International Journal of Biological Macromolecules 186 (2021) 163–173

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

A comprehensive review of the factors influencing the formation of

retrograded starch
Qing Chang a, b, Baodong Zheng a, b, c, Yi Zhang a, b, c, *, Hongliang Zeng a, b, c, *
a
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China
b
Fujian Provincial Key Laboratory of Quality Science and Processing Technology in Special Starch, Fujian Agriculture and Forestry University, Fuzhou 350002, China
c
China-Ireland International Cooperation Centre for Food Material Science and Structure Design, Fujian Agriculture and Forestry University, Fuzhou 350002, China

A R T I C L E I N F O A B S T R A C T

Keywords: The retrogradation of starch is an inevitable change that occurs in starchy food during processing and storage, in
Retrograded starch which gelatinized starch rearranges into an ordered state. The chain length, proportion and structure of amylose
Formation and amylopectin vary in different types of starch granules, and the process is affected by the genes and growth
Internal factor
environment of plants. The internal factors play a significant role in the formation of retrograded starch, while
External factor
the external factors have a direct impact on its structural rearrangement, and the creation of suitable conditions
Mechanism
enables food components to affect the rearrangement of starch. Interestingly, water not only directly affects the
gelatinization and retrogradation of starch, but also serves as a bridge to deliver the influence of other com­
ponents that influence retrogradation. Moreover, there are three mechanisms responsible for forming retro­
graded starch: the migration of starch molecular chains in the starch-water mixed system, the redistribution of
water molecules, and the recrystallization kinetics of gelatinized starch. In this paper, the effects of internal
factors (amylose, amylopectin, food ingredients) and external factors (processing conditions) on the formation of
retrograded starch and the mechanism controlling these effects are reviewed.

1. Introduction texture, appearance, and organoleptic properties. Second, retrograded

Starch is the most important carbohydrate source in the human diet,
and it is the most abundant polysaccharide in plants [1]. The gelatini­
zation and retrogradation of starch during food production and storage
are important processes influencing the texture, taste, digestion, and
functional properties of starchy foods [2]. Different types of foods
require moderate retrogradation of starch (such as vermicelli, etc.),
while others need to suppress retrogradation (such as noodles, etc.) due
to their special texture requirements. Retrograded starch is a kind of
polymer, in which gelatinized starch molecules transform from a
disordered state to an ordered state [3]. During gelatinization, starch
granules swell due to the additional energy supplied and form a high-
energy disordered state [4]. After a cooling treatment, starch chains
interact with other chains or water molecules and rearrange into an
ordered and stable structure [5]. The growing interest in retrograded
starch (more than 662 Science Citation Index [SCI]-indexed articles are
available for the period of 2019–2020) can be attributed to two factors.
First, it displays excellent physicochemical properties, such as thermal
stability, and very low water holding properties, as well as improved
starch contains a part of resistant starch, which is poorly absorbed by the
small intestine but is completely or partially fermented in the colon,
which improves intestinal health [6].
Starch can be fully gelatinized to form a disordered structure at a
certain water content and temperature. When the gelatinized starch is
cooling down, the high-energy disordered amylose and amylopectin
chains gradually recombine into different ordered structures to form
crystals and reach an ordered and stable state. This process is called
starch retrogradation and it is complicated, especially in a complex food
system [7]. This process is affected by various factors and they are
shown in Fig. 1. These factors include the starch constituents (amylose
and amylopectin), other food ingredients (e.g., water, lipids, proteins,
carbohydrates, and salt ions), and processing conditions (temperature,
retrogradation time, and retrogradation pattern). Taking these factors
into account, there are three mechanisms responsible for the retrogra­
dation of starch: the migration of starch molecular chains in the starch-
water mixed system, the redistribution of water molecules, and the
recrystallization kinetics of gelatinized starch. However, none of these
formation mechanisms can fully and accurately describe the changes in

* Corresponding authors at: College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
E-mail addresses: zyifst@163.com (Y. Zhang), zhlfst@fafu.edu.cn (H. Zeng).

