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OMA 2014.10 C: Collaborative Study Manuscript Expert Review Panel Use Only September, 2017

2013 Dietary Starch in Animal Feeds Collab Study Protocol 072513

Accurate and rapid assays for glucose are desirable for analysis of glucose and starch in food and feedstuffs. An established colorimetric glucose oxidase - peroxidase method for glucose was modified to reduce analysis time, and evaluated for factors that affected accuracy. Time required to perform the assay was reduced by approximately 40% by decreasing incubation time and removing steps that do not affect recovery of glucose. Although linear regressions of absorbance and glucose concentrations of standard solutions exceeded R 2 of 0.9997, evaluation of sum of squared residuals, root mean squared error, and significance of the quadratic term indicated that the curves were quadratic in form. Inadequate equilibration of glucose anomers did not appear to be the issue. Historic data suggest that the standard curve is inherently nonlinear. Quadratic curves predicted standard solution glucose concentrations more accurately than did linear forms; predicted overestimations of starch as a percentage of feedstuff dry matter at the midpoint of the standard curve averaged 0.04, 0.48, and 0.92% for quadratic and for linear equations calculated from 5 standard solutions, and for a linear equation calculated from the 0 and most concentrated standard solution, respectively. A hydrophilic antioxidant at levels equivalent to no greater than 10 µ mole ascorbic acid / 0.10 g of air dried sample did not affect absorbance values. 1. Method Test Materials Purified substrates including corn starch, glucose, dextrin, potato starch, and sucrose were analyzed to evaluate recoveries of glucose and starch + maltooligosaccharides and hydrolysis of sucrose when feed matrix provided no barrier to analysis. The feed / food substrates of alfalfa silage, soybean meal, corn silage, split green peas, high moisture ensiled corn grain, wheat flour, and medium grain rice were selected as representative of feeds likely subject to starch analysis, with the first two selected as representative of low starch, the next two as intermediate starch, and the remainder as high starch feeds. Silages and high moisture corn were dried to a constant weight at 55°C in a forced-air oven. The purified substrates and flour were used as purchased and the remaining samples were ground to pass the 1 mm screen of an abrasion (cyclone) mill (Udy Corp., Fort Collins, CO). Average dry matter content of samples was determined with drying for 15 h at 105°C in a forced-air oven. Scope The method is suitable for the determination of the concentration of dietary starch in animal feeds and pet food containing less than 10 µ mol equivalent of antioxidant expressed as ascorbic acid per 0.1 g of air dry sample. Principle Ground or homogenized animal feed and pet food test portions are mixed with acetic acid buffer and heat-stable α -amylase and incubated at 100°C with periodic mixing to gelatinize the starch. After cooling, amyloglucosidase is added and the test mixture incubated for 2 hours at 50°C. The digested mixture is diluted as needed and glucose detected in the resulting test solution using a colorimetric glucose oxidase-peroxidase method. Free glucose is measured simultaneously or in a separate analytical run in each unknown by carrying a second test portion of each test sample through the procedure omitting enzymes and incubating at 100°C for 60 minutes with periodic mixing. Dietary starch is determined as 0.9 times the difference of glucose in the digested test portion minus free glucose in the undigested test portion. AOAC Research Institute ERP Use Only

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