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

Determination of Dietary Starch The analyses for free glucose and enzymatically released glucose + free glucose may be performed in separate analytical runs. An individual run should be completed without allowing samples to sit without processing for any extended period except as described in the protocol. ( 1 ) Accurately weigh two test portions (W E , W F ) of 90 to 100 mg of dried test samples or 500 mg of semi-moist, moist or liquid samples into screw cap glass tubes. Test portion W E is for the analysis of enzymatically released glucose and W F is for the determination of free glucose. In addition to unknowns, weigh test portions (W E , W F ) of D-glucose and purified corn starch which serve as quality control samples. Also include two tubes with no test portion to serve as reagent blanks. ( 2 ) Dispense 30 mL of 0.1M sodium acetate buffer ( a ) into each tube. ( 3 ) To tubes with test portions designated W E and to one of the reagent blanks add a volume of heat-stable, α -amylase ( b ) to deliver ca 1800 to 2100 liquefon units or 8300 to 8400 BAU of enzyme activity (typically 0.1 mL of enzyme as purchased); do not add the amylase to W F and to one of the reagent blanks. Cap tubes and vortex to mix. Note: Vortex tube so that the solution column extends to the cap, washing the entire interior of the tube and dispersing the test portion. ( 4 ) Incubate all tubes for 1 h at 100°C in a forced-air oven, vortexing tubes at 10, 30, and 50 min of incubation. ( 5 ) Cool tubes on bench for 0.5 h. At this point, separate tubes designated for free glucose (tubes containing W F test portions and reagent blank with no enzyme) from the rest of the run. Those designated for free glucose should skip steps (6) and (7) and continue with steps (8) through ( 13 ). ( 6 ) Add 1 mL of diluted amyloglucosidase solution ( c ) to W E test and quality control samples and reagent blank. Vortex tubes. ( 7 ) Incubate tubes for 2 h in a water bath at 50°C, vortexing at 1 h of incubation. (8) Add 20 mL of water to each tube. Cap and invert at least 4 times to mix completely. (9a) Final Test Solution Volume by Sum of Volume Additions: Transfer ca 1.5 mL of test sample solutions to microcentrifuge tubes, and centrifuge at 1000 x g for 10 min. If the sample remains cloudy after centrifugation, centrifuge an additional 10 min at 10,000 x g to clarify the solution before proceeding. Solutions may increase in temperature during centrifugation; allow centrifuged solutions to come to room temperature before preparing dilution. Alternative to centrifugation, filter test solutions through hardened paper filter with 22 µ m retention. (9b) Final Test Solution Volume Using Volumetric Flasks: Quantitatively transfer test sample solutions to 100 mL volumetric flasks with filtration through hardened paper filter with 22 µ m retention. ( 10 ) Prepare dilutions with distilled water as needed (see Study Director’s Comments 4) for solutions from test samples, control samples, and reagent blanks for free glucose and enzymatically released glucose analyses. Dilutions of the reagent blanks should provide solutions with the same dilutions as used with the test solutions, so that the diluted reagent blank solutions AOAC Research Institute ERP Use Only

10 of 32