https://doi.org/10.1016/j.ijbiomac.2021.07.050
Received 15 March 2021; Received in revised form 6 July 2021; Accepted 7 July 2021
Available online 8 July 2021
0141-8130/© 2021 Published by Elsevier B.V.
Q. Chang et al.
the formation of retrograded starch during its processing in a complex
food system. Therefore, this paper reviews our current knowledge of the
internal and external factors influencing the formation of retrograded
starch and its formation mechanism. The benefit of the review is of
significance in predicting and controlling the quality of starchy foods.
2. The composition and structural characteristics of starch
granules
Starch granules exist in plant tissues. They consist of starch chains,
namely amylose or amylopectin chains. The ratio of amylose to
amylopectin varies according to the botanical origin of starch. Except for
high-amylose starch [8] and waxy starch [9], the majority of native
starch has an amylose content between 20% and 30% [10]. In amylose,
glucose residues are linked by α-D-(1–4) glycosidic bonds and form a
linear chain. In addition, Heineck, Cardoso, Giacomelli and da Silveira
[11] found that amylose chains possess a single helix formation. When
suspended in low-temperature aqueous solution, the mobility of
amylose in the amorphous region is enhanced, while their helical
structure is not damaged. Although it is rare in native starch, the double-
helix structure of amylose is common after retrogradation, and is the
basic composition of the long-range ordered structure, i.e., starch crystal
[1]. Amylose chains with a linear structure are better able to rearrange
and form an ordered structure. In addition, the formation of an ordered
structure is closely related to its resistance to digestion [12]. Therefore,
amylose plays an important role in the formation of retrograded starch,
especially the earlier stage of retrogradation. Amylopectin is one of the
largest molecules in the natural world, with an average molecular mass
of up to 1 × 107–1 × 109 Da due to its highly branched structure [13].
Similarly to amylose, amylopectin is constituted by glucose residues,
which are linked by α-D-(1–4) glycosidic bonds and α-D-(1–6) glycosidic
bonds, forming a non-linear structure with many branches. Despite
controversy over the fine structure of amylopectin, it is recognized that
amylopectin chains are basically formed by a host of external chain
segments and internal chain segments. External chains attach to the
internal chains and form a branched structure. The branched-structure
clusters further attach to long chains, and eventually form amylo­
pectin molecules. The normal state of amylopectin branch chains in
native starch granules is a double helix, and this makes a large contri­
bution to starch crystals [14]. Compared to the linear structure of
amylose, the branched structure limits the mobility of amylopectin after
gelatinization, and requires more time to reform an ordered structure
Fig. 1. Factors influencing the formation of retrograded starch.
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International Journal of Biological Macromolecules 186 (2021) 163–173
following retrogradation.
3. Gelatinization and retrogradation characteristics of starch
Gelatinization is the process that occurs before retrogradation, and
the conditions created by gelatinization are critical for retrogradation.
After starch is gelatinized, with the invasion of water molecules and the
collapse of starch granules, the arrangement of the chains changes from
ordered to disordered. This process is directly affected by the amount of
available water, temperature, and the stability of starch structure, as
well as other factors [15,16]. Gelatinization is a continuous process. The
degree of gelatinization increases as the temperature increases over a
certain range, which is related to the stability of certain starches. Once
the gelatinization initiation temperature is reached, the relatively un­
stable structure (the amorphous zone) in starch granules is destroyed
[17]. The amylopectin crystalline zone in native starch has a dense
cluster structure, and therefore its gelatinization temperature is higher
than that of the amorphous zone. Starch in low-temperature aqueous
solution (below the gelatinization temperature) absorbs water but only
swells slightly [18]. It is relatively easier for water to invade the
amorphous zone than the crystalline zone. The spacing between the
starch chains in the amorphous zone increases after the invasion of
water, while the starch crystals remain undamaged. When the starch-
water system is in a higher-temperature environment, the interaction
between short amylopectin side chains is destroyed, which enables
water molecules to plasticize starch chains. In this stage, starch chains
are more mobile, and the birefringence phenomenon of destruction of
native granule starch disappears. Meanwhile, the stretching of the starch
chain causes the clusters to lose their compactness, and the orderly
structure of native starch is destroyed [19].
Gelatinization is a process involving the structural destruction of
starch, while retrogradation is a process of structural reconstruction,
although it does not regenerate the structure of starch back to the con­
dition before gelatinization [15]. Starch gel is an unstable thermody­
namic system, and starch chains in a disordered state after gelatinization
tend to re-polymerize to form a more thermodynamic stable ordered
state [20]. Retrogradation is a continuous process, which can be divided
into short- and long-term retrogradation stages [21]. During retrogra­
dation, starch chains reassociate and form a double-helical structure
during the cooling stage. The double helices are then packed into crys­
tals [5]. As shown in Fig. 2, the double helix of amylose and amylopectin
are shown with arrows, respectively, and re-crystallization can be
Q. Chang et al.
Fig. 2. Steps of crystal growth in the recrystallization process of starch.
divided into three stages, i.e., nucleation (formation of crystal nuclei),
expansion (growth of crystal nuclei), and maturation (perfection of
crystals or further growth of crystals). The occurrence of these stages
depends largely on the temperature [22]. Sievert, Czuchajowska, and
Pomeranz [23] found that when the ambient temperature is close to the
glass transition temperature of starch, the formation rate of a crystal
nucleus is high but the propagation rate of crystals is low. At the crystal
melting temperature, the nucleation rate of starch chains is low, while
the propagation rate is great. The Avrami model is a mathematical
model for studying the crystallization kinetics of starch. The results of
rice starch determined by differential scanning calorimetry (DSC) and X-
ray diffraction (XRD) fit well with the Avrami model with a high
determination coefficient, while the Avrami exponent and rate constant
are significantly different [24]. This is because DSC and XRD evaluate
different structural features of starch, i.e., DSC can be used to determine
the melting enthalpy of starch recrystallized crystals whereas XRD re­
veals the starch crystalline architecture. Starch crystals can be divided
into A- and B-types, and a combination of A- and B-type crystals, namely
C-type [25]. As shown in Fig. 3, the B-type crystal unit is composed of six
double helices that form a hollow hexagon, and the central channel that
is surrounded by the six double helices is filled with 36 units of water in
each full turn. The A-type crystal is similar to the B-type crystal, but the
A-type crystal is polymerized by seven double helices and forms a “solid
hexagonal” structure that is tighter than the B-type crystal [26]. The
Fig. 3. Crystalline type structures in starch.
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International Journal of Biological Macromolecules 186 (2021) 163–173
space between these seven double helices can only be filled with eight
water molecules. The structures of A- and B-type crystal are considerable
similarities by XRD. To some extent, the fiber repeats of both structures
and the lateral distances between helices are virtually identical. The
packing of the helices also shows some similarity, because the A-struc­
ture may be imagined to be built up from the B-structure simply by
inserting another helix in the latter-in place of the water column. The
structure of the A-type crystal unit can also be depicted as a monoclinic
lattice structure. Starch with different types of crystalline structures has
different anti-digestive properties to starch digestive enzymes [27]. The
hexagonal cavity of an A-type crystal is packed with an extra double
helix, which makes it denser than a B-type crystal. Theoretically, the
compacter A-type crystal is more resistant to enzyme activity than the B-
type. However, it has been proven that the A-type crystal does not have a
significantly higher resistance to digestion than the B-type crystal [28].
Kim, Choi, and Moon [29] found no differences in the resistant starch
content in two retrograded starches, which had similar amounts of A-
and B-type crystals, respectively, while the slowly digestible starch
content in the A-type crystalline structure was constantly higher than in
the B-type.
4. Effect of amylose and amylopectin on retrograded starch
Amylose and amylopectin are the main constituents of starch
Q. Chang et al. International Journal of Biological Macromolecules 186 (2021) 163–173

