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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

Final sample solution volume by volumetric addition as written in the procedure = 51.1 mL for enzymatically released starch analysis, 50.0 mL for free glucose analysis. The volume is 100 mL if volumetric flasks are used. Method Comments from the Study Director: 1. Pre-wetting of the pipette tip used to transfer solutions for preparation of dilutions and for addition of reaction solutions to tubes for glucose analysis is recommended to improve accuracy. Discard of the initial aliquot when pipetting from the tube used for centrifugation of the test samples avoids disturbing the pellet. 2. Sample dilutions. — If dietary starch or free glucose concentrations of the samples are unknown, both undiluted and diluted test sample solutions and reagent blanks can be analyzed within the same run to avoid the need to repeat the run. The undiluted and diluted reagent blanks should be used to make corrections for similarly diluted test samples. Control glucose and corn starch samples are always diluted. To ensure that final readings fall within the standard curve, dry test samples at weights of 0.1 g containing > 50% starch are diluted, as are semi-moist, moist or liquid samples containing > 10% starch. Pipetting of 0.5 mL of test solution and 4.5 mL of water or 0.5 mL of test solution and 2.0 mL water and subsequent mixing of the solution is adequate for preparation of the 1 in 10 or 1 in 5 dilutions, respectively. 3. Use of repeating positive displacement pipettes for addition of enzyme solutions and GOPOD reagent is recommended for addition of accurate volumes and mixing of GOPOD reagent with test solutions. Preliminary Studies The preliminary evaluation of the candidate starch method as compared to other starch assays including an AOAC method for starch analysis in grains (extension of AOAC 996.11) was described in JAOACI (2009) Volume 92:42-49. (1) Tables 1 and 2 show the results of starch analyses performed with the assays. The candidate starch method (the “acetate buffer” method) gave greater starch values than the other assays, with approximately 2 percentage units of dry matter more than the extension of the AOAC method ( P = 0.05). Modification: Determination of final sample volume by weight and density or by summation of volumetric additions Subsequent to the preliminary study, we performed an evaluation of method accuracy with final sample volume determined by weight and density or with summation of added volumes. For the summation of added volumes, each volumetric addition was performed using quantitatively accurate laboratory liquid dispensers (bottle top dispensers and positive displacement pipettes) and summing the total of volumes added. The same volumetric tools for liquid additions were used in both approaches. Values for starch recoveries for volume by weight or summation of volumes were evaluated using glucose and corn starch analyzed as two replicate samples per treatment in two runs using 100 units of amyloglucosidase. Expected recovery of glucose is 90% on a starch basis. For glucose recovery, the dilution by weight or summation of volumes did not differ ( P = 0.21; mean + s r : 89.82 + 0.47% and 89.46 + 0.25%, for by weight or by volume, respectively). Recovery of corn starch was 98.44 + 0.66% for volume by weight and 98.04 + 1.37 for summation of volumes and did not differ between treatments ( P = 0.62). One of the starch recovery values was an outlier for the summation of volumes approach (96.16%). Removal of this value gave a mean recovery value AOAC Research Institute ERP Use Only

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