Food Research International 43 (2010) 1959–1974

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Review

Resistant starch: A review of analytical protocols for determining resistant starch and
of factors affecting the resistant starch content of foods
A. Perera a,⁎, V. Meda a, R.T. Tyler b
a
Department of Agricultural and Bioresource Engineering, University of Saskatchewan, Canada
b
Department of Food and Bioproduct Sciences, University of Saskatchewan, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Resistant starch (RS) has drawn considerable attention over the last two decades due to its demonstrated
Received 8 March 2010 and putative positive impacts on health. This has resulted in the development of a variety of analytical
Accepted 8 June 2010 protocols for its determination, hence a comprehensive review of methodologies for analyzing resistant
starch is warranted. Adding to the complexity, the RS contents of starchy materials vary widely and RS levels
Keywords:
are impacted by processing. This review compares commonly used methods for the determination of RS,
Resistant starch
Starch digestibility
briefly reviews the process of starch digestion in the human, and discusses the complications and challenges
Starch determination associated with determining RS in foods.
Starch hydrolysis © 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959
2. Starch digestion in the human . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1960
3. Demonstrated and putative health benefits of RS consumption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1960
4. The rat as a model for starch digestion in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1961
5. Methods of RS determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1962
6. Effect of starch composition on RS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1967
7. Effect of processing on RS levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1969
8. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1972
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1972

1. Introduction susceptible to hydrolysis by amyloglucosidase is referred to as resistant

Polysaccharides in foods consist of starch and plant cell wall
constituents. On the basis of its digestibility, starch was classified by
Englyst and Cummings (1987a) into three groups, namely readily
digestible starch, partially resistant starch and resistant starch. Accord-
ing to this classification, starch that is not accessible to digestive
enzymes, such as that protected by hard to digest coatings in whole and
coarsely-milled grains, starch in raw banana and potato exhibiting B- or
C-type X-ray diffraction patterns, and retrograded amylopectin, is
considered to be partially resistant starch. Retrograded amylose that can
be solubilized in potassium hydroxide and which subsequently becomes
⁎ Corresponding author. Tel.: + 1 306 966 2454.
E-mail address: anula.perera@usask.ca (A. Perera).
starch (RS). This definition of RS was modified later to include ‘the sum
of starch and starch-degradation products that, on average, reach the
human large intestine’ (Englyst, Kingman, Hudson, & Cummings, 1996).
Partially resistant starch and RS as defined by Englyst and Cummings
(1987a) were subsequently reclassified as RS type 1, RS type 2 and RS
type 3 (Englyst, Kingman, & Cummings, 1992; Sajilatha, Singhal, &
Kulkarni, 2006). Enzyme inaccessible starch resulting from structural
rigidity is defined as RS1, and it is calculated as the difference between
the enzyme-extractable glucose contents of a food determined with and
without an homogenization treatment prior to analysis (Sajilatha et al.,
2006). Englyst et al. (1992) reported RS1 as the difference between the
glucose contents of two samples of a food, one after a milling/
homogenization treatment to obtain 0.2-mm-sized particles and the
other after a mincing treatment, and subsequent hydrolysis with
pancreatin and amyloglucosidase for 120 min. It seems that Englyst et al.
(1992) assumed that comminution (milling or homogenization) of

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1960 A. Perera et al. / Food Research International 43 (2010) 1959–1974
foods would yield a consistency and particle size similar to that of
mastication. However, this may not be true for all foods and RS1 values
determined for comminuted samples may differ from those determined
for otherwise identical masticated samples. For example, Åkerberg,
Liljeberg, Granfeldt, Drews, and Björck (1998) evaluated RS in selected
foods including RS1-rich raw banana. Foods were masticated (15 chews
in 15 s) prior to analysis. Åkerberg, Liljeberg, Granfeldt, et al. (1998)
reported RS values (72 g/100 g starch) that were higher than those
reported (53.6 g/100 g starch) by Englyst and Cummings (1986). The
latter group employed the analytical method of Englyst, Wiggins, and
Cummings (1982) where samples were ball-milled. Starch granules in
raw foods which are resistant to digestion comprise RS2. Analytically,
RS2 is measured as the difference in the amount of glucose liberated by
pancreatin and amyloglucosidase, after 120 min of hydrolysis, from two
samples of the same food (having the same particle size), one having
been subjected to a prior boiling treatment and one not. Retrograded
starch formed during the cooling of gelatinized starch is termed RS3
(Englyst et al., 1992). Based on the definitions above, it is clear that the
RS1, RS2 and RS3 contents determined for a particular food sample may
be affected by differences in sample processing (mastication, homog-
enization, grinding, etc.). Cross-linking of starches from wheat, corn,
rice, potato, tapioca, mung bean and oat, using sodium trimetapho-
sphate (STMP), sodium tripolyphospahate (STPP), epichlorohydrin or
phosphoryl chloride (POCl3), produced starch resistant to α-amylase
hydrolysis, which was termed RS4 (Seib & Woo, 1999). These authors
demonstrated that RS levels in wheat starch cross-linked with 12%
STMP/STPP, 2% POCl3 and 2% epichlorohydrin were 75.6, 85.6 and
75.8 g/100 g starch, respectively. Other chemically-modified starches,
e.g. starch etherified with propylene oxide or cross-linked with
phosphorous oxychloride, acid-modified starch and starch oxidized
with sodium hypochlorite, also have been reported to be resistant to in
vitro hydrolysis by α-amylase and amyloglucosidase (Wolf, Bauer, &
Fahey, 1999). The classification of starch has been expanded to include
rapidly digestible starch (RDS) and slowly digestible starch (SDS) in
addition to RS. Starch that is hydrolyzed in vitro within 20 min with α-
amylase and amyloglucosidase is designated RDS (Englyst, Veenstra, &
Hudson, 1996). The category of SDS includes enzyme inaccessible starch
and raw starch that is fully hydrolyzed in vitro during prolonged
incubation (20–120 min) (Englyst et al., 1992; Sajilatha et al., 2006).
Starch that is not hydrolysed after 120 min incubation with pancreatin
and amyloglucosidase is termed RS (Englyst et al., 1992).
2. Starch digestion in the human
As described above, the concept of RS is based on the inability of
digestive enzymes to hydrolyze some physical and chemical forms of
starch in foods in vivo or in vitro. Therefore, it is useful to review the
process of starch digestion in humans. Fig. 1 summarizes the major
organs that participate in the digestion of foods and the enzymes
secreted. Salivary glands that open into the oral cavity produce several
enzymes that initiate carbohydrate digestion, e.g. α-amylase, glycosi-
dases, glucose oxidase, lactate dehydrogenase and β-glucuronidase
(Makinen, 1989). Food masticated and partially hydrolyzed in the
mouth passes through the pharynx and then through the esophagus and
into the stomach. Submucosal glands of the esophagus secrete
bicarbonates and mucin. This is considered a mechanism to protect
the esophagus from HCl refluxed from the stomach (Abdulnour-
Nakhoul et al., 2005). In the stomach, food is mixed with pepsin, gastric
lipase and HCl, which activates pepsin. No significant digestion of starch
takes place in the stomach but contraction of muscles facilitates
comminution of foods (Correa, 1988). Food that is passed into the
small intestine is termed ‘chyme’ and there it is mixed with bicarbonates
and mucin secreted by the Brunner's glands of the small intestine
(DeSesso & Jacobson, 2001). In the duodenum, the first part of the small
intestine, chyme is blended with bile secreted by the gallbladder,
pancreatic α-amylase, and amylolytic and other enzymes secreted by
the epithelial cells of the small intestine, such as enterokinase, sucrase,
maltase and lactase. These enzymes are responsible for the hydrolysis of
carbohydrates into monosaccharides. In addition, proteolytic enzymes
such as trypsin, chymotrypsin, carboxypeptidase and elastase, lipolytic
enzymes such as lipase, phospholipase and cholesterol esterase, and the
DNA and RNA hydrolyzing enzymes ribonuclease and deoxyribonucle-
ase are secreted by the pancreas and mixed with the chyme (Cichoke,
1998; DeSesso & Jacobson, 2001). Chyme that is not hydrolyzed into
absorbable molecules in the small intestine, e.g. starch which has
escaped digestion, along with non-starch polysaccharides, moves into
the colon and undergoes bacterial fermentation. The resulting short-
chain fatty acids are absorbed in the colon and provide 5–10% of the
energy requirement of humans (Nordgaard & Mortensen, 1995). It has
been shown that of the total quantity of starch consumed by ileostomy
subjects, approximately 3% of freshly cooked starch and approximately
12% of cooked and cooled starch escape digestion in the small intestine
and can be recovered in the ileum. All of the RS and the non-starch
polysaccharides in the diet were found in the ileum effluent of tested
subjects (Englyst & Cummings, 1985, 1987b). Approximately 80–90% of
the RS that passes into the human colon is fermented and the remainder
is released with the feces. Individual variations in the microbial
population of the colon can influence significantly the degree of RS
fermentation. The presence of RS in the diet decreases considerably the
bacterial fermentation of non-starch polysaccharides, which is useful as
non-starch polysaccharides increase the bulk of the excreta and improve
its water holding capacity (Cummings, Beatty, Kingman, Bingham, &
Englyst, 1996).
3. Demonstrated and putative health benefits of RS consumption
RS is recognized as a significant contributor to gastrointestinal
health. Undigested starch is fermented by microorganisms in the large
bowel to produce short chain fatty acids, including acetate, propionate
and butyrate (Bird, Brown, & Topping, 2000; Brouns, Kettlitz, & Arrigoni,
2002; Wong, de Souza, Kendall, Emam, & Jenkins, 2006) and isobutyrate,
valerate and isovalerate (Hylla et al., 1998). A high-amylose corn starch
diet containing 46–50% of RS2 significantly increased the colonic
production of acetate, propionate and butyrate in rats. This diet also
increased apoptosis in the colon subsequent to externally induced gene
damage, which suggests an ability of RS to control the initiation of
colonic cancer (Leu, Hu, Brown, & Young, 2009). Butyrate was reported
to increase the level of glutathione, an antioxidant, in colonic mucosa,
improving colonic resistance to toxic agents in the diet (Hamer et al.,
2009). A significant decrease in bile acids (cholic acid, chenodeoxycholic
acid, deoxycholic acid, lithocholic acid, isolithocholic acid and urso-
deoxycholic acid) and total neutral sterols (coprostanol, cholesterol, 4-
cholesten-3-one, campesterol, stigmasterol and β-sitosterol) in the fecal
matter of healthy adults was reported after consuming experimental
foods rich in RS for 4 weeks (Hylla et al., 1998). As the fecal matter of
colon cancer patients carries more bile acids and neutral steroids than
does that of healthy controls (Reddy, Ernst, & Wynderm, 1977), it is
believed that RS has a positive effect on the control and prevention of
colon cancer. Significant expression of a ‘cell cycle regulatory and cell
proliferation gene’ was observed in colorectal cancer patients after their
consumption of 40 g/day of RS for 2–4 weeks. The expression of the
gene was inhibited in control subjects (Dronamraju, Coxhead, Kelly, &
Mathers, 2007).
Consumption of RS has other benefits. A diet rich in dietary fibre and
RS was reported to significantly increase the weight of the pancreas,
liver and intestine, and the length of the intestine, in rats (Correa,
Angelina, Reis, Maria, & de Oliveira Costa, 2009). RS was reported to
decrease the postprandial blood glucose level considerably within the
first 120 min (Kendall et al., 2008). Compared to a diet containing
autoclaved and cooled starch from waxy barley (which would not
undergo significant retrogradation), significantly lower levels of blood
glucose (within the first 120 min) were reported after feeding
 A. Perera et al. / Food Research International 43 (2010) 1959–1974 1961

