AOAC RI ERP E-Book - DS DF

OMAMAN-38 A: Collaborative Study Manuscript Expert Review Panel Use Only September, 2017

magnetic stirrer bar and a 2mag Mixdrive 15 magnetic stirrer apparatus; alternatively incubate in a shaking water bath maintained at 37C at 150 revolutions/min (orbital motion) for exactly 4 h. (Alternatively, If employing the ammonium sulphate suspension of PAA/AMG [PAA (2 KU/mL) /AMG (0.85 KU/mL)] { [C(e)] , alternative}, gently swirl the suspension before use and add 2.0 mL of this suspension and 3 mL of maleate buffer [C(j)] or MES buffer [C(k)] to each bottle and incubate as indicated. (d) Adjustment of pH to approx. 8.2 (pH 7.9-8.4), Inactivation of α -amylase and AMG.— After 4 h, remove all sample bottles from the stirring or shaking water bath, and immediately add 3.0 mL of 0.75 M Tris base solution (reagent 12) to adjust pH to approximately 8.2 (7.9–8.4), at which pH AMG has no activity. Immediately, slightly loosen the caps of the sample bottles, place the bottles in a boiling water bath (nonshaking; 95-100°C), and incubate for 20 min with occasional agitation (by hand). This inactivates both PAA and AMG. With a thermometer, ensure that the final temperature of the bottle contents is >90°C. Checking just one bottle is adequate. (At the same time, if only one shaker bath is available, increase the temperature of the shaking incubation bath to 60°C in readiness for the protease incubation step). (e) Cool and protease treatment.— Remove all sample bottles from the hot water bath and cool to approx. 60°C. Add 0.1 mL of protease suspension [C(f)] with a positive displacement dispenser (solution is thick) and incubate at 60°C for 30 min.

(f) pH adjustment.— Add 4.0 mL of 2 M acetic acid to each bottle and mix. This gives a final pH of approx 4.3. (g) Proceed to step [G(a)] for determination of HMWDF. AOAC Research Institute ERP Use Only (g) Add internal standard.— To each sample, add 1 mL of 100 mg/mL glycerol (or diethyleneglycol) internal standard solution [C(g)].

Method updated 2008--08-10 This copy printed 22 August 2017

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