AOAC RI ERP E-Book - DS DF

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

2013 Dietary Starch in Animal Feeds Collab Study Protocol 072513

Phenol – Toxic in contact with skin or if inhaled. Causes severe burns and eye damage. Suspected of causing genetic defects. May cause damage to organs. Do not breathe dust, fumes. Wear protective gloves, clothing, eye protection, face protection.

It is expected that good quality distilled or deionized H 2

O will be used for reagent preparation and

dilutions. The enzymes used with this method must be validated within laboratory to verify efficacy as well as lack of interference. Recommended validation: analyze 0.1 g samples of purified glucose, sucrose, and purified corn starch. Using AOAC approved methods for determination of dry matters of the samples. On a dry matter basis, the glucose should give dietary starch values of 90 + 2%, starch at 100 + 2%, and sucrose 0.7 + 0.3%. Enzyme preparations must not contain appreciable concentrations of glucose (>0.5%) or background absorbance readings will interfere with test sample measurements. Accuracy of volume additions needs to be verified for a lab to use the summative volume approach which avoids use of volumetric flasks. Accurate volumetric additions are essential in order to use of the sum of volumes to describe final test solution volume. Accuracy of volume additions should be evaluated before the assay by the following procedure: Using 1 to 2 L of distilled water allowed to come to ambient temperature, determine the g/mL density of the water: record the weight of three empty volumetric flasks (volumes between 10 and 100 mL), add the water to bring to volume, and weigh the flasks + water. Calculate water density g/mL as ((flask + water g) – (flask g)) / flask volume mL. Record weights of 5 tubes used for the dietary starch assay. Using the ambient temperature water and the devices used to deliver the liquid volumes used for the enzymatic incubation portion of the assay, deliver the 30, 0.1, 1, and 20 mL volumes to each tube (total of 51.1 mL in each tube). Record the weight of each tube + water. Calculate the grams of water in each tube as (tube + water g) – (tube g). Divide the weight of water in each tube by the determined average density of water to give the volume of water in each tube. The average deviation from 51.1 mL should be no more than 0.5% or 0.25 mL, or 1.0% of 0.5 mL for any individual tube for the summative volume addition approach to be used. If deviations are greater than these, after the addition of 20 mL of water during the dietary starch assay, individual test sample solutions should be quantitatively transferred with filtration through hardened filter paper with a 22 µ m retention into a 100 mL volumetric flasks and brought to volume to fix the test sample solution volume before further dilution and analysis. ( a ) Acetate buffer. — 100mM, pH 5.0 .— Weigh 6.0 g or pipette 5.71 mL of glacial acetic acid and transfer immediately to a flask; quantitatively transfer weighed acid with H 2 O rinses. Bring volume to ca 850 mL. While stirring solution on a magnetic stir plate, adjust pH to 5.0 + 0.1with 1M NaOH solution. Dilute to 1 L with H 2 O. This can be done in an Erlenmeyer flask or beaker that has been made volumetric by weighing or transferring 1 L of water into the vessel and then etching the meniscus line for the known volume. ( b ) Heat-stable α -amylase solution. — Liquid, heat-stable, α -amylase, as purchased contains ca 17400 liquefon units/g or 69800 BAU/g, specific gravity ca 1.2 g/mL (Examples: Product Termamyl 120 L, Novozymes North America, Franklinton, NC; Product Multifect AA 21L, Genencor International, Rochester, NY; origin: Bacillus licheniformis ; pH optima 5.5 – 5.8). Should not contain greater than 0.5% glucose. Used as purchased. If modifications are necessary due to availability of commercial enzymes, the volume used in the procedure should deliver ca 1800 to 2100 liquefon units or 8300 to 8400 BAU of enzyme activity. AOAC Research Institute ERP Use Only

6 of 32