Fig. 1. Enzymes secreted by major organs participating in the digestion of foods in humans. The digestive system, an extension of the environment into the body, also secretes mucus,
acid, bicarbonate and salts to facilitate hydrolysis of foods and absorption of nutrients essential for life.

retrograded high-amylose barley (∼18% RS) to rats (Xue, Newman, &
Newman, 1996). RS3 in the diet has a laxative effect. A supplement of
25 g/day in healthy test subjects increased the daily fecal output above
the normal level with minimal gastrointestinal discomfort (Maki et al.,
2009). A RS-rich green banana diet had beneficial effects on controlling
shigellosis in child patients by decreasing stool volume, number of stools
per day and red blood cells in stools, and increasing levels of acetate,
propionate and butyrate in feces. Despite the expected negative impact
of antibacterial treatments (ciprofloxacin) on short chain fatty acid
production in the colon, acetate, propionate and butyrate production in
child patients increased on the fifth day of feeding green banana
(Rabbani et al., 2009). A RS-supplemented diet significantly increased
populations of lactobacilli, bifidobacteria, staphylococci and streptococ-
ci, decreased the enterobacteria population, and altered microbial
enzyme metabolism in the colon of experimental rats (Silvi, Rumney,
Cresci, & Rowland, 1999).
For a balanced view of the effect on RS on health, it is important to
note that the consumption of high amounts of RS may have some
negative effects on gastrointestinal performance. These include bloat-
ing, borborygmi (noise due to gas movement in the intestine),
flatulence, colic and watery feces (Storey, Lee, Bornet, & Brouns,
2007). Consumption of common bean (Phaseolus vulgaris L.) rich in RS
(12–13 g/100 g raw starch) and soluble dietary fibre introduced
flatulence, bloating, abdominal noise and pain in test subjects, but
symptoms significantly decreased when fermented beans were con-
sumed (Granito, Michel, Frías, Champ, & Guerra, 2005). In a review,
Grabitske and Slavin (2009) explained that diarrhea is an extreme
outcome of ingesting carbohydrates with very low digestibility,
including RS, when the capacity of colonic fermentation is exceeded
by consumption. They also noted that adverse effects are more
prominent with RS3 than with RS2, and that the severity of symptoms
differed with personal attributes such as age, weight, gender, genetics,
health and the composition of the gut microflora. Overall, the benefits of
RS consumption were considered to outweigh any gastrointestinal
discomfort.
4. The rat as a model for starch digestion in humans
As described above, animal models have been used in some RS
research studies and thus it is important to discuss briefly the
dependability and/or applicability of the results obtained. Animal
models used in biomedical research should be either analogous or
1962 A. Perera et al. / Food Research International 43 (2010) 1959–1974
homologous to humans considering the anatomy and physiology of
the organ or system under investigation. Other criteria include, but
are not limited to, the selected animal should be easily available in
substantial numbers, should be easy to handle and manageable with
the resources available, and the life span should be sufficiently long to
carry out the research. In keeping with these criteria, omnivores such
as the rat and the pig are used to simulate the gastrointestinal tract
and liver functions of the human (Chow, 2008). In a comprehensive
review, DeSesso and Jacobson (2001) compared the similarities and
differences between the gastrointestinal tracts of the human and the
rat. Some significant differences are listed here. The human pharynx is
a single tube that is the passageway for both food and air, whereas the
rat pharynx is separated into two compartments for respiratory and
digestive purposes. The human stomach is a single chamber where
food is mixed with enzymes and acid. The rat stomach, although a
single chamber, is functionally divided into two areas. The forest-
omach is the site of bacterial digestion, whereas the glandular
stomach secretes enzymes and acid. Bacterial digestion is absent in
the human stomach. Although the rat is used to imitate digestion in
the human, there is the likelihood of species differences in enzyme
activity. The small intestine has three functionally different regions,
the duodenum, the jejunum and the ileum. Compared to the human,
where the gallbladder secretes bile, the rat liver secretes twice as
much as bile into the duodenum per kg of body weight per day.
Nutrient absorption takes place in the duodenum and the forejejunum
of both rats and humans through diffusion, active transport and
solvent drag mechanisms. Compared to the total length of the
digestive tract, the jejunum of the rat is proportionally longer than
that of the human. The jejunum of the rat represents 90% of the length
of the small intestine, and that of the human, 38%. The transit time of
food through the rat small intestine is slower than that in the human.
Despite the human small intestine being 5.5 times longer than that of
the rat, the transit time of food through the small intestine of the
human and the rat is similar, 3–4 h. The surface area of the human
small intestine is 200 times greater than that of the rat, which is
significant given the difference in their lengths. Water and nutrients
formed through bacterial metabolism are absorbed in the large
intestine. The human large intestine is divided into the cecum,
ascending colon, transverse colon, descending colon, sigmoid colon,
rectum and anus. The sigmoid colon is absent in the rat. The cecum is
responsible for most of the microbial digestion of food in the rat and
represents up to 26% of the length of the large intestine. In contrast,
the human cecum is only 5% of the length of the large intestine. In
addition, the types of microorganisms naturally occurring in the
digestive systems of the human and the rat may be different (DeSesso
& Jacobson, 2001). The differences described above indicate that the
rat model may not provide reliable data on RS digestion in humans,
but it may be useful in understanding the fate of RS within the
digestive tract.
5. Methods of RS determination
Determination of RS in food ingredients and processed foods has
become vital to the provision of nutritional information to consumers
and others. To enable effective use of the research output on RS for food
processing and nutritional applications, analytical procedures for the
determination of RS need to be compared. At present, significant
differences exist among procedures with respect to sample preparation,
the enzymes used, and the establishment of experimental conditions
that mimic gastrointestinal digestion of starch. Ongoing improvements
in analytical methodology are essential, but frequent modifications in
protocols reduce the availability of comparable data to assess the
efficacy of methods and the nutritional quality of foods. Added to this,
food analytes used in the various procedures have differed in their
genetic origin, composition and processing history. This paper reviews
current methods of analysis of RS and difficulties related to comparing
data generated by different methods.
In vitro methods for the analysis of RS in foods are based
conceptually on the gastrointestinal digestion of starch in foods.
Enzyme hydrolysis is a common feature of all methods, combined
with chemical hydrolysis or gravimetric isolation. Differences in
sample preparation are significant and range from mechanical
methods (milling, grinding and homogenization) to mastication.
The type of RS quantified is dependant on the protocol. Most methods
are focused on the determination of total RS, but specific methods
have been developed to quantify RS1, RS2 and RS3.
Methods for the determination of RS proposed by Englyst et al.
(1982) and Englyst et al. (1992) are the basis for this discussion.
Significant modifications proposed by other authors are compared and
contrasted. As proposed by Englyst et al. (1982), RS and other non-
starch polysaccharides are first separated from the enzyme-hydrolyz-
able starch. Then, RS is solubilized in alkali and thereby separated from
the other enzyme resistant polysaccharides. Even though Englyst et al.
(1982) labelled this fraction as RS, the analytical protocol used reveals
