AOAC RI ERP E-Book - DS DF

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)] { [ [see 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  -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 12pH 11.0) C(l ) 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; 9598 -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 C(m) to each bottle and mix. This gives a final pH of approx 4.3. 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 )]. ).

(g) Proceed to step [ G(a )] for determination of HMWDF. ).

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

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