granules. Starches isolated from different botanical sources have
different ratios of amylose to amylopectin, which has a large impact on
the starch properties. Amylose and amylopectin have different mobil­
ities, depending on their having a linear or multi-branched structure [5].
After being plasticized by water during gelatinization, amylose has a
better rearrangement ability than amylopectin, because its linear
structure requires relatively little space for rearrangement and reset­
tlement. It is therefore easier for amylose to form a double helix or
crystal than amylopectin. In contrast, amylopectin has a large number of
branches, and its chain distributions are disordered after gelatinization,
which makes it difficult to rearrange and recover its ordered structure.
Amylopectin can also retrograde, although this process requires a longer
time than for amylose [30]. Therefore, the short-term retrogradation of
starch (from the first few hours to several tens of hours) is generally
attributed to the re-arrangement of amylose, while the long-term
retrogradation is attributed to the re-arrangement of amylopectin
[21]. In addition, it is believed that the retrogradation of amylopectin
can be divided into two processes: the inter chain repolymerization of
the double helix structure and the packaging of the double helices of
starch chains.
As shown in Table 1, the ratio of amylose to amylopectin in certain
starches is a critical feature when investigating its retrogradation. Liu
et al. [31] found that the retrograded maize starches showed a typical B-
type XRD pattern, and it was easier to retrograde for starch with a high-
amylose maize starch that had 79.05% amylose content rather than a
normal maize starch with 25.43% amylose content. This may be related
to the ordered crystalline structure formed by amylose, which was
determined by XRD. In addition to the ratio of amylose to amylopectin,
the retrogradation of starch is also influenced by other properties of
starch chains, such as the degree of branching and chain length. Li et al.
[32] used the 1,4-α-glucan branching enzyme (GBE) to treat corn starch,
and found a decline in the amylose content and an increase in the
amylopectin content, which led to a decrease in retrogradation. The
main reason for this phenomenon was found to be the transglycosylation
activity of GBE. Vamadevan and Bertoft [30] also investigated the
impact of different structural types of amylopectin on retrogradation,
and found that the retrogradation of amylopectin was affected by the
external chain length and inter-block chain length. Long external chains
contributed to long, stable double helices, while long inter-block chains
influenced the flexibility of the amylopectin backbone, which was the
pre-requisite procedure for the formation of a double helix by external
chains. Wu et al. [33] used glucan 1,4 α-maltotriohydrolase and pul­
lulanase to hydrolyze and debranch gelatinized normal maize starch and
retrograded the hydrolysate at 4 ◦ C for 48 h to improve the formation of
resistant starch type 3. The results showed that the retrogradation of
starch was improved when there was an increase in the proportion of
medium-length chains with a DP of 30–130 by HPSEC-MALLS-RI chro­
matogram, and the resistant starch content was up to 40.8 ± 0.7%.
In addition to the above factors, starch molecular fine structures,
including the molecular size and chain-length distributions of amylose
and amylopectin are main factors influencing the retrogradation of
starch [34]. Retrograded properties are affected not only by the amylose
content, but also by the molecular size, and the molecular sizes of the
whole (branched) amylose and amylopectin components [35]. When
considering the starch molecular fine structure on retrogradation, mo­
lecular size and chain-length distributions often need to be considered at
the same time [34]. In general, for amylose with a long or short chain
length, due to steric hindrance or the degree of dispersion, the molecules
are not easy to polymerize to form resistant starch. In contrast, amylose
molecules with a short to medium chain length are easy to regenerate to
form resistant starch. Larger amylose molecules, a higher amount of
amylose short to medium chains, shorter amylose medium chains, and
longer amylopectin medium chains would result in a slower digestion
rate for both the fast- and slow-digestible starch fraction. The short-term
retrogradation of rice starches was positively correlated with the
amount of amylose short to medium chains, while it was negatively
correlated with the amylose molecular size [36]. Amylopectin is not
easy to retrograde due to its branched structure, but studies have shown
that the amylopectin of normal starches with an external A and B1 chain
population, DP of peak maximum at ≥15.5 glucose units, smaller
amylopectin molecules, and longer amylopectin internal chains will
increase the long-term amylopectin retrogradation rate [37].
5. Influence of food ingredients on the retrograded starch
5.1. Water content
Water plays a key role in the gelatinization and retrogradation of
starch, with starch gelatinization and retrogradation only occurring
when the water content reaches a certain level. During gelatinization,
water can contribute to the uncoiling of the double helix and promote
the movement of a single chain, while a low water content may lead to
the incomplete gelatinization of starch [38]. In native starch, many
starch chains are originally combined with each other by hydrogen
bonds to form a double helix, which limits the mobility of starch chains
[39]. For gelatinization, heating and water are indispensable. Heating
damages the hydrogen bonds between starch chains, while water, as a
plasticizer, can preferentially combine with the depolymerized starch
chain, and thus enhance the mobility of starch chains [40]. The presence
of water also makes the starch granule swell; thus, providing a larger
space for the movement of starch chains. The retrogradation of starch
occurs when the gel cools down, and the rearrangement of starch chains
is mainly driven by van der Waals forces. This process is also accom­
panied by a heat release and the reformation of hydrogen bonds between
starch chains [41]. When starch is gelatinized in water at different starch
to water (S/W) ratios, the space between the starch chains varies as the
water content changes. This means that the retrogradation situation will
be completely different. When the enthalpy of the starch-water system