that it measures RS3 only. Briefly, in this method (Fig. 2) 100–200 mg of
homogenized wet sample or ground dry sample is mixed with sodium
acetate buffer at pH 5.4 and heated for 1 h at 100 °C to gelatinize starch.
Having included homogenization and boiling steps, this protocol
eliminates the contribution of RS1 and RS2 to the final RS value. An
enzyme mixture containing α-amylase, pullulanase and amyloglucosi-
dase is added and hydrolysis is carried out for 16 h at 40 °C. Absolute
ethanol is added to terminate enzyme activity and to precipitate non-
hydrolyzed starch. The pellet is collected after centrifugation and
washed twice with 80% ethanol. The residue is dried with acetone and
then treated with 2 M potassium hydroxide for 30 min at room
temperature to solubilize retrograded starch (RS3). An aliquot of alkali
digest is mixed with 2 M acetic acid and amyloglucosidase and the
contents are incubated at 65 °C for 1 h. After cooling and centrifuging,
neutral sugars (glucose, galactose, mannose, xylose and arabinose) in
the supernatant are analyzed by gas–liquid chromatography (GLC). The
quantity of glucose detected by GLC represents the amount of RS (RS3)
in the sample.
The above procedure was modified later by the authors (Englyst
et al., 1992) to measure total glucose, free glucose, total RS, and RS1, RS2
and RS3 as separate fractions. The determination of total RS is carried
out in step-wise fashion. Total glucose (TG) and free glucose (FG)
contents are determined, as explained in Fig. 3.1, in order that total
starch (TS) may be calculated as TS= (TG − FG) × 0.9. In this protocol
for TS determination, RS1 and RS2 are eliminated by mincing/milling/
homogenizing the food sample and subsequently heating the sample at
100 °C for 30 min, respectively. Any retrograded starch that contributes
to RS3 is eliminated by treating the sample with 7 M potassium
hydroxide at 0 °C. The rapidly digestible starch (RDS) and slowly
digestible starch (SDS) contents of the sample are determined
separately and total RS is calculated as RS = TS − (RDS + SDS)
(Fig. 3.2). In this method, 0.8–4.0 g of sample is minced only to
minimize structural changes. The low viscosity of samples leads to the
release of considerably higher amounts of glucose during enzyme
hydrolysis, especially in the absence of other polysaccharides, than is
the case with the in vivo digestion of starch. Thus, guar gum is added to
increase the viscosity. Glass beads are added to samples to ensure
proper mixing and to break cell walls during enzyme hydrolysis. For the
determination of RDS and SDS, samples are equilibrated with 20 mL of
acetate buffer at 37 °C and then starch is hydrolyzed with an enzyme
mix containing pancreatin as a source of α-amylase (30,000 BPU/g),
amyloglucosidase (400 AGU/mL), and invertase. (3000 EU/mL). Englyst
et al. (1992) proposed that enzyme hydrolysis be carried out at 37 °C
instead of at 40 °C, as previously used in the Englyst et al. (1982)
protocol, to better mimic human physiological conditions of digestion. A
significant reduction in the duration of enzyme digestion is used in the
Englyst et al. (1992) protocol. Instead of 16 h of sample digestion with
 A. Perera et al. / Food Research International 43 (2010) 1959–1974
Fig. 2. Main steps in the Englyst et al. (1982) protocol for the determination of enzyme resistant starch (RS3). The presence of an enzyme resistant fraction of starch in foods was first
observed by the authors during the measurement of non-starch polysaccharides in foods.
pancreatin, as per Englyst et al. (1982), the Englyst et al. (1992) protocol
uses sequential removal of aliquots of enzyme digest at 20 min (as the
rapid release of sugars ended at 20 min of digestion with pancreatin)
and at 120 min from the beginning of the digestion. The rationale to
terminate enzyme hydrolysis at 120 min is that the release of glucose
reached a plateau at 120 min. The glucose content of the 20-min
enzyme digest (G20 fraction) represents RDS and is calculated as RDS=
(G20 − FG) × 0.9. The difference in glucose contents between the 120-
min and 20-min digests represents SDS and is calculated as SDS = (G120
− G20 − FG)× 0.9. Instead of directly measuring glucose originating
from RS as in Englyst et al. (1982), this method indirectly estimates total
RS as RS = TS− (RDS + SDS). As 37 °C is the highest temperature at
which a sample is treated in this protocol, any RS2 in the food sample
would not be hydrolyzed by α-amylase. RS1 also is preserved in the
unhydrolyzed fraction as the sample is only minced and no milling or
homogenization is employed. As a result, this indirect measurement of
RS includes any RS1, RS2 and RS3 in the sample.
Englyst et al. (1992) devised protocols for determination of RS1,
RS2 and RS3. By definition, RS1 is the fraction of starch that is
inaccessible to enzymes due to physical impediments, e.g. large-sized
particles and cell walls. The analytical procedure for RS1 determina-
tion (Fig. 3.3a) differs from that of Englyst et al. (1982) (Fig. 2) with
respect to the method of reduction of particle size. One portion of the
sample (A) is minced once and the other portion (B) is either milled or
homogenized, as appropriate, to obtain particles b 0.2 mm in
diameter. Then, using the protocol for the determination of RDS and
SDS (Fig. 3.2), glucose released after 120 min of enzyme hydrolysis
(G120) is determined for each portion. RS1 is calculated as RS1 = [G120
(B) − G120(A)] × 0.9. Starch in raw foods and foods cooked with little
water (preventing complete gelatinization of starch) contribute to
RS2. The method for the determination of RS2 (Fig. 3.3b) compares
the amount of glucose released enzymatically from a cooked sample
to that from a raw sample (Englyst et al., 1992). One portion (C) of a
raw food sample is heated in 0.1 M sodium acetate buffer, pH 5.2, in a
boiling water bath for 30 min, while another portion (D) is used
directly without a heat treatment. The samples are hydrolysed with
amylase, amyloglucosidase and invertase and the glucose released
1963
after 120 min (SDS) is determined using the procedure outlined in
Fig. 3.2. RS2 is calculated as RS2 = [G120(C) − G120(D)] × 0.9.
Determination of RS3 is limited to samples containing retrograded
starch (Englyst et al., 1992) and the protocol is similar to that of
Englyst et al. (1982) in many ways. In contrast to the protocol for the
determination of RDS and SDS for the estimation of total RS (Fig. 3.2),
where α-amylase, amyloglucosidase and invertase are used, the
protocol for RS3 determination uses α-amylase and pullulanase at
40 °C. After removing the enzyme-hydrolyzable starch from the
sample, retrograded starch is solubilized in 4 M potassium hydroxide
at room temperature, whereas Englyst et al. (1982) used 2 M
potassium hydroxide. However, this concentration is lower than
that used (7 M) in the protocol for the determination of TG outlined in
Fig. 3.1. The temperature at which retrograded starch is solubilized in
potassium hydroxide also is different in the two procedures; 0 °C for
TG and room temperature for RS3 determination (Englyst et al., 1992).
Goñi, García-Diza, Mańasb, and Saura-Calixto (1996) cautioned
about the possible effect of sample preparation in RS protocols as
drying, cooling and conditions of storage can alter the level of RS in
foods. Their method of RS determination is summarized in Fig. 4. In
this procedure, a 100-mg sample is milled or homogenized before use
and by doing so RS1 is eliminated. Sample pH is decreased to 1.5 with
HCl–KCl buffer to simulate gastric pH. As a major modification to the
procedure of Englyst et al. (1992), hydrolysis with pepsin at 40 °C for
1 h is introduced. Sample pH is adjusted to 6.9 with tris–maleate
buffer to simulate conditions in the small intestine. Instead of using a
cocktail of enzymes as in Englyst et al. (1992), samples are hydrolysed
with α-amylase only for 16 h at 37 °C. As this method is focused on
the determination of RS (not SDS and RDS), products from enzyme
hydrolysis are discarded at this point. As 40 °C is the highest
temperature to which the sample is exposed, any RS2 in raw foods
is included in the unhydrolyzed fraction. The pellet is dispersed in 4 M
potassium hydroxide at room temperature for 30 min to solubilize
RS3. Dextrin in the alkali digest is hydrolyzed to glucose with
