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Rice Science, 2021, 28(1): 31í42

Review

Physiochemical Properties of Resistant Starch and Its

Enhancement Approaches in Rice

DING Yi1, #, WANG Maike1, #, SHEN Yi 1, SHU Xiaoli1, WU Dianxing1, SONG Wenjian1, 2
(1State Key Laboratory of Rice Biology / Key Laboratory of the Ministry of Agriculture for the Nuclear Agricultural Sciences,
Zhejiang University, Hangzhou 310058, China; 2Agricultural Technology Extension Center, Zhejiang University, Hangzhou 310058,
China; #These authors contributed equally to this study)

Abstract: With changes in food preferences and life styles, more and more attentions have been focused
on healthier food. Nowadays, resistant starch (RS) which can resist digestion in the human intestine has
been recognized and accepted. High RS diet shows great benefit for the human health, and breeding
high RS rice variety is a great target for realizing dietary intervention. To provide guidance for RS
improvement in rice, this review summarized the unique physiochemical properties of RS and the
possible approaches, i.e. genetic regulation, for enhancing RS content in rice, and proposed the potential
ways to obtain rice variety with high RS content.
Key words: rice; resistant starch; starch modification; physiochemical property

Starch is one of the main nutrients in rice grains and
the major determinants for rice quality. It can be
classified chemically into amylose, a linear and flexible
polymer, and amylopectin, a branched polymer of
amylose chains (Li and Gilbert, 2018). Starch particles
are mainly composed of crystalline and non-crystalline
areas, and also a sub-crystalline structure between
crystalline and amorphous (Hizukuri, 1985). Amylose
is a single helical structure located in the amorphous
region of the crystalline structure of starch granules,
and amylopectin is a double helical structure located
in the crystalline region (Buléon et al, 1998). The
supramolecular structure of starch granule is closely
related to its physicochemical and digestive characteristics
(Chen et al, 2016). Based on its digestive properties,
starch can be categorized into three groups: rapidly
digestible starch (RDS), slowly digestible starch (SDS)
and resistant starch (RS) (Bello-Perez et al, 2020).
RS was first recognized as a complicating factor
when determining total dietary fiber levels with the
Prosky method (Englyst et al, 1987). Since incomplete
digestion and absorption of starch in the small intestine
has been relatively recognized as a normal phenomenon,
interest in non-digestible starch fractions has been
rising (Cummings and Englyst, 1991). Subsequent
research had found that RS is digested not as rapidly
as common starch. It owns some unique functions
besides some biological benefits like traditional fiber
(Asp et al, 1988; Haralampu, 2000), e.g. smoothing
postprandial blood glucose, preventing colon cancer.
With the global epidemic incidence of obesity and
type II diabetes, and the enhancement of people living
standard and quality, more studies are focusing on RS
and high RS diets. RS in rice has been observed to be
significantly and negatively correlated with glycemic
index (GI) (Kumar et al, 2018). As the staple food,
rice is an ideal dietary intervention food. Enhancing
RS content in rice is important for fulfilling the intervention.
Focusing on the physicochemical properties of RS and
its improvements in rice, we summarized the recent
studies on RS in rice and proposed potent approaches
for producing high RS rice variety.

Received: 7 March 2020; Accepted: 6 July 2020

Corresponding author: SONG Wenjian (swj@zju.edu.cn)
Copyright © 2021, China National Rice Research Institute. Hosting by Elsevier B V
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Peer review under responsibility of China National Rice Research Institute
http://dx.doi.org/
http://dx.doi.org/10.1016/j.rsci.2020.11.005
32
Classification and measurement in vitro of RS
RS is the portion of starch and products of starch
degradation that escapes digestion in the small
intestine of healthy individuals, following which it is
fermented by microorganisms in the colon (Englyst et
al, 1992; Cheng and Lai, 2000). It is subdivided into
five types: RS1, RS2, RS3, RS4 and RS5 (Table 1).
RS1 is physically inaccessible starch, presenting in
undamaged food matrices. It has been reported in
whole-grain foods, unbroken grains, seeds and some
dense food fragments. RS1 can be disrupted during food
processes and may be omitted when starches/ flours
rather than whole foods were used in the quantification
of RS (Oladele, 2017). RS2 always exists in starch
that has not been gelatinized, so it is useful for
products that needn’t gelatinization, like popcorn. RS1
and RS2 are two typical modes, and most of them can
be inverted into digestible starch after food production.
