantages, it is actually not suited for determinations of starch granule sizes in the presence of a granule shape distribution. Most of the concurrent laser diffraction instruments rely on the Mie light-scattering theory18 for uniform spherical particles and the modified Mie theory18 for some other shapes of uniformity. The technique is, therefore, inherently very sensitive to particle shapes, and not entirely suited even for certain shapes of uniformity19, let alone for starch granules having a set of shapes of varying proportions. The ESZ technique measures the electric field disturbance proportional to the volume of the particle passing through an aperture. It provides granule volume sizes, as well as the number and volume distribution information, etc., at high resolutions. Since the ESZ technique is independent of any optical properties of particles including color, shape, composition or refractive index, and results are very reproducible, it is particularly suited for determining size distributions of starch granules having a set of shapes.
Starch granule sizes have also been defined by using many parameters. They were often simplistically described by average diameters, which in some cases were the arithmetic means of the microscopically measured maximal lengths of 2D images12,20, or averages of equivalent sphere diameters3. In other cases, the granule size distributions were specified by using size ranges21,22, the distribution mean volume or mean diameter (sphere equivalent, weighted by number, volume, or surface area) assuming a normal distribution14,23,24,25,26. These descriptors of starch granule sizes from various analyses are of a vastly different nature, and not strictly comparable. It could be very misleading if these &#8220;sizes&#8221; of starch granules from different species or even the same tissues of the same species were directly compared. Furthermore, the spread (or shape) parameter of the assumed normal distributions, i.e., the standard deviation &#963; (or graphic standard deviation &#963;g) measuring the width of the distribution (i.e., the spread of the sizes), has been ignored in most studies.
To resolve the aforementioned critical issues facing starch granule sizing analyses, we outlined a procedure for reproducible and statistically valid determinations of granule size distributions of starch samples using the ESZ technique, and for properly specifying the determined granule lognormal size distributions using an adopted two-parameter multiplicative form27 with improved accuracy and comparability. For validation and demonstration, we performed replicate granule sizing analyses of sweetpotato starch samples using the procedure, and specified the lognormal differential volume-percentage volume-equivalent-sphere diameter distributions using their graphic geometric means <img alt="Equation 1" src="/files/ftp_upload/61586/61586eq1.jpg" /> and multiplicative standard deviations s* in a <img alt="Equation 1" src="/files/ftp_upload/61586/61586eq1.jpg" /> x/ (multiply and divide) s* form.

<table border="0" cellpadding="0" cellspacing="0" style="width:1001px;" width="1001">
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		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Analytical beaker</td>
			<td style="width:211px;">Beckman Coulter Life Sciences</td>
			<td style="width:171px;">A35595</td>
			<td style="width:359px;">Smart-Technology (ST) beaker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aperture tube, 100 &#181;m</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>A36394</td>
			<td>For the MS4E, , 1000 &#181;m</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Disposable transfer pipettor,</td>
			<td>Fisher Scientific (Fishersci.com)</td>
			<td style="width:171px;">13-711-9AM</td>
			<td style="width:359px;">Other disposable transfer pipettors with similar orifice can also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Fisherbrand Conical Polypropylene Centrifuge Tubes, 50 ml</td>
			<td>Fisher Scientific (Fishersci.com)</td>
			<td>05-539-13</td>
			<td>Any other similar types of tubes can be used.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:261px;">Glass beakers, 150 to 250 ml</td>
			<td>Fisher Scientific (Fishersci.com)</td>
			<td>02-540K</td>
			<td style="width:359px;">These beakers are used to contain methanol for washing the aperture tube and stirer between runs.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">LiCl</td>
			<td style="width:211px;">Fisher Chemical</td>
			<td>L121-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Methanol</td>
			<td>Fisher Chemical</td>
			<td>A412-500</td>
			<td>Buy in bulk as the analysis uses a large quantity of methanol.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Mettler Toledo ML-T Precision Balances</td>
			<td>Mettler Toledo</td>
			<td style="width:171px;">30243412</td>
			<td style="width:359px;">Any other precision balance with a readablity 0.01 g to 1 mg will work.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">Multisizer 4e Coulter Counter</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>B23005</td>
			<td style="width:359px;">The old model, Multisizer 3 can also be used with slight adjustment of parameters. The 4e model comes with a 100 &#956;m aperture tube. Other aperture tubes of different diameter can be purchased separately from the company.</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;">Ultrasonic processor UP50H</td>
			<td>Hielscher Ultrasound Technology</td>
			<td>UP50H</td>
			<td style="width:359px;">Other laborator sonicator having a low-power (&#60;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.</td>
		</tr>
	</tbody>
</table>

analysis and specification of starch granule size distributions

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.