Table 1
Effects of amylose and amylopectin on the retrograded starch.
Factor Starch source Amylose content Retrograded conditions Results References

Ratio of amylose to High-amylose maize starch (HAM);
amylopectin amylopectin maize starch (APM);
normal maize starch (NM).
Degree of branch Native corn starch, corn starch after
treatment of 1,4-α-glucan branching
enzyme.
Ratio of amylose to 17 starch samples with different
amylopectin amylose content and chain structure
Degree of Normal maize starch treated by
polymerization of glucan 1,4 a-maltotriohydrolase
branch chain (AMTS) for various duration.
70.34%, 0%, 22.49% S/W = 1:2 g/mL; T =
for HAM, APM, NM, 4 ◦ C; ST = 5, 10, 15 days.
respectively.
S/W = 6:100 g/mL; T =
4 ◦ C; ST = 1, 3, 5, 7, 14,
21 and 28 days.
S/W = 1:2 g/mL; T =
4 ◦ C; ST = 10 days.
26.4% for normal S/W = 4:50 g/mL; T =
maize starch 4 ◦ C; ST = 2 days.
RS content for 15 d: retrogradated HAM [31]
(17.35%) > retrogradated NM (15.76%) >
retrogradated APM (13.02%)
Treatment of 1,4-α-glucan branching [32]
enzyme increases the degree of branch and
retards retrogradation.
Retrogradation is influenced by both [30]
external chain of amylopectin and its
internal chain.
Retrogradation is increased by the change of [33]
branch chain lengths, especially medium-
length chains (DP 30–130). RS content (up
to 40.80%).

S/W, starch: water; T, temperature; ST, storage time.

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Q. Chang et al. International Journal of Biological Macromolecules 186 (2021) 163–173

reduces, starch chains are likely to re-tangle as the water interacts with
other free starch chains. Excessive amounts of water make it difficult for
the inter-chain hydroxyl groups to interact through hydrogen bonds,
with a large distance between starch chains. In contrast, if the gelati­
nization process occurs with a low water content, the amylose in the
amorphous state of the original starch cannot be sufficiently dissolved,
and part of the crystalline structure will be maintained [38]. This is
because the insufficient water content may inhibit the leaching of
amylose, and thereby curb the formation of the crystal nucleus, as well
as the expansion and maturation of the crystal. Longton and Legrys [42]
reported that the recrystallization of starch only occurs over a certain
range of moisture content (20–90%). The degree of recrystallization of
starch increases to some extent with an increase in the water content
(from 20 to 50%), and then decreases with a further increase in the
water content (from 50 to 90%). As the water content increases, the
space available for the movement of starch chains and their mobility
increases and when the moisture content is higher than a certain limit,
the excessive distance increases the difficulty of cross-polymerization

Table 2
Effects of food ingredients on the retrograded starch.
Factors Additive Starch source Retrograded conditions Results References