RS3, coming from retrograded starches, is applied
widely in food products (Haralampu, 2000) and is the
main type exhibiting the functional properties of RS
for human. RS3 shows low digestibility, increased
complexing ability with iodine and fatty acid, which
makes it to be an ideal encapsulating material for drug
delivery (Khan et al, 2019). RS4 is a kind of modified
starch with changes in starch structure through
chemical methods (Ashwar et al, 2017b). Different
modi¿cations, i.e. physical, chemical, enzymatic and
genetic modifications, have been employed for the
preparation of RS. As the contributions of RS to
human health are recognized, artificially increasing
RS4 content is an important research direction. RS5 is
a newly defined type, and refers to a kind of amylose-
lipid complexed starch (Bird and Topping, 2008). It
can be evaluated in both amorphous amylose-lipid
complex and crystalline complex, which is produced at
lower and higher temperatures respectively (Hasjim et al,
2013). RS5 has some specific advantages compared to
other RS types, e.g. better thermal stability than RS2
when heated; less formation processes than retrograded
amylose (RS3) or chemically modified starch (RS4),
Table 1. Description of different classifications of resistant starch (RS).
Classification Description
RS1 Physically inaccessible
RS2 Natural uncooked granules
RS3 Natural retrograded granules
RS4 Chemically modified
RS5 Amylose-lipid complexed
Adapted from Englyst et al (1992), Bird and Topping (2008), and Hasjim et al (2013).
Rice Science, Vol. 28, No. 1, 2021
and its structure can be recovered simultaneously during
the cooling process after dissociated by heat (Hasjim
et al, 2013).
There are two widely used methods for measuring
RS in vitro, which are enzymatic-gravimetric method
(Prosky method) (e.g. AOAC991.43, AACC32-07)
and enzymatic-chemical method (Englyst et al, 1992;
Göni et al, 1996) (e.g. AOAC2002.02, AACC32-40).
Both methods include lipid extraction, protein and
carbohydrate enzymatic digestion, but the remaining
non-digestible composition was measured by gravimetry
in the Prosky method and by chemical analysis in the
enzymatic-chemical method (Kontraszti et al, 1999;
Haralampu, 2000). With the gravimetric method, the
RS values represent a range of materials formed during
food processing, which contain not only undigested
starch but also other non-digestible fibers, and the
result is acceptable only for analytes free of non-starch
polysaccharides. While with the enzymatic- chemical
method, the non-digestible composition is treated with
KOH to extract RS from the fibre-rice matrix. Produced
dextrins are hydrolyzed to glucose by amyloglucosidase
and the released glucose is determined (Perera et al,
2010). In the procedure proposed by Englyst et al
(1992), RS is defined as the starch portion that cannot
be hydrolyzed after 120 min and determined as [RS =
Total starch – (RDS + SDS)], among which, RDS and
SDS are measured after incubation with pancreatic
amylase and amyloglucosidase at 37 ºC for 20 min and
a further 100 min. While in the protocol developed by
Göni et al (1996), RS is measured after hydrolysis for
16 h at 40 ºC. Based on these two methods, some
modifications i.e. different buffers, pHs and combinations
of enzymes, have been proposed, but the procedure of
removing hydrolyzable starch with enzymes remains
unchanged (Perera et al, 2010).
Physiochemical properties related to RS
Some physiochemical properties of starch, such as granule
size and available specific surface, granule integrity
and porosity, crystalline structure, amylose/amylopectin
Example
Whole grains
Banana starch
Cooked rice
Crosslinked starch
Aqueous starch with butterfat
DING Yi, et al. Properties of RS and RS Improvement in Rice
ratio, proteins and lipids on the granule surface (Copeland
et al, 2009; Dona et al, 2010), determine the starch
digestibility and influence the formation of RS. Deepening
the understanding of the relationship between these
properties and the content of RS might open some
new ways to improve RS content.
Starch granules
Rice starch granules are generally pentagonal and
angular. The starch granule in high-RS3 mutant is
significantly higher percentage of oval-shaped granules
and bigger oval size than that in the wild parent (Yang
et al, 2006). High amylose rice, which also shows
high RS2 content like, Goami 2, has semi-compound
and elongated starch granules (He and Wei, 2020).
Physical, chemical or enzymatic modifications would
bring some impacts on the starch granules. The micro-
structure of rice starches treated with dual autoclaving-
retrogradation shows a continuous dense network with
irregular shape without granular structure, which is
largely responsible for its increased resistance to enzyme
attack due to the increased crystal density (Ashwar et al,
2016). Phosphorylated rice starch by cross-linking
with sodium trimetaphosphate/sodium tripolyphosphate
(STMP/STPP) retains the integrity of granules, except
some fissures appeared on the surface of some granules
and some large starch granules presented (Ashwar et al,
2017a). Granular structures of rice starch treated by
autoclaving-cooling or Neisseria polysaccharea amylosucrase
(NpAS) are displaced by irregular continuous matrix
structures (Kim et al, 2013).