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...

Watch this Scientific Journal Video about Analysis and Specification of Starch Granule Size Distributions at JoVE.com

Analysis and Specification of Starch Granule Size Distributions

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. &#160;

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 ...

This technique enables a reproducible and statistically valid determination of starch granule sizes and provides a proper description of statistically distributed granule sizes. This technique determines starch granule volumes independent of their shapes and uses statistically valid granule sample sizes for size distribution and provide improved specification of granule size distributions. This method is applicable to plant and food science research studies that involve starch granule sizes in addition to any other starch applications that require granule size information.Before beginning an analysis, dissolve 25 grams of lithium chloride in 500 milliliters of methanol and select an aperture tube with a particle diameter range covering the known granule size range of the starch samples to be analyzed. To set up a standard operating method, start the analyzer software and in the main menu, click SOP and create SOM wizard and select the appropriate settings, then click edit the SOM and select the appropriate settings. To select a preferences file, under SOP, click create preferences wizard and select the appropriate preference settings, then click create SOP wizard, enter a description and author, and select the SOM and preferences files to create and save the SOP.To set up the analyzer for an analysis, turn on the analyzer and verify the ready status in the analyzer software. Fill the electrolyte jar with the previously prepared electrolyte and empty the waste jar as necessary. Install and secure the selected aperture tube according to the guide in the user's manual and push the lock release clip to unlock the assay platform.Manually lower the platform to the bottom and place an analytical beaker containing 100 milliliters of electrolyte onto the platform. Move the stirrer to the stirring position and manually raise the platform to the self-locking upper position to immerse the aperture tube and stirrer in the electrolyte. Click fill on the bottom instrument toolbar to automatically fill the system with electrolyte and click flush to automatically flush the system, then click SOP and load in SOM to load the standard operating method and click sample and enter sample info to enter the sample information for the run.To prepare a starch methanol sample sizing suspension, weigh one or two 0.5 gram samples from each of the two or three replicate starch extracts and add each aliquot to five milliliters of methanol in a 50 milliliter conical centrifuge tube. Use several pulses of low-intensity ultrasound from an ultrasonic processor to fully disperse the starch granules and use a disposable transfer pipette to apply about 200 microliters of the first starch methanol suspension to the 100 milliliters of lithium chloride methanol electrolyte under constant stirring in the beaker, then close the sample compartment door and click preview to start a preview run. In the status panel, verify that the dynamically displayed concentration bar is green and shows a 5 to 8%nominal concentration range for the suspension.Click stop to stop the preview and click start to start the analysis. The analyzer will automatically complete the run once the total count of size granules, which is displayed along with the runtime on the status panel in a run, reaches the total count as set by the control mode of the SOM. To perform a technical repeat run using the same starch electrolyte suspension, click start or repeat on the bottom toolbar.At the end of the analysis, empty the beaker, rinse it with methanol, and refill it with 100 milliliters of fresh electrolyte solution for analysis of the next sample. If an SOM was used to control the runs, use the create preferences wizard to select the preferences settings as desired for viewing, printing, and statistical analyses of the results. To overlay the results from multiple runs on a single graph, in the overlay window, select the results to be overlaid.Click add to move the files to the selected files box and click OK to overlay the selected results on a single graph. To add a file to an open overlay, click run file and open for overlay to access the overlay window, select the desired file, and click to add. Click file, file tool, and average to open the average window and navigate to and select multiple desired result files in the file box.Click add to move the files to the selected files box and click OK to average the selected results for display in a single graph. To include an additional result file in an average distribution, click run file and open and add to average to open the add to average window and add the file of interest. The new average will appear on the graph in the run window.To obtain the graphic geometric mean and standard deviation for specifying the average distribution, click calculate and average statistics to open the statistics summary window to display the means and standard deviations of the data. The third row of the table displays the graphic geometric mean and standard deviation for specifying the average distribution. And the second column shows the statistics from averaging the distributions.Here, differential volume percentage volume equivalent sphere diameter distributions for the four replicate sizing analyses of representative sweet potato starch samples and their average distributions are shown. In this figure, the average cumulative number and volume percentage size distributions of the four replicate sizing analyses, which were transformation views of the average differential volume percentage size distribution, can be observed. Comparing the cumulative number and volume percentages of these starch granule samples revealed that the granules with smaller volume equivalent sphere diameters accounted for much larger percentages of the total count than the total volume.It is important to minimize starch granule aggregates in the starch samples and assay suspensions and to attain a 5 to 8%nominal concentration range for starch electrolyte suspension. Assays for characterizing physical, chemical, and thermal properties of starch samples and for correlating granule sizes with starch synthesis characteristics in source plants can also be performed.

Research

JoVE Journal

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