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interfering enzymes or sugars etc).
AOAC Method 996.11 has also been adapted to run at pH 5. This was evaluated after I had discussions
with Mary Beth Hall in 2007 (or 2008). The method works fine at pH 5 and both enzymes are active and
stable at this pH. The change to do both incubations at pH 5 is convenient. However, in our hands, the
same analytical values were obtained for a number of starch containing samples when sam both
incubations were run at pH 5 as compared to running the alpha-amylase incubation at pH 7 (as per
996.11). It is known that a small amount of maltulose can be formed on hydrolysis of starch by alpha-
amylase at pH 7 or above. However, this occurs in the industrial hydrolysis of starch which is performed
at a starch concentration of approx. 30% w/v. Starch analyses are performed at a starch concentration
of just 0.03% (1,000-fold lower concentration).
Page 4, line 79. “the use of mildly…excludes the use of alkali or DMSO and thus excludes resistant
starch from inclusion in the dietary starch fraction”. This is exactly what is measured in AOAC Method
996.11, unless there is a requirement to also measure RS. So where is the difference? Also, how can
the author be sure that RS is not hydrolysed in the gut of horses or chickens (pigs will be much the
same as humans).
Page 4, line72. Dietary starch is defined and includes glycogen. Of course these methods also measure
glycogen and maltodextrins, but glycogen is unlikely to be in an animal ration, and maltodextrins would
be rare (perhaps some in distillers grains).
Page 6, lines 119-121. Pure corn starch gave a recovery of 99.3%, but in the interlab results, this
averaged at just 89,4%. Why?
Page 7, point (6). In our laboratory, we have not experienced non-linear color formation with GOPOD
reagent over t he range 0 – 1.2 absorbance units. Is this a problem with enzyme purity?
Page 8, point (8). Ease of use/efficiency. The advantages claimed are exactly the same advantages as
described in AOAC Method 996.11. Where is the difference?
Page 9, lines 182-184. Method uses the same temperature for AMFG and glucose analysis. This is
already done in 996.11.
Page 9. Lines 195-197. Enzyme purity.
Enzymes must be free of glucose, but it is essential that they are also free of other enzymes active on
other glucose containing polymers e.g. beta-glucan. Industrial AMG preparations are highly
contaminated with beta-glucanase and to a lesser extent beta-glucosidase. This requirement should be
highlighted.
Page 10, lines 214-215. For the participants in the interlab, did you state purity requirements for
glucose oxidase and peroxidase. It is essential that high purity enzymes are used. Glucose oxidase is
commonly contaminated with catalase and this results in instability and fading of the color formed in
this reaction.
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