M.B. Hall, University of Florida, (Aug, 1998) Modified Holm et al., (1986)

Sample: Contains up to 100 mg starch; that do not contain large quantities of free glucose, fructose, or sucrose. Not to be used on samples pre-extracted with aqueous ethanol. Samples should be ground through a 1 mm screen or finer.
 
1.Accurately weigh samples (75 - 100 mg of pure starch, up to 200 mg of feed samples) in duplicate into 100 ml beakers.
         Add 20 ml of high quality distilled water to the sample and stir with a magnetic stir bar.
 
2. Add 0.1 ml Termamyl heat-stable ?-amylase (Sigma A3306 heat-stable ?-amylase may be substituted for Termamyl) to sample and water, and
         stir with a magnetic stir bar.

3.Cover beaker with aluminum foil and place in a 92 - 93C water bath for 1 hour. Remove beakers from water bath and cool on bench (~15 minutes).
 
4. Filter samples through glass wool plugs in funnels into 100 ml volumetric flasks. Rinse beakers, then funnels and glass wool thoroughly with             distilled water. Bring filtered solutions to volume. Mix solutions thoroughly through repeated inversion and shaking of capped or stoppered flask.
 
5.Pipette a 1 ml aliquot of each sample into individual 50 ml volumetric flasks (size of flask can be varied depending upon concentration of starch in sample for sample to fall within standard curve).
 
6.Add 8 ml of 0.1 M sodium acetate buffer (pH ~ 4.5) to each flask.
 
7.Add 50 l of amyloglucosidase (Sigma, A-3514 from A. niger in ammonium sulfate; do not use an amyloglucosidase containing glucose, such as that used for total dietary fiber analysis). Cap flask with foil or Parafilm. Gently swirl flask.
 
9.Incubate flasks in 60C water bath for 30 minutes, gently swirling every 10 minutes.
 
10.Bring samples to volume with high quality distilled water. Cap and mix well by inversion.
 
11.Assay the gelatinized, hydrolyzed sample for glucose to determine starch content.
 

NOTES: ---Before analysis for glucose using a spectrophotometric method, the sample must be clear, not cloudy or with suspended particles -- these will inappropriately increase the absorbance. After the sample is brought to its final volume (step 10), an aliquot can be centrifuged briefly to remove suspended particles. The clear solution can be used for the glucose analysis.

--- A reagent blank which includes only water, enzymes, and buffer should be carried through the assay, analyzed for glucose, and used to make corrections to the sample absorbance values.

Reference:

Holm, J., I. Bjorck, A. Drews, and N.-G. Asp. 1986. A rapid method for the analysis of starch. Starch/Starke 38:224-226.