2.3. Methods for resistant starch analysis
As RS is not a distinct chemical fraction but rather a sub-fraction
of total starch, the amount that is recovered by different methods
has the potential to vary depending on the sample preparation techniques
required by each method and the enzymes and incubation
conditions that are applied. In addition to the ingredients and foods
identified for this study, additional flour ingredients were investigated
as these can contain RS in the form of starch granules (RS2).
Two methods for total RS were evaluated, a version of the Englyst
starch digestibility method with direct RS determination (Englyst,
Englyst, Hudson, Cole, & Cummings, 1999) and AOAC 2002.02.
For the Englyst starch digestibility method, samples containing
500 mg carbohydrate were treated with an acid-protease solution
to simulate gastric conditions. This was followed by incubation
with an excess of pancreatic amylase and amyloglucosidase for
2 h with controlled pH, temperature, horizontal mixing and viscosity.
This hydrolyses available starch, with the remaining RS fraction
dispersed by alkali and hydrolysed for measurement as glucose.
For the AOAC 2002.02 method, 100 mg samples were treated
with a low concentration of pancreatic amylase and amyloglucosidase
for 16 h with mixing. The reaction was stopped and RS precipitated
by the addition of ethanol to form a 50% solution. The RS in the
washed residue was dispersed with 2 M potassium hydroxide, buffered,
hydrolysed with amyloglucosidase and measured as glucose.
In addition, each of these procedures was modified to incorporate
an initial boiling treatment in order to provide a determination
of RS3 only. This was compared with the direct RS3
measurement from the NSP + RS3 method.