AOAC RI ERP E-Book - DS DF

OMAMAN-38 B: Collaborative Study Protocol Expert Review Panel Use Only September, 2017

4.524 (v%). Can be prepared by mixing 5 volumes of 2 propanol with 95 volumes of denatured ethanol formula SDA-3A[100 volumes of 95% EtOH combined with 5 volumes of methanol]. (b) EtOH (or IMS), 78 % .— Place 179 mL water into 1-L volumetric flask. Dilute to volume with 95 % EtOH or IMS. Mix. (c) Acetone , reagent grade. (d) Stock PAA plus AMG powder .— PAA (40 KU/g) plus AMG (17 KU/g) as a freeze-dried powder mixture. https://secure.megazyme.com/Rapid-Integrated-Total- Dietary-Fiber-Assay-Kit Note: One Unit of AMG activity is the amount of enzyme required to release one µ mole of D -glucose from soluble starch per minute at 40°C and pH 4.5; one Unit of PAA activity is the amount of enzyme required to release one µ mole of p -nitrophenyl from Ceralpha reagent per minute at 40°C and pH 6.9; AOAC Method 2002.01). PAA/AMG preparations should be essentially devoid of β -glucanase, β -xylanase and detectable levels of free D -glucose. Stable for > 4 years at −20°C. (e) PAA (4 KU/5 mL)/AMG (1.7 KU/5 mL) .— Immediately before use, dissolve 1 g of PAA/AMG powder in 50 mL of sodium maleate buffer (50 mM, pH 6.0 plus 2 mM CaCl 2 ) and stir for approx. 5 min. Store on ice during use. Use on the day of preparation. [ Alternatively: Some individuals are allergic to powdered PAA and/or AMG. In this instance, engage an analyst who is not allergic to prepare the powdered enzymes as an ammonium sulphate suspension as follows: gradually add 5 g of PAA/AMG powder mix (PAA 40 KU/g plus AMG 17 KU/g; reagent 4) to 70 mL of cold, distilled water in a 200 mL beaker on a magnetic stirrer in a laboratory hood and stir until the enzymes are completely dissolved (approx. 5 min). Add 35 g of granular ammonium sulphate and dissolve by stirring. Adjust the volume to 100 mL with ammonium sulphate solution (50 g/100 mL) and store at 4 o C. (This preparation contains PAA at 2 KU/mL and AMG at 0.85 KU/mL). Stable at 4 o C for 3 months]. (f) Protease suspension (50 mg/mL, ~ 6 Tyrosine U/mg).— Stabilised suspension in 3.2 M ammonium sulphate. -Swirl gently before use. Dispense using a positive displacement dispenser. Protease must be devoid of α -amylase and AOAC Research Institute ERP Use Only

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