Beside starch granule types, starch granule diameter
and molecular weight of amylopectin and amylose
both show negatively correlation with the digestibility
of starch (Sandhu and Lim, 2008). The smaller
granules are more susceptible to enzyme digestion,
which may be attributed to a larger specific surface
area increasing enzyme binding rate (Zaman and
Sarbini, 2016). Generally, the diameter of rice starch
granule ranges from 3 to 8 ȝm. After heating-moisture
treatment (HMT), rice starch granule size is increased
(Wang et al, 2018). Micro-particle size of cross-linked
phosphorylated rice starch (RS4) reaches up to
45.53–49.29 ȝm, which is bigger than the normal rice
starch granules (Ashwar et al, 2018). However, Jeong
and Shin (2018) found that RS4 is only 0.001 to 0.1
ȝm, which makes them stably though not swelling in
an aqueous medium. Ding et al (2019) found that
granule size shows a negative correlation with RS3
and no significant correlation with RS2.
33
Apparent amylose
There is a certain relationship between amylose
content and RS content. Noda et al (2003) suggested
that the apparent amylose content (AAC) in raw rice
starches is negatively correlated with digestibility.
Higher content of amylose show lowers the digestibility
of starch (Sajilata et al, 2006). Generally, high-
amylose starches present high content of RS (Shu et al,
2006; Shi and Gao, 2011; Gani et al, 2017), and AAC
is positively correlated with RS (RS2 or RS3) (Zhang
et al, 2007; Chung et al, 2011; Bao et al, 2017; Zhou
et al, 2018). The high-RS (RS3) rice mutant is
characterized by significantly higher AAC (Yang et al,
2006). AAC can be a strong predictor of RS (Chen M
H et al, 2017). Reduction in digestibility after retrogradation
is more effective in rice with high AAC than that with
low AAC or waxy rice (Alhambra et al, 2019), as the
hydrogen bonds presenting in the glucose chains
connecting amylose make them more resistant to
enzymatic activity (Zaman and Sarbini, 2016).
Although most of the studies showed that there is a
positive correlation between AAC and RS, Kim et al
(2013) reported that AAC is not associated with RS
content of NpAS-treated starches, and phosphorylated
rice starches with higher RS content have significantly
lower amylose content than their respective native
starches with lower RS content, which might be due to
some of amylose molecules are leached from the
starch granules during the phosphorylation process
(Ashwar et al, 2017a). What’s more, Ashwar et al
(2016) found that retrogradation shows no positive
effects on RS producing, because the portion of
amylose complexes with lipid during cooking and
cooling cannot combine by the iodine and be detected
with common iodine staining method.
Chain length distributions of amylopectin
Chung et al (2011) and Zhou et al (2018) found that
the chains of degree of polymerization (DP) 12–24
have positive correlation with RS (RS3 or RS2), and
the chains of DP 25–36 have negative correlation with
RS3. The proportion of DP 6–12 is negatively correlated
with RS2 and SDS, and positively correlated with
RDS (Chung et al, 2011). Similar results are found in
rice ae mutant which has very high RS2 content, in
which the proportion of short chains (DP ” 12) is
markedly lower, whereas the proportion of long chains
(DP • 37) is greatly elevated (Nakamura et al, 2015).
It strongly suggests that besides amylose, intermediate
34
chains and long chain amylopectin molecules may
play an important structural role in rendering the
starch digestibility, and elevations in the proportion of
intermediate chains may be associated with the
increases of RS in rice (Butardo et al, 2011). Ryu et al
(2010) found that after enzyme modification, the most
abundant branch-chain length of amylopectin in waxy
corn starch is increased from DP 13 to DP 24, and RS
level is increased from 6.9% to 29.0%. The amylosucrase-
modified starches have lower and higher proportion of
DP 6–12 and DP 13–36 respectively when compared
to the native starches, regardless of AAC of native
starches (Kim et al, 2013).
However, the DPs of modified starches by citric
acid, lactic acid or acetic acid are significantly
reduced with the increment of RS content as compared
to those of native starches (Hung et al, 2016). What’s
more, Shu et al (2007) found that the increased contents
of short chains with 8 ” DP ” 12 and decreased
intermediate and long chains with DP • 24 are clearly
associated with the increases of RS3 in rice. A small
increase in DP 10–20 and a small decrease in DP
35–50 in high RS rice mutant b10 might also
contribute to the increment in RS3 when compared to
the common rice (Zhou et al, 2016). The inconsistent
results about the relationship between amylopectin
chain length distribution and RS content may be due
to different starch origins, different RS types and
different measurement procedures used. Tsuiki et al
(2016) found that RS3 is positively correlated with
AAC when RS3 content is lower than 4%, and is
positively correlated with the amount of amylopectin
long chains when 15% ” RS ” 35%. Ding et al (2019)
reported that RS3 shows significantly positive
correlation with the amount of chains with DP < 12
and significantly negative correlation with the amount
of chains with 13 < DP < 24 and DP > 37, while RS2
shows a significant positive correlation with the
amount of chains with 25 < DP < 36.
