Biochemistry
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Analysis and Specification of Starch Granule Size Distributions
Chapters
Summary March 4th, 2021
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.
Transcript
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.
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