Water content; Starch nanoparticles (SNP) S/W = 1:1, 1:2, 1:5 g/mL; Largest retrogradation occurs when S/ [43]
environmental obtained from Proso millet T = 4 or 25 ◦ C; ST = 15, W = 1:2 in 25 ◦ C and 4 ◦ C
condition starch. 30, 60, 120, and 240 min.
Water content; type Waxy rice starch (TKW5, S/W = 1:4, 1:7, 1:10 g/ Water content more significantly [44]
of starch TCSW2); Low-amylose rice mL; T = 4 ◦ C; ST = 0, 1, 3, affects non-waxy starch than waxy
starch (TK9, TCS10); High- 5, and 7 days. starch. RS content (under 10%).
amylose rice starch (TCS17).
Water content Lotus seed starch S/W = 5:95, 10:90, 20/ S/W = 3:7 lead to largest degree of [7]
80, 30/70, 40:60, 50:50 retrogradation.
g/g; T = 4 ◦ C; ST = 14
days.
Type of lipid Glycerol, AA = 10%; three Rice flour S/W = 3:7 g/mL; T = (1) Glycerol and three emulsifiers [51]
emulsifiers (GMS, DATEM and 4 ◦ C; ST = 7 days. inhibited the retrogradation of rice
DMG), AA = 0.5 and 1.0%. starch; (2) retrogradation was inhibited
in a larger extent when the amount of
additive increase.
Adding amount of GMS; concentration of GMS solution Normal maize starch (NMS), Starch - GMS gels (8% w/ Addition of GMS decreases the [52]
lipid = 1, 2, and 3%. waxy maize starch (WMS), v); T = 7 ◦ C; ST = 10 retrogradation of NMS and WMS.
and high amylosemaize days.
starch (HAMS).
Proteins with Albumins, glutenin, globulins, Wheat starch S/W = 1:10 g/mL; T = Glutenin retards retrogradation of [55]
different chain gliadin, AA = 0, 0.5, 1.0, 1.5, and 4 ◦ C; ST = 1 day. wheat starch, while other 3 proteins
lengths 2.0 g per 10 g starch. promote it. Promoting effect varies
according to the type and additive
amount of protein.
Glutenin Wheat glutenin isolated from wheat Wheat starch S/W = 1:10 g/mL; T = Glutenin retards the retrogradation of [56]
flour; AA = 15% of starch weight. 4 ◦ C; ST = 24 h. wheat starch.
Polypeptides with Different proteins obtained from Maize starch S/W = 1:10 g/mL; T = All polypeptides can prohibit the [57]
different amino hydrating soy protein by microbial 4 ◦ C; ST = 1 day. retrogradation of maize starch, but the
acids proteases (acidic, alkaline and degree of inhibiting effect varies with
neutral proteases) the type of additive.
Proteins Protamex-hydrolyzed rice bran Rice starch. 5 g mixtures of rice and PRBPH-1 retards retrogradation of [58]
protein at 1 h (PRBPH-1); AA = 0%, PRBPH-1 with 10 g gelatinized rice starch
3%, 6%, 9%, 12% of starch. water; T = 4 ◦ C; ST = 1,
3, 5, 7, 14, 21 and 28
days.
Proteins Antilisterialgrass carp protein Rice starch Misture of starch and AGCPH reduces the degree of [59]
hydrolysate (AGCPH), starch AGCPH: water =1:2 g/g; crystallinity and retrogradation of rice
weight: additive weight = 100:0, T = 4 ◦ C; ST = 1, 7and 14 starch
97:3, 94:6, 91:9, and 88:12 g/g. days.
Types of glucose, fructose and maltose, AA = Pueraria lobata starch S/W = 1:4 g/mL; T = At 4 ◦ C, glucose, fructose and maltose [69]
carbohydrate; 5% − 22 ◦ C, 4 ◦ C and 28 ◦ C; inhibit the retrogradation of starch; at
temperature ST = 1, 2, 4, 7, 14, 21 − 22 ◦ C, maltose still retard the
days. retrogradation, while glucose, fructose
promote the retrogradation.
Types of Sodium alginate, xanthan gum, guar Wheat flour 140.0 g water with 200 g (1) Addition of guar gum and sodium [70]
carbohydrate gum, native corn starch, acetylated and 18 g sugar, 38 g other alginate retard retrogradation; (2)
starch, oxidized corn starch, addictive; T = 4 ◦ C; ST = partial replacement of wheat flour with
hydroxypropylated corn starch. 10 days. modified starch can retard the
retrogradation of wheat starch.
Degree of Inulin with different length: FS (DP Wheat starch. S/W = 1:10 g/mL; T = Inulin inhibits amylose retrogradation [67]
polymerization ≤ 10), FI (DP of 2–60) and FXL (DP − 22 ◦ C, 4 ◦ C and 25 ◦ C; and accelerates amylopectin
(DP) of ≥ 23); AA = 2.5% to 20% of ST = 7 days. retrogradation.
carbohydrate suspension.
Amount of Pullulan; AA = 0%, 1.4%, 2%, 6%, Normal rice starch Mixture of starch and Pullulan inhibits the retrogradation of [21]
carbohydrate and 10% of starch weight. pullulan: water = 1:2 g/ amylose and amylopectin.
mL; T = 4 ◦ C; ST = 1, 3, 5,
7, 14, 21, and 28 days.
Amount of Flaxseed gum, AA = 0, 0.1, 0.2, 0.3, Maize starch S/W = 1:10 g/mL; T = Flaxseed gum inhibits the [71]
carbohydrate 0.4% of suspension. − 20, 4 and 20 ◦ C; ST = retrogradation.
12 days.