amyloglucosidase at 60 °C for 45 min. Glucose content is determined
using a colorimetric assay. Based on the analytical procedure of Goñi
et al. (1996), the presumed definition for RS is starch that is not
1964 A. Perera et al. / Food Research International 43 (2010) 1959–1974
Fig. 3.1. Summary of the protocol for the determination of total starch (TS) as per Englyst et al. (1992). The free glucose (FG) and total glucose (TG) contents are measured to estimate
the total starch content of the sample.
hydrolyzed by α-amylase within 16 h at body temperature, which is
in contrast to the Englyst et al. (1992) definition where RS is starch
that is not hydrolyzed by pancreatin, amyloglucosidase and invertase
within 120 min.
Other modifications to the Englyst et al. (1992) method have been
proposed but the essence of the method (to remove hydrolyzable starch
with enzymes) remained unchanged. Chung, Lim, and Lim (2006)
modified the Englyst et al. (1992) protocol to eliminate redundancy and
to introduce a reduction in sample size (to 0.5 g). Instead of adding guar
gum separately to each sample tube with subsequent dissolution in
acetate buffer, Chung et al. (2006) prepared a solution of guar gum in
HCl (to increase its solubility) and then added aliquots to each sample.
The volume of sodium acetate is decreased to 5 mL by increasing its
molar concentration to 0.5 M (20 mL of 0.1 M sodium acetate is used in
the Englyst et al. (1992) protocol). Chung et al. (2006) deleted invertase
from the enzyme mix. The digests collected (0.5 mL) after 20 and
120 min were mixed with 4 mL of 80% ethanol, which replaced the
20 mL of 66% ethanol in the Englyst et al. (1992) protocol. RS was
calculated as the difference between total starch and starch not
hydrolysed at 120 min as in Englyst et al. (1992).
The Megazyme® kit for RS determination is widely used in
analytical laboratories and is the basis of both AOAC method 2002.02
and AACC Method 32–40 (Megazyme, 2008). The procedure is
summarized in Fig. 5. This method, in many ways, is similar to that
of Englyst et al. (1982). Samples are ground to pass a 1-mm sieve
which generates a coarse meal, thus RS1 is accounted for in this
method. The Megazyme® protocol eliminates the initial boiling of
samples in acetate buffer and the use of pullulanase. Instead, it
employs a mixture of α-amylase (3 Ceralpha Units/mg) and
amyloglucosidase (3300 U/mL) to hydrolyze starch in raw or
processed food samples. Hydrolysis is carried out for 16 h at 37 °C
as proposed by Englyst et al. (1982). As a result, RS2 in raw foods
would not be hydrolyzed during this procedure. After inactivation of
enzymes with 99% ethanol, glucose in the sample is removed by two
washings with 50% ethanol. Similar to Englyst et al. (1982), the pellet
collected from digestion is treated with 2 M potassium hydroxide to
extract RS3 from the fibre-rich matrix. Dextrins thus produced are
hydrolyzed to glucose with amyloglucosidase. The incubation time
with amyloglucosidase is reduced to 30 min at 50 °C (Megazyme,
2008) from 1 h at 65 °C (Englyst et al., 1982). The Megazyme®
method uses the glucose oxidase-peroxidase colorimetric assay to
determine the glucose concentration in the final hydrolysate, as did
Englyst et al. (1992). The Megazyme® protocol for RS does not enable
the determination of SDS and RDS.
Eerlingen, Crombez, and Delcour (1993) proposed enzymatic-
gravimetric isolation of RS from wheat starch pre-gelatinized at
121 °C and subsequently stored at 0, 68 and 100 °C. In this method,
samples are placed in boiling phosphate buffer, pH 6, and hydrolyzed
with Termamyl® (heat stable α-amylase) at 100 °C for 30 min. After
cooling to room temperature, the pH is adjusted to 4.5 with 2%
phosphoric acid. Samples are hydrolyzed with amyloglucosidase at
60 °C for 30 min and then centrifuged. The pellet is washed with
deionized water, dispersed in phosphate buffer at pH 7.5 and
incubated with protease at 42 °C for 4 h. The residue after digestion
is collected by filtration and oven-dried at 80 °C. The weight of the
enzyme-insoluble residue represents the RS content of the starch
 A. Perera et al. / Food Research International 43 (2010) 1959–1974
Fig. 3.2. Englyst et al. (1992) protocol for the determination of rapidly digestible starch (RDS) and slowly digestible starch (SDS) as the preliminary step in the estimation of RS in a
food sample. The procedure for the estimation of total starch (TS) is explained in Fig. 3.1.
sample. Faraj, Vasanthan, and Hoover (2004) also isolated RS
gravimetrically, but introduced a preliminary hydrolysis with lichi-
nase and β-glucosidase. Then, samples are treated with a fungal
protease at 42 °C, Termamyl® at 100 °C and amyloglucosidase at
50 °C, respectively. RS is collected by drying the enzyme resistant
residue. It is important to note that the gravimetric separation of RS is
acceptable only for analytes free of non-starch polysaccharides.
Other modifications have been made to the sample preparation
step of RS determination with the intent of more closely simulating in
vivo digestion. Åkerberg, Liljeberg, Granfeldt, et al. (1998) and
Åkerberg, Liljeberg, and Björck (1998) employed chewing of food
samples to initiate hydrolysis by salivary amylase. According to these
authors, the number of times a food sample is chewed has a significant
effect on the end result. Briefly, the chewed food sample (15 chews in
15 s) and subsequent mouth wash are expectorated into a beaker
containing pepsin in Na/K phosphate buffer. Then, the pH is adjusted
to 1.5 with HCl and the sample is incubated for 30 min at 37 °C to
simulate gastric conditions. Sample pH then is adjusted to 5 with
sodium acetate buffer (0.5 M, pH 5) and sodium hydroxide.
Subsequently, starch hydrolysis is carried out with a mixture
containing MgCl2, CaCl2, 2-propanol, pancreatin and amyloglucosi-
dase for 16 h at 40 °C. Then, 95% ethanol at 60 °C is added in excess,
leading to the formation of a precipitate at room temperature. The
precipitate, collected by filtration, is dehydrated with 95% and 99%
ethanol and solubilized in 2 M potassium hydroxide. The total starch
content of the alkali digest is determined and expressed as RS. Muir
and O'Dea (1992) also employed chewing for sample preparation.
1965
Sample pH is then adjusted to 2 and the sample is hydrolysed with
pepsin for 30 min at 37 °C. After neutralizing the pH with sodium
hydroxide, pancreatin and amyloglucosidase are added to samples in
acetate buffer at pH 5 and incubated for 6 h at 37 °C. According to the
authors, this reduction in hydrolysis time (6 h instead of 16 h) is
intended to represent better the movement of food through the small
intestine. The Muir and O'Dea (1992) protocol significantly deviates
from the approaches already discussed as the pellet collected after
pancreatin hydrolysis (at 37 °C) is further treated with thermostable
α-amylase (Termamyl) at 100 °C, presumably to hydrolyse RS2. The
pellet recovered after Termamyl hydrolysis is boiled with dimethyl
sulfoxide (DMSO) for 1 h. The supernatant from Termamyl hydrolysis
(RS2) and the digest from DMSO treatment (RS3) are mixed and the
glucose content of the mixture is determined. Introduction of DMSO
in lieu of potassium hydroxide for solubilization of RS3 is a novel
feature of the Muir and O'Dea (1992) protocol. Saura-Calixto, Goňi,
Bravo, and Maňas (1993) determined RS in dietary fibre. Instead of
using pancreatin as the source of α-amylase, they used heat stable α-
amylase at 100 °C to eliminate traces of starch. The pellet from
enzyme digestion is sequentially hydrolyzed using a protease and
amyloglucosidase for 35 min at 60 °C. The residue is collected and
dehydrated with ethanol and acetone, and subsequently dispersed in
2 M potassium hydroxide at room temperature. After adjusting the pH
to 4.75 with acetate buffer, dextrins in the solution are hydrolyzed
with amyloglucosidase at 60 °C for 35 min. The glucose oxidase-
peroxidase system is used to measure glucose in the supernatant
collected after centrifugation.
1966 A. Perera et al. / Food Research International 43 (2010) 1959–1974
 A. Perera et al. / Food Research International 43 (2010) 1959–1974
Fig. 4. Goñi et al. (1996) protocol for the determination of resistant starch (RS) in foods.
It is important to note that very little information is available on
the in vivo digestibility of foods for which in vitro RS data is available.
