32
Basic method for the determination of RS was proposed by Englyst et al. (1982).
Briefly, in this method 100–200 mg sample is mixed with sodium acetate buffer at
pH 5.4 and heated for 1 h at 100 °C to gelatinize the starch. An enzyme mixture
(α-amylase, pullulanase and amyloglucosidase) is added and hydrolysis is carried
out at 40 °C for 16 h. Absolute ethanol is added to precipitate nonhydrolyzed starch
and to terminate enzymatic activity. The pellet is collected by centrifugation and
washed with 80% ethanol twice. The residue is dried with acetone and then treated
with 2 M KOH for 30 min at room temperature to solubilize the starch. An aliquot
of alkali digest is mixed with 2 M acetic acid and amyloglucosidase and then incubated at 65 °C for 1 h. After cooling and centrifugation, neutral sugars (glucose,
mannose, galactose, arabinose and xylose) in the supernatant are analyzed by GLC
(gas–liquid chromatography). The amount of glucose detected by GLC represents
the amount of resistant starch (RS3) in the sample.
The above procedure was later modified by Englyst et al. (1992) to better mimic
human gastrointestinal conditions. The authors proposed that enzymatic hydrolysis
can be carried out at 37 °C instead of at 40 °C. Also significant reduction in the
duration of enzymatic digestion is used. Instead of 16 h of sample digestion with
enzymes, the protocol uses sequential removal of aliquots of enzyme digest at
20 min and at 120 min from the beginning of the digestion. The rationale to terminate enzymatic hydrolysis at 120 min is that the release of glucose reached a plateau
at this time interval. The starch fraction digested within 20 min of incubation is
classified as RDS, the starch fraction digested between 20 and 120 min is classified
as SDS, and the remaining fraction that is not further digested is RS.
Goni et al. (1996) further modified the procedure by decreasing the sample pH to
1.5 with HCl–KCl buffer to simulate gastric pH. Hydrolysis with pepsin at 40 °C for
1 h is carried out. To simulate conditions in the small intestine sample pH is adjusted
to 6.9 with tris–maleate buffer. Instead of using a group of enzymes, hydrolysis is
carried out with α-amylase only for 16 h at 37 °C. Glucose content is determined by
the colorimetric method.
The Megazyme assay kit is widely used in analytical laboratories for the determination of RS and is the basis of both AACC Method 32–40 and AOAC method
2002.02 (Megazyme 2008). In this method samples are ground to a coarse meal
which can pass a 1-mm sieve. Initial boiling of samples in acetate buffer and the use
of pullulanase is eliminated. Instead a mixture of α-amylase and amyloglucosidase
is employed to hydrolyze starch in raw or processed food samples. Hydrolysis is
carried out at 37 °C for 16 h. The incubation time with amyloglucosidase is 30 min
at 50 °C. This method uses the glucose oxidase-peroxidase colorimetric assay
(GOPOD) to determine glucose concentration in the final hydrolysate. The
Megazyme protocol for RS determination is not applicable to the determination of
SDS and RDS.
Other modifications have been made to the procedure of RS determination particularly in the sample preparation step with the intention of simulating the in vivo
digestion. However it is obvious that significantly different levels of RS would be
detected in similar foods because of wide variations in analytical protocals, including
differences in the enzymes used and in their activity, concentration and sequence of
application, and dissimilarities in the conditions of experimental protocals.
B. A. Ashwar et al.
Basic method for the determination of RS was proposed by Englyst et al. (1982).
Briefly, in this method 100–200 mg sample is mixed with sodium acetate buffer at
pH 5.4 and heated for 1 h at 100 °C to gelatinize the starch. An enzyme mixture
(α-amylase, pullulanase and amyloglucosidase) is added and hydrolysis is carried
out at 40 °C for 16 h. Absolute ethanol is added to precipitate nonhydrolyzed starch
and to terminate enzymatic activity. The pellet is collected by centrifugation and
washed with 80% ethanol twice. The residue is dried with acetone and then treated
with 2 M KOH for 30 min at room temperature to solubilize the starch. An aliquot
of alkali digest is mixed with 2 M acetic acid and amyloglucosidase and then incubated at 65 °C for 1 h. After cooling and centrifugation, neutral sugars (glucose,
mannose, galactose, arabinose and xylose) in the supernatant are analyzed by GLC
(gas–liquid chromatography). The amount of glucose detected by GLC represents
the amount of resistant starch (RS3) in the sample.
The above procedure was later modified by Englyst et al. (1992) to better mimic
human gastrointestinal conditions. The authors proposed that enzymatic hydrolysis
can be carried out at 37 °C instead of at 40 °C. Also significant reduction in the
duration of enzymatic digestion is used. Instead of 16 h of sample digestion with
enzymes, the protocol uses sequential removal of aliquots of enzyme digest at
20 min and at 120 min from the beginning of the digestion. The rationale to terminate enzymatic hydrolysis at 120 min is that the release of glucose reached a plateau
at this time interval. The starch fraction digested within 20 min of incubation is
classified as RDS, the starch fraction digested between 20 and 120 min is classified
as SDS, and the remaining fraction that is not further digested is RS.
Goni et al. (1996) further modified the procedure by decreasing the sample pH to
1.5 with HCl–KCl buffer to simulate gastric pH. Hydrolysis with pepsin at 40 °C for
1 h is carried out. To simulate conditions in the small intestine sample pH is adjusted
to 6.9 with tris–maleate buffer. Instead of using a group of enzymes, hydrolysis is
carried out with α-amylase only for 16 h at 37 °C. Glucose content is determined by
the colorimetric method.
The Megazyme assay kit is widely used in analytical laboratories for the determination of RS and is the basis of both AACC Method 32–40 and AOAC method
2002.02 (Megazyme 2008). In this method samples are ground to a coarse meal
which can pass a 1-mm sieve. Initial boiling of samples in acetate buffer and the use
of pullulanase is eliminated. Instead a mixture of α-amylase and amyloglucosidase
is employed to hydrolyze starch in raw or processed food samples. Hydrolysis is
carried out at 37 °C for 16 h. The incubation time with amyloglucosidase is 30 min
at 50 °C. This method uses the glucose oxidase-peroxidase colorimetric assay
(GOPOD) to determine glucose concentration in the final hydrolysate. The
Megazyme protocol for RS determination is not applicable to the determination of
SDS and RDS.
Other modifications have been made to the procedure of RS determination particularly in the sample preparation step with the intention of simulating the in vivo
digestion. However it is obvious that significantly different levels of RS would be
detected in similar foods because of wide variations in analytical protocals, including
differences in the enzymes used and in their activity, concentration and sequence of
application, and dissimilarities in the conditions of experimental protocals.
B. A. Ashwar et al.