Crystal structure
Starch digestibility is also related to starch granule
crystal, and the crystalline regions prevent or hinder
the access of digestive enzymes to the glucose chains
(Polesi et al, 2017). Starch crystal structure can be
classified into type A, B and C according to the
diffraction peaks of X-ray spectrum. Type A crystalline
usually has strong diffraction peaks at 15º, 17º, 18º
and 23º, type B has strong peaks at 5.6º, 17º, 22º and
24º, and type C is a mixed of type A and B (Imberty
Rice Science, Vol. 28, No. 1, 2021
and Perez, 1988). V-type, identified as the complex of
amylose with iodine, fatty acid, emulsifiers or butanol
and other substances, has different strong diffraction
peaks according to the guest molecules (Tan and Kong,
2020). Amylose-lipid complexes have strong peaks at
around 20º (Lu et al, 2019). Native rice starch is
traditionally classified as an A crystalline type (A
polymorph) as other cereal starches. Rice crystalline
polymorph shifted from A to C or B with genetic
modification has been demonstrated to reduce the in
vitro digestibility of cooked white rice grain (Butardo
et al, 2011). Rice starch treated with pullulanase and
the RS isolated from cooked rice display a mixture of
B- and V-type, which show more resistance to starch
hydrolysis by Į-amylase (Shi and Gao, 2011). High
amylose transgenic rice starch is mainly C-type starch
crystal structure, and would change to B-type starch
crystals after heating to 75 ºC, while the original
parent changed from type A to indefinite morphology
(Wei et al, 2010). Heat treatments of rice starch result
in the disappearance of A-type and the formation of
V-type crystallite, and due to long pressure cooking,
little B-type is observed (Wei et al, 2011). Parboiling
also results in the formation of Vh type pattern (Cheng
et al, 2019). However, pores have recently been observed
to be present on the surface of raw B-type rice starches.
The reduced enzymatic hydrolysis of B-type rice starch
granules may be determined by a higher proportion of
long chain amylopectin (Dhital et al, 2015).
The high-RS (RS3) mutant is characterized by
significantly reduced crystallinity (Yang et al, 2006).
RS4 derived from rice starches also shows decreased
crystallinity (Ashwar et al, 2017a). Esterification can
cause a decrease in the degree of crystallinity of rice
starch (Ye et al, 2019). Rice starch after HMT only
shows a slight reduction in the peak intensities without
crystalline changes (Wang et al, 2018), whereas
debranched rice samples with higher RS have higher
relative crystallinity (Shi and Gao, 2011).
Pasting and other gel properties
Peak viscosity (PKV) and break-down value (BDV)
obtained by Rapid Viscosity Analyzer (RVA) are
significantly negatively correlated with AAC (Shu et al,
1998; Bao, 2017). It is assumed that RS would also
have significant correlation with RVA parameters. The
high-RS (RS3) rice mutant is characterized by
significantly reduced paste viscosity, onset temperature,
peak temperature, final temperature and enthalpy of
gelatinization ('H) when compared with the original
DING Yi, et al. Properties of RS and RS Improvement in Rice
parent (Yang et al, 2006). All RVA parameters of dual
autoclaving-retrogradation treated rice starches, except
peak time and pasting temperature (PT), are significantly
lower than those of native ones (Ashwar et al, 2016).
PT, setback value (SBV) and cool paste viscosity
(CPV) are negatively correlated with RDS, and
positively correlated with SDS and RS2 (Chung et al,
2011). Benmoussa et al (2007) reported that BDV of
raw starches is positively correlated with RDS and
negatively correlated with SDS of cooked and stored
rice cultivars. wx ae double mutant displays a higher
PT and PKV, and a slow increase of blood glucose in
rats when fed with wx ae starch (Kubo et al, 2010). PT
is a strong predictor of RS3 (Chen M H et al, 2017).
Hot peak viscosity (HPV) and coo paste viscosity
(CPV) are positively correlated with the content of
RS2, while significantly negatively correlated with the
content of RS3 (Ding et al, 2019). Zhou et al (2018)
also reported that the RS3 content shows negative
correlation with HPV and CPV. RS4 microcapsules of
crosslinked rice starch with STMP/STPP present
significant higher transition temperatures and 'H than
those of native rice starch, indicating crosslinking
enhances the thermal stability of rice starch (Ashwar
et al, 2018). Besides, hardness of rice paste gel shows
negative relation to RS. The gel formation capability
of rice is improved by supplementing the enzyme-
modified RS. With 20% substitution of amylosucrase
modified RS for common rice starch, gel hardness of
rice paste is increased up to 60%, and gel cohesiveness
seems to decline significantly, while springiness is
kept intact (Doan et al, 2019).