S/W, starch: water; T, temperature; ST, storage time; AA, additive amount; GMS, glycerol monostearate; DATEM, diacetyl tartaric esters of mono- and diglycerides;
DMG, distilled monoglycerides.

167
Q. Chang et al.
between starch chains due to excessive dilution, eventually leading to a
decrease in crystallinity. As shown in Table 2, different studies have
shown the maximum recrystallization concentration of different
starches, which may be related to factors, such as starch type, environ­
mental conditions, and the different stages of retrogradation. In an
experimental study by Gong Li, Xiong, and Sun. [43], short-chain
amylose was debranched from amylopectin-rich waxy proso millet
starch, and then used to investigate the effect of the amylose/water ratio
on the recrystallization of amylose. An amylose/water ratio of 1:2 (w/w)
remarkably increased the crystallinity of short chain amylose compared
with the ratios of 1:1 and 1:5. Hsu, Lu, Chang, and Chiang [44] found
that the amount of water added significantly influenced the retrogra­
dation of cooked rice flour, which led to a difference in the digestibility
of retrograded starch. Chen et al. [7] found that the moisture content
had no effect on the crystal type of lotus seed retrograded starch and all
samples showed B-type crystal by XRD and solid-state 13C cross-
polarization and magic-angle spinning nuclear magnetic resonance
(13C CP/MAS NMR). Water contents of 70% and 80% were the best
condition for the recrystallization of lotus seed starch as determined by
XRD and fourier transform infrared (FT-IR). It was related to the water
distribution by low-field 1H NMR, in which bound water was related to
the hydrated starch chains, while channel water in the B-type crystal
represented the micro-crystalline structure. In addition, the effects of
fat, protein, sugar, and other factors on starch gelatinization and
retrogradation have a large influence on the structure of the retrograded
starch, such as the starch complex, while in some cases their affect is
manifested through the distribution and availability of water.
5.2. Lipids
Lipid can co-exist with starch to form a lipid-starch complex [45].
Starch is isolated from plant tissue, which always contains lipids. In
addition, lipids are commonly added to processed starchy food. Lipids
can form starch-lipid complexes with single starch chains (mainly
amylose) through a hydrophobic effect. At the high homogenization
pressure (70–100 MPa), the XRD and DSC results revealed that a V-type
crystalline polymorph was formed between lotus seed starch and glyc­
erin monostearate [46]. In starch paste, most amylose exists in a single-
helical form before the retrogradation process begins. When they co-
exist with starch, lipids may enter the hydrophobic cavity of single-
helical starch chains due to their hydrophobicity by various treatment
methods, such as heat-moisture, ultrasound, hydrostatic pressure, and
high pressure homogenization [47]. Lipids, such as fatty acids, possess
both hydrophilic and hydrophobic groups, and the latter group can drive
the lipids to move close to the internal hydrophobic cavities of single-
helical starch chains. The length of lipid chains has a large impact on
the stability of lipid-starch complexes. Long-chain lipids have lower
hydrophilia than short-chain lipids, which creates a strong force that
prevents the lipid-starch complexes being damaged under the influence
of external factors, such as heating. Previous studies have also found that
lipids may form complexes with amylopectin as well as amylose [48]. It
has been assumed that the outer chains of amylopectin can interact with
lipids in the same way as the interaction of lipids and amylose, and thus
curb the process of retrogradation. With respect to digestibility, the
lipid-starch complex is a kind of resistant starch, which can be defined as
resistant starch type 5 [49].
In terms of the influence of lipids on the retrogradation of starch gel,
most studies have shown that lipids curb the retrogradation of starch.
The extent of the negative effects of lipids on the retrogradation of starch
is influenced by the type of starch, properties of the lipids, and the
environmental conditions under which retrogradation occurs [50]. As
shown in Table 2, Prakaywatchara, Wattanapairoj, and Thir­
athumthavorn [51] found that glycerol combined with any other
emulsifier (e.g., glycerol monostearate (GMS), diacetyl tartaric esters of
mono- and diglycerides (DATEM), or distilled monoglycerides (DMG))
could curb the retrogradation of rice flour. The inhibiting effect varied
168
International Journal of Biological Macromolecules 186 (2021) 163–173
according to the type and level of additive. On the other hand, Garcia
and Landi Franco [52] found that adding GMS curbed the retrogradation
of normal maize starch and waxy maize starch, but had no significant
effect on high amylose maize starch (HAMS). The inhibitory effect of
GMS on the retrogradation of different starches can only be seen with
certain amounts of additive. According to these phenomena, the inhib­
itory effect of lipids on the retrogradation of starch may occur through
three possible mechanisms.
(1) Lipids could affect the process of gelatinization and indirectly
curb retrogradation. Partial gelatinization is disadvantageous for
retrogradation [53]. Starch chains initially form lipid-starch
complexes with the endogenous lipids in native starch. Water
penetration into the granules may be restrained, while starch
granule swelling and solubilization in cold water is hampered
[54]. The swelling of starch granules becomes more difficult, and
the mobility of starch chains is reduced.
(2) During retrogradation, free single chains can preferentially form
single helical complexes with lipids [49]. This inhibits the release
of amylose and the formation of inter-chain hydrogen bonds be­
tween amylose chains, and thus restricts the retrogradation
process.
(3) The formation of a crystal nucleus by amylose is a key process
that may affect the further growth of the crystal. The formation of