From a nutritional point of view, this is a drawback. Grinding and
homogenization would certainly produce food particles much smaller
than those resulting from chewing. Englyst et al. (1992) compared
chewing of samples against mincing and reported that differences in
results were more related to the source of starch than to the method
of mincing. Individuals differ in their chewing habits and may not
chew the same number of times as in an experimental procedure.
Therefore, available RS1 may vary from person to person as its level
depends on particle size.
It is important to consider possible variability in RS data associated
with the technique employed to sample the enzyme hydrolysate,
because the colorimetric glucose assay is sensitive to inaccuracies in
sample volume. Englyst et al. (1992) proposed drawing of 0.5-mL
volumes of the hydrolyzate after 20 and 120 min to determine RDS
and SDS, respectively. Then, RS is computed as the difference between
total starch and enzyme-hydrolyzed starch. Chung, Liu, and Hoover
(2009) reduced the volume of sample drawn to 0.1 mL. It has been
observed that undigested food particles interfere with pipetting
(especially at the 20-min sampling point) more frequently than one
might expect. This would significantly influence the reproducibility of
the colorimetric assay of glucose. The advantage of using gas–liquid
chromatography to measure glucose is such errors can be corrected by
using an internal standard, as did Englyst et al. (1982). In this
connection, the Megazyme® protocol eliminated variability related to
sample volume by employing the total volume of the digest after
potassium hydroxide treatment for centrifugation and then drawing
Fig. 3.3. Summary of protocols for the determination of RS1 (a) and RS2 (b) according to Englyst et al. (1992). RS1 represents the fraction of starch inaccessible to enzymes due to
large particle sizes. RS2 represents the enzyme resistant starch in raw foods.
1967
an aliquot for color development. The availability of RS controls from
Megazyme® is useful for understanding experimental errors.
It is reasonable to expect that variations in analytical procedures,
including differences in the enzymes used and in their concentration
and sequence of application, and dissimilarities in the conditions of
experiments, would significantly alter the levels of RS detected in
similar foods. RS values of selected foods as reported by some authors
(Åkerberg, Liljeberg, & Björck, 1998; Chung et al., 2006; Englyst et al.,
1992; Faraj et al., 2004; Muir & O'Dea, 1992; Szczodrak & Pomeranz,
1991; Tribess et al., 2009) are presented in Table 1. Englyst and Hudson
(1996) reported RS values of a large number of different food items, but
the non-availability of information on handling and storage pre- and
post-processing prevent any useful comparison. The analysis of resistant
starch in Englyst et al. (1982) is limited to a few sources including white
bread, whole wheat bread and corn flakes (0.8%, 0.8% and 2.9% RS,
respectively, on a dry weight basis) because the main focus of this study
was to propose a method to determine non-starch polysaccharides in
foods, not RS.
6. Effect of starch composition on RS
Amylose and amylopectin are the two major polysaccharides in
starch, both having glucose as the fundamental building block. Linear
chains of glucose in both amylose and amylopectin are linked through α
(1→ 4) bonds. Amylopectin is heavily branched and chains are linked
through α (1→ 6) bonds to form a complex structure. Amylose shows a
low degree of branching (Bertoft, 2004). Conventionally, amylose in
starch is recognized by the deep blue color of the complex formed with
1968 A. Perera et al. / Food Research International 43 (2010) 1959–1974
Fig. 5. Summary of the Megazyme method of determination of resistant starch (RS) (Megazyme, 2008).
I2. In this structure, poly-iodine occupies the interior of the amylose
helix consisting of six glucose units per turn and in almost circular cross-
section (Rundle, 1947). A steady blue color (at 570 nm) corresponds to a
degree of polymerization (DP) ≥ 45 or a helix with 7–8 turns. Amylose
chains with 12 b DPb 45 produce a range of colors from red to purple,
respectively (Bailey & Whelan, 1961). In the amylopectin structure,
areas where branching begins and those having only linear chains are
clearly separated, producing amorphous and crystalline layers, respec-
tively. It is believed that amylose is confined mostly to the amorphous
layers (Donald, 2004). Two models are postulated for the biosynthesis of
amylose. Debranching of pre-amylopectin to produce pre-amylose and
subsequent elongation of glucan chains by granule bound starch
synthase I (GBSSI) to form amylose is a widely accepted model. The
other model proposes the synthesis of amylose through elongation of
short chains of malto-oligosaccharides by GBSSI (Chibbar, Ganeshan, &
Båga, 2007; Smith, 2001; van de Wal et al., 1998).
German, Blumenfeld, Guenin, Yuryev, and Toistoguz (1992) dem-
onstrated that gelatinized amylose aggregates to form a gel network
during cooling, whereas amylopectin acts as an agent of aggregation
and a network filler. Hydrolysis of amylose gels is dependant on gel
strength, since diffusion of α-amylase is decreased in strong gels that
form resistant starch (Cairns, Sun, Morris, & Ring, 1995). The ratio of
amylose to amylopectin, their chain lengths and the presence of lipid
influence the degree of retrogradation of starch from cereals and
pulses (Chung & Liu, 2009a). In lipid-rich starches, gelatinized
amylose preferentially combines with lipid at a relatively high
temperature (during cooling) before retrogradation begins and
impedes retrogradation of the remainder of the amylose. On the
other hand, small amounts of amylose-lipid complexes act as
nucleation sites for amylose chains. During retrogradation, long
chains of amylose in potato and amylopectin in amylomaize form
weak associations at low temperatures (exothermic peak at ∼30 °C)
perhaps due to the obstructions caused by branched chains (Chung &
Liu, 2009a). Variations in the composition of cereal starch, in terms of
the amylose to amylopectin ratio, are governed by the genome and
their genetic potential to undergo mutations (Rahman et al., 2007).
For example, mutations in wheat that lead to null genes for GBSSI
resulted in amylose-free endosperm starch (Vrinten & Nakamura,
2000). Similarly, inhibition of the expression of GBSSI in potato
produced amylose-free starch. On the other hand, rice varieties high
in GBSSI were reported to produce endosperm rich in amylose (Wang
et al., 1995). Differences in the GBSSI gene have been observed to
produce differences in the amylopectin levels in japonica and indica
rice (Umemoto, Yano, Satoh, Shomura, & Nakamura, 2002).
The influence of genetics on the RS contents of pulses is presented
in Table 2. Three cultivars of pea, 1674-13, 1215-33 and 1329-12,
contained different levels of RS when analyzed using the same
protocol. Two cultivars of lentil, CDC Meteor and CDC Robin, had
similar levels of RS (Chung, Liu, Hoover, Warkentin, & Vandenberg,
2008). Similarly, three cultivars of common bean, Majesty, Red
Kannern and AC Nautica, had similar levels of RS (Chung et al.,
2008). Significant differences in RS levels were observed among
chickpea cultivars (Myles, FLIP 97-101C, 97-Indian2-11) (Chung, Liu,
Donner, et al., 2008).
Genotype-specific differences in the amylose and amylopectin
ratios of sorghum and related differences in RS were reported.
According to Sang, Bean, Seib, Pedersen, and Shi (2008), starch from
heterowaxy sorghum (14% amylose) had the highest amount of RS
(23.7%) compared to waxy sorghum starch (0% amylose; 8.4% RS) and
normal sorghum starch (23.7% amylose; 17.9% RS), indicating the
significance of the amylose to amylopectin ratio with respect to the RS
level. In another study of four different hybrids, aeae × aeae (high
amylose); fl1fl1 × fl1fl1; aeae (female) × fl1fl1 (native) and aeae
(pollen) × fl1fl1 (female), starch from self pollinated aeae
produced the highest amounts of RS (55.2%) compared to the other
hybrids (1–6% RS) (Yao, Paez, & White, 2009). Genotypic variations
influenced RS levels in thermally processed low- (23-Am), and high-
amylose (67-Am) pea (Pisum sativum L). Autoclaved (1.35 bar for 1 h)
 A. Perera et al. / Food Research International 43 (2010) 1959–1974 1969