Non-starch polysaccharides, lipids and proteins
Non-starch polysaccharides (NSPs) added to starch
containing products during processing can improve
the texture, water mobility, stability and viscosity, and
slow down the diffusion of digestive enzymes into the
food matrix and the absorption of nutrients (Bai et al,
2017). Adding 30% NSPs (agar etc.) to rice starch can
substantially enhance the enzyme resistance of such
mixture paste (Sasaki and Kohyama, 2011). However,
the influences of NSPs on starch properties depend
both on the types of rice and the NSPs. Endogenous
NSPs, especially the hemicellulose, play an important
role in RS3 formation and rice cooking quality.
Operation on NSPs for RS improvement is feasible, as
NSPs may also be an alternative operating factor for
obtaining rice with both lower glycemic index (GI)
and improved palatability (Sun et al, 2018).
35
Rice endosperm lipid fractions especially those
starch lipids also show a pronounced contribution to
RS3 formation and the texture of paste gel (Zhang et al,
2019). Lipids of rice can be classified into non-starch
lipids and starch lipids, which are majorly located in
the embryo and aleurone layer, and occur on the
surface (surface starch lipids) or inside the granule
(true starch lipids), respectively. The rice endosperm
lipid fractions mostly consist of lysophospholipids and
free fatty acids (Morrison, 1988). Removals of
endogenous lipids significantly increase the starch
digestibility through improving starch swelling and
gelatinization (Ye et al, 2018a). The high-RS (RS3)
mutant is also characterized by significantly higher
crude lipid content, which will form amylose-lipid
complexes and enhance the resistance to be digested,
and endow a specific aroma to the rice (Yang et al,
2006). Zhou et al (2016) also observed increased levels
of total lipid and amylose-lipid complex in the starch
of high RS3 rice mutant b10. Amylose-lipid complexes
formed by degraded starch chains have higher thermal
stability and greater thick crystalline lamellae than
common starch molecules (Wang et al, 2018).
However, studies on starch-protein interactions and
their influences on starch digestibility in rice are
scarce. Only Ye et al (2018a) found that removals of
proteins in rice endosperm increase the starch digestibility
of rice. Endogenous proteins of rice may also impair
digestion because protein and starch will interact
within food matrix, but the influences are not as so
pronounced as lipids. The endogenous proteins and
lipids can inhibit the contact between digestive enzyme
and starch granule through attaching to the surfaces of
the starch granules, thus decreasing the digestion (Ye
et al, 2018a). It suggests rice variety with relatively
higher protein and lipid contents may be helpful for
keeping stable postprandial blood glucose levels.
Healthier rice with low GI may be bred by operating
lipid and protein contents and compositions.
Approaches for resistant starch improvement
With the increased interest in RS and deep understandings
of the physiochemical properties related to RS, some
manual methods including enzymatic, physical, chemical
and genetic modifications have been conducted to
improve RS content and even carry out different types
of RS conversion.
Chemical modification
Modification of starch introduces new functional
36
groups to promote structural changes and affects its
physicochemical properties. Chemical modification has
been one of the most effective approaches to produce
RS4. Many types of chemically modified starches have
been prepared using oxidation, etherification, esterification,
hydroxypropylation and cross-linking approaches
(Masina et al, 2017).
Among the organic acids used, citric acid has the
most impact on starch characteristics and RS formation,
followed by lactic acid and acetic acid (Hung et al,
2016). Citric acid esterification is preferred due to the
mild acid treatment, nutritional safety, non-toxicity
and economics (Sánchez-Rivera et al, 2017). Several
studies reported that treatment of starches with citric
acid solution at high temperature increases the SDS
and RS contents (Shin et al, 2007). Citric acid
esterification significantly increases the level of RS
and enhances the resistance of esterified rice starch to
digestion due to cross-linking and esterification within
the starch molecules (Ye et al, 2019). The RS contents
of acid treated rice starches are in a range of 30.1%–
39.0%, significantly higher than those of native rice
starches (6.3%–10.2%) and heat-moisture treated rice
starches (18.5%–23.9%) (Hung et al, 2016). DPs of
the rice starches treated with citric acid, lactic acid or
acetic acid are significantly reduced compared to
those of the native starches (Hung et al, 2016). The
swelling power and solubility of starch citrate are both
lower than unmodified starch. Turbidity measurements
suggest there is a slower rate of re-association in starch
citrates during storage (Ye et al, 2019).
The application of STMP/STPP as a cross-linker
can improve the integrality and stability of starch. Cross-
linked rice starches have higher content of RS compared
to their native starches (Ashwar et al, 2017a). The RS
content in STMP/STPP cross-linked phosphorylated
rice starch increases to 45.35% from 4.42% in the
native starch (Ashwar et al, 2018). Annealing rice
starch before cross-linking with STMP/STPP can
improve the RS level of cross-linked starch. Also, acid
hydrolysis of the cross-linked starch can also be
helpful for developing RS4 (Song et al, 2011). Besides,
previous acid hydrolysis of waxy rice starch before
cross-linking with STMP can also improve RS level
and produce RS4 nanoparticle (Jeong and Shin, 2018).