a lipid-amylose complex reduces the amount of available
amylose, which contributes to the formation of the crystal nu­
cleus, and restricts the further retrogradation of starch.
5.3. Proteins
Protein is a common component in most starchy foods, and can affect
the starch properties during processing and storage. Currently, protein is
commonly added to food to prevent the retrogradation of starch [55].
However, not all proteins can curb the retrogradation of starch, because
whether the effect of a protein on starch is positive or negative is largely
dependent on the properties of the protein, as well as various other
factors. Proteins with different properties can have different influences
on the retrogradation of starch. A couple of examples are listed in
Table 2. Lian, Guo, Wang, Li, and Zhu [55] isolated pure starch and
proteins with different chain lengths (albumin, globulin, gliadin, and
gluten) from flour, and mixed a single protein with starch at different
ratios, and then gelatinized them. In the subsequent retrogradation, the
addition of albumin, globulin, and gliadin improved the retrogradation
of starch, while gluten curbed retrogradation due to its long chain,
which formed many hydrogen bonds with water, and therefore
decreased the amount of available water. The addition of gluten created
an alkaline environment, which was disadvantageous for the retrogra­
dation of starch. In contrast, there were several reasons why the other
three proteins enhanced the retrogradation of starch. First, the addition
of specific proteins may result in the amount of available water reaching
a point that is suitable for the retrogradation of starch. The methyl exists
in albumins, globulins and gliadins, but not in glutenins. Glutenin also
retards the retrogradation of wheat starch [56]. Some of the water in
gelatinized starch moves from starch to protein due to the competition
for water between methyl groups with a high hydration capacity and
starch. Second, the addition of specific proteins changes the electro­
negativity of starch. When the starch links with globulins, the electro­
negativity of starch will intensify, which might substantially increase the
nucleation rate of wheat starch. Lian Zhu, Wen, Li, and Zhao [57] hy­
drolyzed soy protein to create polypeptides that were composed of
different amino acids, and added them to the maize starch in different
proportions. The results showed that most polypeptides had a negative
effect on the retrogradation of starch, while the others had only a minor
effect on the retrogradation of starch. Protamex-hydrolyzed rice bran
protein [58] and antilisterial grass carp protein hydrolysate [59] also
reduce the retrogradation of rice starch. Most other studies have also
Q. Chang et al.
shown that the addition of protein curbs the retrogradation of starch.
The mode by which a protein co-exists with starch is a contributing
factor that affects the retrogradation of starch [60]. In the first mode,
starch and protein do not mix with each other and both form an inde­
pendent enriched phase in solution. The second mode occurs when the
interactions between the two biopolymers are favored. For example,
when starch and protein carry the opposite charge, due to the electro­
static interaction or Van der Waals' forces, they are likely to interact with
each other and form co-polymers. In the third mode, instead of forming
independent enriched phases or interacting with each other, starch and
protein constitute a single-phase mixture. This process is rare, only
occurring when the mixing process is exothermic, but several examples
have been reported. Salehifar, Seyyedain Ardebili, and Azizi [61]
studied the influence of protein on the retrogradation of bread and found
that the enthalpy of recrystallization decreased with an increase in the
amount of protein, which resulted in a decline in the degree of retro­
gradation. Through a water absorption experiment, it was found that
flour with protein can absorb more water than the same amount of
starch; thus, it was speculated that the addition of protein can absorb
more water when a sufficient supply was available, which may dilute the
absolute concentration of starch, reducing the possibility of inter-chain
entangling of starch chains, and thus restraining the retrogradation of
starch. Niu, Han, Cao, Liu, and Kong [62] studied the effect of adding
porcine plasma protein hydrolysates on the long-term retrogradation of
corn starch and found that the addition of porcine plasma protein hy­
drolysates effectively slowed the retrogradation of corn starch. With an
increase in the amount of porcine plasma protein hydrolysates, the
gelatinization enthalpy of samples decreased. This may be because the
porcine plasma protein hydrolysates were prone to interact with water
molecules and could compete with starch to capture the available water.
Without sufficient water, there may be an incomplete gelatinization of
starch and a lack of space for the rearrangement of starch chains in the
retrogradation process. Kong, Niu, Sun, Han, & Liu [63] found that when
porcine plasma protein hydrolysates were mixed with starch, the protein
could combine with starch granules, and thus prevent water from
entering. This raised the temperature required for gelatinization. How­
ever, the increase in gelatinization enthalpy was largely due to the
destruction of the amino acid and water combination. In the process of
gelatinization, the available water was shared between the amino acid
and starch, resulting in less water being available for the gelatinization
of starch. In addition, according to Tang, Yan, Gu, Yayuan, and Cai [64],
the recrystallization of amylopectin required the participation of water,
and when less water was available the recrystallization of starch would
be limited during retrogradation. Therefore, it was evident that protein
can affect either the gelatinization or retrogradation of starch by
changing the availability and distribution of water. In addition, the