Table 1
Comparison of resistant starch contents of foods as determined by different methods.

Food Description RS Description of hydrolysis Author

White bread 1% Pancreatin, amyloglucosidase Englyst et al. (1992)

2% Chewing, pepsin, pancreatin, amyloglucosidase, Åkerberg, Liljeberg, and Björck (1998)
MgCl2, CaCl2, 2-propanol
Wholemeal bread 1% Pancreatin, amyloglucosidase Englyst et al. (1992)
70% barley wholemeal and 30% 6% Chewing, pepsin, pancreatin, amyloglucosidase, Åkerberg, Liljeberg, Granfeldt, et al. (1998)
white wheat flour MgCl2, CaCl2, 2-propanol
Wheat based products Wheat flour 0.085% Termamyl, protease, amyloglucosidase Tharanathan and Tharanathan (2001)
(Chapati, Poori and Phulka) Wheat flour and water at 2.5:1; salt 0.67–1.0%
and ground nut oil
Cornflakes 3% Pancreatin, amyloglucosidase Englyst et al. (1992)
6.5% Pepsin, pancreatin, amyloglucosidase, Muir and O'Dea (1992)
Termamyl
Rice Whole grain 11.8% Pepsin, pancreatin, amyloglucosidase, Muir and O'Dea (1992)
Termamyl
Retrograded native rice starch 9.3% Pancreatin, amyloglucosidase Chung et al. (2006)
Gelatinized rice starch 8.6% Pancreatin, amyloglucosidase Chung et al. (2006)
Barley Extruded flour 0.05% Lichinase, β-glucosidase, fungal protease, Faraj et al. (2004)
(100 °C, 20% moisture) Termamyl, amyloglucosidase
Raw, pure starch 4.1% Heat stable α amylase, amyloglucosidase Szczodrak and Pomeranz (1991)
Autoclaved, high-amylose starch 7.1% Heat stable α amylase Szczodrak, and Pomeranz (1992)
Green banana Dried flour at 52–58 °C 41–59% Amyloglucosidase, pepsin and amylase Tribess et al. (2009)
(AOAC method)
Biscuit (1:1 banana and wheat flour) 15% Pancreatin, amyloglucosidase Englyst, Kingman, et al. (1996)
Green pea (65-Am) Boiled seeds 33.4% Chewing, α-amylase, amyloglucosidase Skrabanja et al. (1999)
Autoclaved seeds 32.6%
Pea Boiled and frozen 6.3% As per Englyst et al.(1992) Bravo (1999)
Canned (autoclaved) 4.9%

seed of 23-Am (7.6% RS on a starch basis) had a lower level of RS than
did boiled seed (12.85% RS on a starch basis), whereas autoclaved
(32.6% RS) and boiled (33.4% RS) seed of 65-Am had similar levels of
RS (Skrabanja, Liljeberg, Hedley, Kreft, & Björck, 1999). Three
genetically-modified potato lines with different amylose contents
were compared for their RS contents after similar gelatinization and
retrogradation treatments (Leeman, Karlsson, Eliasson, & Björck,
2006). RS contents increased with increasing amylose content. For
example, line 527-1 contained 1% amylose and 0.1% RS, Prevalent, 23%
amylose and 5.3% RS, and line 342, 64% amylose and 26% RS. According
to Åkerberg, Liljeberg, and Björck (1998), bread made from barley rich
in amylopectin but low in amylose (3%) contained a lower amount of
RS (∼ 2.5%) than did bread made from high (44%) amylose barley
which contained ∼10% RS. The effect of amylose/amylopectin chain
length on the levels of RS formed is a complex phenomenon.
Comparing wild-type rice with mutants rich in RS, Shu et al. (2007)
concluded that the level of RS in cooked rice could not be explained on
the basis of amylose content alone. Instead, RS content was
determined by the degree of polymerization (DP) of the side chains
of amylopectin. Rice with high levels of RS had increased amounts of
short chains (DP ≤ 12), and decreased amounts of intermediate
(13 ≤ DP ≤ 36) and long (DP ≥ 37) chains. Cooked waxy maize starch
with higher amounts of amylopectin long chains (DP N 13) than short
chains (DP b 13) produced increasing amounts of RS over the cooling
phase at 4 °C, and after 160 h the RS level reached approximately 45 g/
100 g starch (Zhang, Sofyan, & Hamaker, 2008). Waxy maize starch
with higher amounts of amylopectin short chains (DP b 13) than long
chains (DP N 13) produced very low levels of RS (b5 g/100 g starch)
during the cooling phase. Ao, Simsek, Zhang, Venkatachalam, Reuhs
and Hamaker (2007) reported that partial hydrolysis of α-1,4 linkages
of side chains of pre-gelatinized maize starch using β-amylase and
maltogenic α-amylase and re-forming α-1,6 linkages using transglu-
cosidase resulted in products that were higher in RS compared to
untreated maize starch. Cairns, Morris, Botham, and Ring (1996)
studied the degree of polymerization (DP) of retrograded pea starch
gels and reported three levels (on a number average basis), with DP
107–108 and 26–28 being the most common types, and 5, a minor
type, indicating the contribution of long chains to gel formation. Using
potato starch, Eerlingen, Deceuninck, and Delcour (1993) demon-
strated that the DP of amylose (chain lengths from 40 to 610) was not
related to the DP of RS (19–26) formed. Escarpa, González, Maňas,
García-Diz, and Saura-Calixto (1996) adjusted amylose to amylopec-
tin (extracted from potato starch) ratios to observe changes in RS
contents. In an autoclave (115 °C, 2 bar, 20 min), samples of amylose,
amylopectin and their mixtures were gelatinized with water at a ratio
of 1:20. An amylose to amylopectin ratio of 100:0 produced 36.5% RS,
and when the ratio was decreased to 50:50, the RS content decreased
to 21.5%. When the ratio was changed to 0:100 (amylose: amylopec-
tin), RS decreased further to 7.5%.
These data clearly show that the RS contents of pulse and cereal
starches, and of their starch pastes, vary with the genetic factors that
influence amylose and amylopectin content and their chain lengths.
Therefore, comparison of analytical protocols for determination of RS
should consider the genetic origin of analytes, as comparison of RS
values of food products having similar starch ingredients but with
different genetic make-ups may lead to invalid conclusions.
7. Effect of processing on RS levels
Available moisture and temperature influence starch gelatinization
and retrogradation (Evans & Haisman, 1982; Liu & Thompson, 1998;
Eerlingen, Crombez, et al., 1993; Eerlingen, Deceuninck, et al., 1993).
Retrogradation of amylopectin occurs well at 2 °C forming elastic gels
which melt above 100 °C (Ring, 1985). Amylose gels retrograde better at
higher temperatures (Eerlingen, Crombez, & Delcour, 1993). Storage of
gelatinized, amylose-rich wheat starch at a low temperature (0 °C)
favours nucleation of the gel network; however, propagation of the
network is weak due to the low mobility of molecules. The reverse is
true during high temperature (100 °C) storage, where nucleation of the
gel network is weak but propagation is favoured due to the increased
mobility of molecules. During cooling an ‘ordered structure’ develops as
double-helical regions of several amylose chains align parallel to each
other, or single amylose chains fold to produce parallel areas, where
folded regions are amorphous and the lamellae are crystalline. For
example, the gel strength of potato starch is related to the storage
temperature. Considering storage temperatures between 5 and 50 °C,
1970 A. Perera et al. / Food Research International 43 (2010) 1959–1974

Table 2
Resistant starch concentrations in pulses as influenced by genetics, processing method and RS analytical protocol.

