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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

in which α - and β -anomers were reported to have equilibrated by 40 min into the 35°C incubation (5). The quadratic terms of all standard curves were significant. For glucose standards prepared in 0.2% benzoic acid solution the time between preparation of the standards and their analysis and reading did not affect the 0.1:3.0 standard solution:GOPOD reagent samples (time by glucose concentration, P = 0.53, reduced model time P = 0.22; least squares means for absorbance: 0.557, 0.560, and 0.560 for 1, 2, and 3 d after standard preparation, standard error of the difference: 0.0009)(Table5). The use of another GOPOD reagent (GOPODa) that used a greater number of units of glucose oxidase, fewer units of peroxidase, and used 4-hydroxybenzoic acid rather than phenol gave similar results to the previously tested GOPOD assay (Table 6). All standard curves produced with GOPODa (7) were more quadratic than linear, as determined on the basis of significance of the quadratic term, and reduction in root mean squared error and sum of squared residuals between the linear and quadratic forms of the curves. Although the original assay reported a linear response in absorbance through 200 µ g glucose/mL (5), the nonlinear nature of the relationship of the absorbance per unit of glucose and non-zero intercept of the linear equations indicate that this is perhaps not the best model (Table 7). In agreement with the original study, the relationship between absorbance and glucose concentration in the present study became grossly nonlinear and in violation of Beer’s Law (absorbance response plateaued or declined with increasing glucose concentrations) at approximately 1500 µ g glucose/mL for the 0.1:3.0 ratio of sample solution:GOPOD (data not shown). The original basis for presuming linearity of the responses at glucose concentrations less than 200 µ g glucose/mL likely lies in the very high R 2 for the linear form of the curves, and that the absorbance per unit of glucose values differ in the fourth or fifth decimal place. While the quadratic form seems to fit better than the linear form, we do not necessarily consider it to be the ‘true’ or ‘best’ form of the relationship. The quadratic form is presented as a clear improvement over presumed linearity. SPECIFICITY The dietary starch method gave a very low starch values for sucrose (0.17 + 0.00% of sample dry matter), alpha-cellulose (0.03 + .02% of air dry sample) and isolated oat beta-glucan (0.31 + 0.09% of air dry sample) indicating that run conditions and enzyme preparations used did not appreciably hydrolyze these feed components, particularly sucrose that has been shown to interfere with starch analysis (8). Use of separate free glucose determinations allowed correction for free glucose and background absorbance associated with each sample. The final detection method, the glucose oxidase – peroxidase method, is specific for glucose, which limits interference from other carbohydrates. Effect of Antioxidants on Glucose Detection Addition of ascorbic acid to tubes containing glucose and subject to the dietary starch assay showed a linear decrease in absorbance at additions of greater than 10 µ moles of ascorbic acid (for 0.1:3.0 sample:GOPOD, effect of ascorbic acid on absorbance for 0 through 10 µ mol of ascorbic acid, P = 0.30; effect of ascorbic acid for 10 through 50 µ mol: linear P <0.01; Table 8). The effect was relatively small through 20 µ mol of ascorbic acid. Investigations into the antioxidant content of foodstuffs (9) showed that most of the high starch or leafy vegetable foods had hydrophilic antioxidant values that would be equivalent to less than 10 µ moles of ascorbic acid per 0.1 g of dry matter. Exceptions included foods high in phenolic compounds (e.g., beets, red OAC Research Institute ERP Use Only

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