Acetylation can make 6- to 10-fold increases in the
RS4 content of rice starches, whereas AAC, syneresis,
pasting parameters and thermal properties are decreased,
and polyhedral granules with rough surfaces are presented
in the acetylated rice starch (Ashwar et al, 2017b).
Rice Science, Vol. 28, No. 1, 2021
Enzymatic modification
Some enzymatic methods have been used to make
debranched starch which in turn affects the content of
RS. In the past years, enzymatic treatment is preferred
to be used in production of modified starch due to its
environmentally friendly or biobased nature (Gurung
et al, 2013). Amylosucrase from Neisseria polysaccharea
is a bacterial glucosyltransferase that translocates
glucosyl units of sucrose to acceptor molecules, i.e.
starch molecules by introducing Į-1,4-glycosidic bond
to elongate the chains at the non-reducing ends.
Amylosucrase modification can increase the RS content
regardless of AAC, which likely results from structural
modification of amylopectin (Kim et al, 2013). Amylosucrase
enzymatically-modified RS can be applied as a texture
enhancer and a functional ingredient to develop
structurally strengthened rice starch gels (Doan et al,
2019). ȕ-amylase treatment can also increase the RS
content and reduce the hydrolysis of in vitro digestion
of extruded rice starch, especially for the extruded
starch with the maximum degree of gelatinization. The
increased RS might due to the increased density of
branch points (Į-1,6) in the amorphous regions after
enzyme treatment (Ye et al, 2018b). The hydrolysis of
Į-1,6-linkages is the rate-limiting step in the
hydrolysis of amylopectin and the changes in chain
length distributions appear to facilitate retrogradation
(Ao et al, 2007).
Debranched rice starch by pullulanase shows type V
crystalline rather than A-type, like native starch.
Molecular rearrangement occurs and degree of order
in starch granules increases, which may result in
enhanced solubility, water holding capacity and RS
content (Cao et al, 2020). The maximum RS content
reaches up to 28.61% at 55 ASPU/g for 12 h, which
matches the enhancement of AAC (Shi and Gao,
2011). Enzymatic molecular structure modification
followed by HMT is also shown to successfully lower
the digestibility of both normal and waxy rice starch
(Kim et al, 2016). The debranching starch followed by
octenyl succinylation (DBOS) is found to have higher
RS content and a lower extent of starch digestion
through forming emulsions that show resistance to
digestion with a degree of lipid release (Jain et al,
2020). DBOS starch is a potential ingredient for
developing functional products with good satiety.
Enzymatic approaches might be important for
developing healthier starch-based products with less
likelihood to induce diabetes and other chronic
diseases (Jain et al, 2020).
DING Yi, et al. Properties of RS and RS Improvement in Rice
Physical modification
HMT and other heating treatments
HMT is performed at temperatures above the
gelatinization temperatures with insufficient moisture
to gelatinize (below 35%). It has been reported as a
green technique to alter the molecular, crystalline and
granule structure of starch, and thus can regulate the
granule swelling, amylose leaching, gelatinization parameters,
viscosity and enzyme susceptibility (Hoover, 2010; da
Rosa and Dias, 2011; Jiranuntakul et al, 2011), and
has been conducted to improve both SDS and RS
levels of the starch (da Rosa and Dias, 2011).
HMT can simultaneously disorder and reassemble
the rice starch molecules to form densely packed
starch fractions, which is conducive to convert some
fractions of RDS into SDS and RS. HMT (18%, 60
min) promotes an increase in RS content and HMT
(16%, 60 min) causes an increase in SDS in the rice
flour (Silva et al, 2017). Particularly, HMT rice starch
has as high as 25.0% (SDS + RS) when the moisture
content is less than 30% (Wang et al, 2018). Sometimes,
HMT is combined with other approaches, e.g. parboiling
either before or after HMT. Dual autoclaving-
retrogradation treatment can also result in significant
increase in RS yield (Ashwar et al, 2016).
Compared to HMT, dry heat treatment (DHT) is a
simple and safe physical modification that does not
destroy starch granule structure but changes the
physicochemical properties, e.g. decreased RDS content
and predicted GI (Sun et al, 2014; Oh et al, 2018a),
and has received a great deal of attention recently. The
in vitro starch digestibility of DHT starches obtained
with a convection oven is negatively correlated with
the heating temperature (Oh et al, 2018a). DHT of
various combinations of hydrocolloids and starches
can also retard starch digestibility, especially for high
amylose starch mixed with xanthan gum (Oh et al,
2018b). DHT can be useful for enhancing the physical
and nutritional properties of high amylose rice starch
(Oh et al, 2018a).