Starch source Treatment RS Method of RS analysis Author

Pea Raw, flour Cultivar — 1674–13 13.3% AACC method 32–40 (2000) with modifications Chung, Liu, Hoover, et al. (2008)
Cultivar — 1215–33 14.7%
Cultivar — 1329–12 10.1%
Pea Starch Cultivar — 1674–13 12.6% AACC method 32–40 (2000) Chung, Liu, Donner, et al. (2008)
Cultivar — 1215–33 8.1%
Cultivar — 1329–12 9.7%
Pea Raw Cultivar — 1674–13 10% Englyst et al. (1992) with modifications Chung, Liu, and Hoover (2009)
Annealed (15 °C; 70% moisture; 24 h) 10.9%
Heat-moisture treatment 13.3%
(100 °C; 30% moisture; 2 h)
Heat-moisture treatment 14.5%
(120 °C; 30% moisture; 2 h)
Common bean Raw flour Cultivar — Majesty 36% AACC method 32–40 (2000) Chung, Liu, Pauls, et al. (2008)
Cultivar — Red Kanner 35.5%
Cultivar — AC Nautica 32.4%
Starch Cultivar — Majesty 21.3%
Cultivar — Red Kanner 21.9%
Cultivar — AC Nautica 17.2%
Lentils Raw flour Cultivar — CDC Meteor 14.9% AACC method 32–40 (2000) with modifications Chung, Liu, Hoover, et al. (2008)
Cultivar — CDC Robin 14.4%
Lentils Starch Cultivar — CDC Meteor 13% AACC method 32–40 (2000) Chung, Liu, Donner, et al. (2008)
Cultivar — CDC Robin 13.2%
Lentils Raw Cultivar — CDC Meteor 9.1% Englyst et al. (1992) with modifications Chung, Liu, and Hoover (2009)
Annealed (15 °C; 70% moisture; 24 h) 11.4%
Heat-moisture treatment 13.2%
(100 °C; 30% moisture; 2 h)
Heat-moisture treatment 14.7%
(120 °C; 30% moisture; 2 h)
Lentil starch-gelatinized Native Cultivar 1544-8 12.8% Englyst et al. (1992) with modification Chung et al. (2010)
Annealed with 70% moisture for 14.1%
24 h at 50 °C
Heat-moisture treatment 17.5%
(30% moisture + 24 h at ambient
temperature + 120 °C for 24 h)
Annealed and then heat-moisture 18.5%
treated
Heat-moisture treated and then 19.6%
annealed
Chickpea Raw flour Cultivar — Myles 6.4% AACC method 32–40 (2000) with modifications Chung, Liu, Hoover, et al. (2008)
Cultivar — FLIP 97-101C 4.7%
Cultivar — 97-Indian2-11 3.1%
Chickpea Starch Cultivar — Myles 8.4% AACC method 32–40 (2000) Chung, Liu, Donner, et al. (2008)
Cultivar — FLIP 97-101C 18.4%
Cultivar — 97-Indian2-11 13.3%
Moth bean Raw 12.2% Goñi et al. (1996) Bravo et al. (1998)
Freshly cooked 3.9%
Cooked; stored at 4 °C for 24 h 4.8%
Horse gram Raw 26.4% Goñi et al. (1996) Bravo et al. (1998)
Freshly cooked 5.2%
Cooked; stored at 4 °C for 24 h 5.8%
Black gram Raw 19.7% Goñi et al. (1996) Bravo et al. (1998)
Freshly cooked 3.4%
Cooked; stored at 4 °C for 24 h 4%
Navy bean starch-gelatinized Native 14.8% Englyst et al. (1992) with modification Chung et al. (2010)
Cultivar AC Compass Annealed with 70% moisture for 24 h at 16.4%
50 °C
Heat-moisture treatment 19.8%
(30% moisture + 24 h at ambient
temperature + 120 °C for 24 h)
Annealed and then heat-moisture 20.5%
treated
Heat-moisture treated and then 21.9%
annealed

the higher the storage temperature, the higher the gel strength,
provided the starch content is N50% (Nakazawa, Noguchi, Takahashi,
& Takada, 1985). X-ray diffraction studies showed that two different
types of crystals were formed during retrogradation of wheat starch at
40 and 95 °C. At higher temperatures, wheat starch retrograded to form
structures with a large number of lamellae and having large particle
sizes. Resistance to α-amylase increased as the number of lamellae in
crystals increased (Zabar, Shimoni, & Bianco-Peled, 2008). Lamellae do
not form along the entire length of the amylose chain, rather about 24
glucose molecules of an amylose chain participate in lamella formation
(Eerlingen, Crombez, et al., 1993; Eerlingen, Deceuninck, et al., 1993).
These helices are thought to be formed through hydrogen bonding (Wu
& Sarko, 1978). As retrogradation proceeds, double helices are
organized into ‘hexagonal cells’ (Haralampu, 2000). Through their
influence on gelatinization and retrogradation, differences in the
conditions of food processing, such as changes in moisture, temperature
and duration of heating and subsequent cooling, influence the RS
content of foods.
 A. Perera et al. / Food Research International 43 (2010) 1959–1974 1971

Recent publications describing the influence of processing method
on variations in the RS contents of pulses and pulse products are
summarized in Table 2. For three cultivars of pea, 1674-13, 1215-33 and
1329-12, refined starch contained less RS than did the meal (Chung, Liu,
Hoover, et al., 2008), presumably due to the elimination of structural
impediments to amylase hydrolysis, i.e. a reduction in RS1 levels, during
starch extraction. This observation also was applicable to starch isolated
from the common bean cultivars Majesty, Red Kanner and AC Nautica
(Chung, Liu, Pauls, et al., 2008). For the chickpea cultivars Myles, FLIP 97-
101C and 97-Indian 2-11, starch contained more RS (Chung, Liu, Donner,
et al., 2008) than did the meal (Chung, Liu, Hoover, et al., 2008). In these
studies, pulse flours were prepared with a cyclone mill and starch was
isolated by the same procedure in each case. However, the analytical
procedures used to determine RS in chickpea starch and meal differed
slightly, making it difficult to draw conclusions from the observations.
Raw moth bean, horse gram and black gram had considerably higher RS
values than did cooked beans. When the cooked pulses were stored at
4 °C for 24 h, RS contents increased slightly (Bravo, Siddhuraju, & Saura-
Calixto, 1998). The cotyledons of some pulses, e.g. bean (Phaseolus
vulgaris L.), contain mammalian and insect α-amylase inhibitors as part
of the seeds' defence mechanism (Moreno, Altabella, and Chrispeels,
1990), the presence of which may result in very high levels of resistant
starch in assays of raw flours and meals.
The effect of processing on the RS content of some cereal products is
presented in Table 3. Han and Lim (2009) reported significantly higher
RS values for japonica brown rice as the moisture content of soaked rice
reached 20%, compared to rice containing 30% moisture prior to cooking.
As reported by Chung et al. (2006), native waxy rice starch had the
highest level of RS (∼9%), whereas gelatinized waxy rice starch (∼3%)
and retrograded, gelatinized waxy rice starch (3–3.8%) had the lowest
levels of RS. The observation of higher RS levels in native waxy starch
than in gelatinized and retrograded waxy starches is expected, as all or
some of the RS1 and RS2 in native waxy starch may be converted to
enzyme hydrolysable starch during heating, and the formation of RS in
the high-amylopectin waxy starches would not be extensive (Eerlingen
& Delcour, 1995; Russel, Berry, & Greenwell, 1989). This idea is
supported by the observation that waxy rice starch, partially gelatinized
at 60 or 70 °C, had RS contents (8.6 and 7.7%, respectively) similar to that
of native waxy starch. The RS contents of retrograded gels of wheat, corn
and potato were increased significantly when starch was used instead of
flour (as expected), but the opposite was reported for rice (García-
Alonso, Jiménez-Escrig, Martín-Carrón, Bravo, & Saura-Calixto, 1999).

Table 3
Resistant starch concentrations in cereal starches as influenced by processing.

Starch source Treatment RS Method of RS analysis Author

Japonica brown rice Pre-soaked in water at 25 or 50 °C to reach 20% moisture + cooked 29.2–32.4% Englyst et al. (1992) Han, and Lim (2009)
Pre-soaked in water at 25 or 50 °C to reach 30% moisture + cooked 20.7–25.9%
Waxy rice Native waxy rice starch 9.3% Englyst et al. (1992) Chung et al. (2006)
Gelatinized starch 3.0%
Partially gelatinized starch at 60 °C for 5 min 8.6%
Partially gelatinized starch at 70 °C for 5 min 7.7%
Pastry wheat flour Extruded at 20% moisture; 150/200/250 rpm; 40–120 °C; stored at 4 °C/0 days 0.48–0.52% Megazyme® assay Kim et al. (2006)
Extruded at 20% moisture; 150/200/250 rpm; 40–120 °C; stored at 4 °C/7–14 days 1.21–1.35%
Extruded at 40% moisture; 150/200/250 rpm; 40–120 °C; stored at 4 °C/0 days 0.63–0.67%
Extruded at 40% moisture; 150/200/250 rpm; 40–120 °C; stored at 4 °C/7–14 days 1.52–1.86%
Extruded at 60% moisture; 150/200/250 rpm; 40–120 °C; stored at 4 °C/0 days 2.54–2.65%
Extruded at 60% moisture; 150/200/250 rpm; 40–120 °C; stored at 4 °C/7–14 days 3.55–4.25%
Corn Acid modified with 1.64 M HCl at 40 °C for 4 h + gelatinized 5% AOAC 991.43 (1998) Koksel, Masatcioglu,
+ autoclaved + lyophilized Kahraman, Ozturk, and
Basman (2008)
Acid modified with 1.64 M HCl at 40 °C for 4 h + gelatinized 12%
+ autoclaved + Stored at 95 °C for 48 h + lyophilized
Corn Normal corn starch 19.7% Englyst et al. (1992) modified as Chung, Hoover, and Liu
per Chung, Liu, et al. (2009) (2009)
Annealed with excess water for 24 h at 50 °C 18.3%
Heat-moisture treatment 16.9%
(30% moisture + 24 h at ambient temperature + 120 °C for 24 h)
Annealed with excess water for 24 h at 50 °C + Heat-moisture treatment 17.3%
(30% moisture + 24 h ambient temperature + 120 °C for 24 h)
Heat-moisture treatment 19.7%
(30% moisture + 24 h ambient temperature + 120 °C for 24 h)
+ annealed with excess water for 24 h at 50 °C
Corn Native corn starch 1.3% AOAC 991.43 (1985) Brumovsky, Brumovsky,
Fretes, and Peralta (2009)
Heat-moisture treatment 1.5%
— 30% moisture + 100 °C for 60 min
30% moisture + 120 °C for 60 min 4.2%
Wheat Native wheat starch 1.0%
Heat-moisture treatment 1.9%
— 30% moisture + 80 °C for 60 min
30% moisture + 100 °C for 60 min 1.6%
High-amylose corn Autoclaved at 120 °C + Stored at 4 °C for 24 h (repeated twice) 30% Goñi et al. (1996) Zhao and Lin (2009)
Autoclaved at 120 °C + Stored at 4 °C for 24 h (repeated twice) 39%
+ hydrolysed with 0.1 M citric acid
Corn (normal) Treated with acid (HCl) and methanol at 25 °C for 30 days ∼12% Englyst et al. (1992) Lin et al. (2009)
Treated with acid (HCl) and methanol treatment at 25 °C for 30 days ∼38%
+ annealed at 50 °C for 72 h with water
Corn (normal) Gamma irradiation (60Co) 0 kGy 19.7% Englyst et al. (1992) Chung and Liu (2009b)
10 kGy (2 kGy/h) 22.2%
10 kGy (0.67 kGy/h) 23.0%
10 kGy (0.40 kGy/h) 24.7%
1972 A. Perera et al. / Food Research International 43 (2010) 1959–1974
Green banana starch is a rich source of RS but the amounts varied
significantly (41–59%) with the method of drying employed during
starch processing (Tribess et al., 2009).
Heat-moisture treatments (30% moisture, 24 h at ambient temper-
ature, heated at 100 or 120 °C for 2 h) were applied to corn (Table 3) and
lentil and pea (Table 2) starches, with and without a pre-gelatinization
treatment (Chung, Liu, & Hoover, 2009). The heat-moisture treatment
increased RS levels considerably. For example, native corn, lentil and pea
starches had RS contents of 5, 9 and 10% respectively; corresponding
values were increased to 12%, 15% and 15% after the 120 °C heat-
moisture treatment. In another experiment, a heat-moisture treatment
(30% moisture, 24 h at ambient temperature, heated at 120 °C for 24 h)
decreased the level of RS in corn starch (16.9%) compared to that of
native, normal corn starch (19.7%). A subsequent annealing treatment
(sample to water ratio of 3:7, 50 °C, 24 h) compensated for the lower RS
content (Chung, Hoover, & Liu, 2009). Heating of corn starch at 30%
moisture for 40 or 60 min at 100 °C or 120 °C yielded products with 1.3
and 1.5% RS, respectively, at 100 °C and with 1.8 and 4.2% RS,
respectively, at 120 °C, illustrating the significance of the combined
time and temperature effect on RS levels (Brumovsky, Brumovsky,
Fretes, & Peralta, 2009). Annealed and/or heat-moisture-treated pulse
starches which were subsequently gelatinized produced higher
amounts of RS than did gelatinization of native starches (Chung, Liu, &
Hoover, 2010). For example, gelatinized, native lentil and navy bean
starches contained 12.8 and 14.8 g RS/100 g starch, respectively. Lentil
starch annealed (70% moisture, 24 h, 50 °C) and subsequently heat-
moisture treated (30% moisture, 120 °C, 24 h), and heat-moisture-
treated lentil starch which was subsequently annealed, exhibited RS
levels of 18.5 and 19.6 g/100 g starch, respectively, after gelatinization
(Table 2). Corresponding values for navy bean after gelatinization were
20.5 and 21.9 g/100 g starch (Table 2). The effect of extrusion pelletizing
on RS was determined with barley, wheat, oat, corn and rice. Dough was
cooked at 95 °C for 1 h before extrusion and then dried at 100 °C for 1 h.
For all grain types used, with the exception of rice, a significant
reduction in RS was reported after extrusion (Hernot, Boileau, Bauer,
Swanson, & Fahey, 2008). The RS level of extruded pastry wheat flour
increased as the initial moisture level of the feed (20, 40 or 60%) and
post-process storage time at 4 °C (0, 7 or 14 days) increased (Kim,
Tanhehco, & Ng, 2006) (Table 3). High-amylose corn starches (Hylon VII
and Gelose 80; 70 and 80% amylose and 60 and 46% RS, respectively)
subjected to extrusion cooking (35 and 50% moisture, 100 and 140 °C,
150 and 750 s−1 shear) exhibited significantly reduced RS concentra-
tions (14 and 17%, respectively). This was thought to be due to enhanced
opportunities for amylase action after shear damage to starch granules
(Htoon et al., 2009). Both a pre-gelatinization treatment of corn starch
with HCl at 40 °C (Koksel et al., 2008) and a post-retrogradation
treatment of high-amylose corn starch with citric acid (Zhao & Lin,
2009) increased the level of RS (Table 3). Wheat and corn starch cooked
at 100 °C for 10 min during an in vitro digestibility assay had RS levels of
9 and 11.4%, respectively; similar levels of RS were seen in wheat
(10.9%) and corn (13.3%) starch autoclaved three times at 121 °C for
15 min and then cooled within 15 min (Hickman, Janaswamy, & Yao,
2009). Starch autoclaved as above, and subsequently dried, when
subjected to β-amylase hydrolysis at pH 5.5 and 55 °C for 20 h exhibited
significantly higher levels of RS, 23.1 and 29.9% in wheat and corn starch,
respectively (Hickman et al., 2009). Commercially available canned
beans had lower levels of RS than did canned bean flour prepared after
drying at 50 °C (Osorio-Díaz et al., 2002). RS levels in corn starch
subjected to acid–methanol treatment only or acid–methanol treatment
combined with annealing at 50 °C for 72 h were 12 and 38 g/100 g
starch, respectively (Lin, Wang, & Chang, 2009). Gamma irradiation at
10 or 50 kGy (dose rate of 2 kGy/h at room temperature) increased the
RS content of corn starch from 19.7% to 2.2 and 25.1%, respectively
(Chung and Liu, 2009b).
It is apparent that even minor differences in the conditions under
which starch gelatinization and retrogradation are achieved can lead
to significant differences in the RS contents of foods and food
ingredients. Therefore, meaningful comparison of analytical proce-
dures for RS determination requires analysis of foods processed under
identical conditions. Comparison of analytical procedures for RS using
currently available data is difficult as the analytes used are often
dissimilar.
8. Conclusion
Analytical procedures for RS vary with respect to the sample
preparation method, particle size of samples, nature of the prelimi-
nary heat treatments for raw analytes (if any), enzymes and buffers
employed, incubation temperatures, sampling of the enzyme digests,
chemicals and conditions of hydrolysis, and the glucose determina-
tion method. The effects of these changes on the RS values obtained
generally cannot be compared as the analytes used were not identical
in most cases. Genetic differences with respect to the source of starch,
type of processing (meal vs. starch) and conditions of processing
(partial vs. complete gelatinization), chemical modification of
samples, and storage conditions significantly influence RS values.
Therefore, the concept of RS in a food material is a subjective
parameter and any generalizations made for a particular food item or
category must be made with caution.
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