Convective microwave heat-treated rice at 180 ºC
for 10 min also gives the maximum RS and shows
higher efficiency of converting digestible starch to RS
in rice than other dry heat treatment methods like
fluidized bed drying, pan roasting and tray drying
(Kanagaraj et al, 2019). Higher temperature in hot-air
drying can also slightly increase RS content in cooked
rice (Donlao et al, 2018). Additionally, other physical
methods such as irradiation are used to increase the
amount of RS. As the disruption of amorphous and
37
crystalline regions of the granule by gamma radiation
is dependent on the dose applied, a radiation dose of 1
kGy is ideal for increasing RS content with minimal
influences on other properties, while the highest dose
(5 kGy) decreases the AAC, PKV, water absorption
and volume expansion (Polesi et al, 2017).
Cooking methods
In general, rice grain is eaten as cooked rice to provide
energy to humans. To date, the highest RS content
(mainly RS3) of cooked rice reported is less than 5%
(Ritudomphol and Luangsakul, 2019). Therefore, it is
of great practical significance to give the concerns
over the effect of different cooking methods on the RS
content.
Conventional parboiling has been observed to lower
the digestibility of rice. Cooked rice with the majority
of cooking methods shows significant higher starch
digestibility than uncooked rice in many studies.
Steaming produces the highest SDS among all studied
varieties (Toutounji et al, 2019). Conventional cooking
or steaming method shows no positive effect for
increasing RS content (Chiu and Nutrition, 2013;
Reed et al, 2013). Stir-frying leads to the lowest starch
hydrolysis rate and the highest RS content. Parboiling
is suggested as an effective method for increasing the
RS content of rice (Reed et al, 2013; Tamura et al,
2016; Tian et al, 2018). Chiu and Stewart (2013)
reported that cooking rice with rice cooker, conventional
oven or pressure cooker has no difference in the RS
content of freshly cooked rice, except that Jasmine
rice prepared by pressure-cooker has lower RS content.
Li et al (2014) found that freshly prepared cooked rice
with a rice cooker is digested slower than that
prepared in the microwave. Pressure cooking for 10
min can increase the RS content in japonica paddy
rice from 1.95% to 7.95%. RS and SDS can be further
increased by almost 3.5 times by HMT after or before
parboiling (Cheng et al, 2019).
Cooking temperature also affects RS content.
Extrusion cooking is a continuous high-temperature
and short time process (Singh et al, 2007). With the
increase of cooking temperature, RDS is increased
significantly for all the tested rice (waxy, low- and
high-AAC), whereas SDS and RS are decreased at a
certain degree. But the cooking time has few effects
on starch digestibility (He et al, 2018). Ritudomphol
and Luangsakul (2019) also found that higher SDS
and RS and lower GI can be obtained by cooking at
lower temperature with a higher ratio of water or
shorter time, and cooking at 82 ºC with 1.9-fold water
38
volume is the optimal cooking condition. Using
high-amylose content (about 28%) brown rice via
soaking in 30–40 mg/mL citric acid solution, retort-
type cooking (121 ºC, 30 min, 15 psi), and drying can
produce RS-enriched cooked rice (Kim et al, 2020).
Besides different cooking methods and conditions,
the addition of other substances during the cooking
process also affects the RS content. A combination of
retrogradation and addition of lipids while cooking are
effective in decreasing digestibility (Sudhair et al, 2015).
Pullulan can also suppress the complete gelatinization
of starch and induce a significant increase of SDS and
RS contents, from 21.24% to 38.11% with a concomitant
decrease of the RDS content when 0.50% pullulan is
added to rice during cooking (Chen et al, 2017). The
addition of NSPs can also hamper the digestion of
starch (Maki et al, 2012; Foschia et al, 2013; Tamura
et al, 2014), and addition of ghee (clarified butter) to
rice during cooking results in lower GI and increased
RS content, while vegetable oils have no such
pronounced effects (Bhupinder et al, 2015). What’s
more, mixing rice with vegetables causes significant
reduction in GI, especially with fenugreek and
cauliflower (Kumar et al, 2020). Heating rice starch in
an acidic solution is shown to significantly increase its
RS content (Shin et al, 2007; Hung et al, 2016).
Genetic modification
With genome-wide association analysis of 235
varieties, Wx is the major gene associated with the GI
value of rice starch (Fitzgerald et al, 2011). Wx gene
controls the RDS and RS3, and isoamylase II (ISA2)
gene also controls the RDS (Kong et al, 2015). Besides
Wx gene, starch synthase IIa (SSIIa), debranching enzyme
isoamylase I (ISAI) and ADP-glucose pyrophosphorylase
small subunit (AGPase I), are also found to be
associated with RS3 content (Bao et al, 2017). High
RS3 content in rice is found to be the defect of starch
synthase IIIa (SSIIIa). SSIIIa is the major gene
responsible for RS formation in rice, but it is also
regulated by Wx gene (Zhou et al, 2016). Expression
of GBSSI shows positive correlation to RS2 content
(Kumar et al, 2018). pfkB, GBSS1, SSIIa, Į-amylase,
GBSSIIa and several genes related with amylopectin
biosynthesis and degradation are associated with RS2
(Parween et al, 2020).
RS manipulation has primarily being targeted
through starch manipulation (Li et al, 2019). Various
mutagenesis and gene modification approaches have
been explored to produce high RS varieties in rice. A
serial of rice mutants, like the RS111 mutant with a
Rice Science, Vol. 28, No. 1, 2021
high RS3 content (7.54%), have been successfully
developed by space mutagenesis (Yang et al, 2006;
Shu et al, 2007). Three high amylose rice varieties,
Goami 2, Goami 3 and Goami 4, have been developed
by N-methyl-N-nitrosourea (MNU) mutagenesis, which
also show high RS2 content ranging from 11.87% to
13.69% (Yoon et al, 2013). Through crossing between
Zhehui 7954 and high RS mutant R102, rice line
Yitang 1 with high RS3 content has been bred (Zhang
N et al, 2011).
Rice lines carrying mutations in SSIIIa and/or
BEIIb both display a high RS3 content (Tsuiki et al,
2016; Zhou et al, 2016). A mutation of BEIIb co-
segregates with RS2 content in rice (Yang et al, 2012;
Dhital et al, 2015). Targeted mutagenesis of BEIIb in
rice by CRISPR/Cas9 produces homozygous beIIb
mutants, which show significantly increased RS2 content
(Sun et al, 2017). Using RNA silencing-mediated down-
regulation of BEIIb can also produce modified rice
with high levels of RS2 (Wei et al, 2010; Butardo et al,
2011; Zhu et al, 2012). Steamed rice of BEIIb-deficient
mutant lines also contains considerably higher RS3 than
the other mutant lines, and new mutant lines by
introducing SSIIa and/or GBSSI from an indica rice
cultivar into a japonica rice-based BEIIb-deficient
mutant line has the same or slightly higher RS3
content than that of the beIIb parent line (Itoh et al,
2017). Freshly cooked grains of ami-BEIIb contain
10-fold higher RS3 content than that of its parent
Nipponbare, and also considerably higher than that of
IR36 ae mutant (Butardo et al, 2011). Simultaneous
repression of the SSIIa and SSIIIa through RNAi
results increase in amylopectin chains of DP 7–11, and
deceases in those of short (DP 5–6) and long (DP
12–23) (Zhang G Y et al, 2011), which might be the
main cause for high RS. However, high RS content
production also companies with high amylose content
in these mutants or gene modified rice. High amylose
content usually brings the deteriorating cooking and
eating quality and undesirable palatability, like low
toughness and low elasticity. The expression of other
starch synthase related genes is also altered in this
modified rice compared to their native parents, and the
high amylose accumulation in ssIIIa background is
possibly posttranslational regulated (Zhou et al, 2016).
Novel key regulators may contribute to RS formation
through influencing the flux between starch, sugar and
nitrogen metabolism (Parween et al, 2020). That gives
us some considerations that the expression of multiple
starch-synthesizing enzymes may need to be down-
regulated simultaneously to obtain very high RS rice
DING Yi, et al. Properties of RS and RS Improvement in Rice
grains (Butardo et al, 2011) with acceptable palatibility.
Operations on other genes participating in carbon and
nitrogen metabolisms might also be alternative.
Perspective
This review focused on the physiochemical properties
related to RS in rice, mainly those determining the
ability of starch to be digested and the approaches to
improve RS content. Due to the inconsistency of
starch origins, RS types and measurement procedures
among different studies, some results show contradiction.
Some studies found there are negative correlations
between RS with granule size, and AAC, while the
others found there are positive correlations between
RS and granule size or AAC. What’s more, NSPs and
lipids also show influences on both RS formation and
rice palatability. Some high RS/AAC rice has also
been obtained through regulating genes related to
amylose and amylopectin synthesis, e. g., BEIIb, Waxy.
Additionally, adjusting cooking methods are practical
and convenient to increase the content of RS3 in
cooked rice. The interactions between intrinsic and
extrinsic factors need further investigated. Enhancing
our understanding on physicochemical properties and
regulation of RS formation in rice will assist in developing
new rice varieties with improved digestibility. It is
prospective that high RS rice with acceptable
palatability will be bred in the future by genetic
cooperation on starch or NSPs or together, thereby
maximizing its unique functions in daily healthy life.
ACKNOWLEDGEMENTS
This work was supported by the Chinese Ministry of Agriculture
(Grant No. 2016ZX08001006), and Science Technology Department
of Zhejiang Province, China (Grant Nos. 2016C02052-6,
C02058-4, 2017C02019 and 2018C02055).
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