formation of a physical barrier is another way in which the starch
retrogradation is affected by protein. This is typically the case for whole
foods and baked products, where the storage proteins can form a matrix
filled with the starch granules [65]. Protein bodies closely surround
starch granules from adlay seed and act as a physical barrier against
starch gelatinization. Removal of protein leads to increase in gelatini­
zation enthalpy and breakdown viscosity [66].
5.4. Small molecular sugar and non-starch polysaccharides
The effect of carbohydrates on the retrogradation of starch can be
positive [67], negative [21], or even negligible [68]. As shown in
Table 2, the effects of carbohydrates on the retrogradation of starch vary
according to the properties of the different types of carbohydrate and
starch, as well as the conditions under which retrogradation occurs. Guo
and Du [69] found that adding glucose, fructose, and maltose could
affect the retrogradation of starch, but the extent of the influence
differed according to the temperature at which retrogradation was
conducted. At 4 ◦ C, all three types of sugar curbed the retrogradation of
starch to a different extent. The degree of influence followed the order of
169
International Journal of Biological Macromolecules 186 (2021) 163–173
fructose > maltose > glucose. At − 22 ◦ C, the retrogradation of starch
was promoted with the addition of glucose or fructose. However, Kang,
Reddy, Park, Choi, and Lim [70] also found that a mixture of different
starches can affect their retrogradation. The degree of polymerization
(DP) of a carbohydrate is also an important factor that determines its
effect on the retrogradation of starch. Luo et al. [67] studied the influ­
ence of inulins (which have different DPs) on the retrogradation of
wheat starch. The results showed that adding synanthrin could raise the
gelatinization temperature of starch, and the retrogradation of starch
was totally curbed. A low-concentration of synanthrin can inhibit the
short-term retrogradation of amylose, while the retrogradation of
amylopectin is improved by synanthrins, especially synanthrins with a
low DP. In addition, the amount of carbohydrate added also directly
affects its influence on the retrogradation. Chen, Ren, Zhang, Tong, and
Rashed [21] and Feng, Yang, Sun, Xu, and Zhou [71] showed that the
greater the amount of foreign carbohydrate added, the more significant
the inhibitory effect of the carbohydrate on the retrogradation.
Several hypotheses have been proposed to explain how sugars affect
the retrogradation of starch. On one hand, carbohydrate can prevent the
gelatinization of starch. In the starch-sugar-water system, sugar can
bond with starch molecules and form a starch-sugar-starch structure,
which stabilizes the structure of starch and prevents it from being
damaged when gelatinization occurs. Taking carrageenan as an
example, the presence of carrageenan may reduce the available water
for the swelling of starch, and the swelling of starch granules in carra­
geenan dispersions has been shown to decrease with an increasing
concentration of carrageenan [72]. This shows that gelatinization is a
process where starch and carbohydrates compete for water. On the other
hand, the retrogradation is related to its syneresis. Syneresis is the water
separated from a starch gel because of the gel network shrinking due to
the reconstituting of leached starch molecules. Non-starch poly­
saccharides can promote or inhibit the syneresis of starch retrograda­
tion. Sage seed gum significantly decreased the syneresis of native wheat
starch during storage at 4 ◦ C [73], while xanthan gum promoted the
syneresis of waxy potato starch [74]. Finally, both sugar and water can
interact with starch, plasticize the starch chains, and increase their
mobility. Sugar may replace the water molecules that interact with
starch chains, but the plasticizing effect of some sugars is less than that
of water. These plasticizers reduce retrogradation compared with equal
amounts of other plasticizers, such as water [75].
5.5. Salt ions
Salt ions have a significant effect on the gelatinization, retrograda­
tion, thermal properties, and physicochemical properties of starch.
Oosten [76] mentioned that starch was a weakly acidic substance that
could release hydrogen ions under certain conditions. As shown in
Fig. 4, starch can release hydrogen ions into the solvent until an equi­
librium potential is attained. The dynamic equilibrium eventually causes
the starch to be negatively charged, while the aqueous solution is
positively charged and weakly acidic due to the increased hydrogen ion
concentration. For example, when salt (NaCl) is dissolved in solution, as
a strong electrolyte it ionizes into cations and anions. The negatively
charged starch then tends to attract and bind with the cation. This
process changes the repulsive force of the inner layer of the starch and
changes the pH of the solvent, which may interfere with the properties of
the starch. The degree of influence on the salt ions and starch varies with
the type of ion and starch, their concentration, and environmental
conditions. For example, Guo and Du [69] found that the effect of NaCl
addition on the retrogradation of starch had different effects depending
on the retrogradation temperature. At 4 ◦ C, Na+ replaced hydrogen ions
and interacted with starch chains and created a Donnan potential, which
enhanced the mutual repulsion between starch chains, thereby inhibit­
ing the re-association of starch [77]. At − 22 ◦ C, NaCl was ionized into
Na+ and Cl− in the maximally freeze-concentrated solution, resulting in
the water molecules being unable to maintain a tetrahedral structure,
Q. Chang et al. International Journal of Biological Macromolecules 186 (2021) 163–173

Fig. 4. Conformations of starch chains in water and NaCl solutions.

and thereby reducing the interaction of water and starch [78]. Under
these conditions, it is easy to remove the effect of the plasticization of
water from the starch chain, and thus form hydrogen bonds; hence, the
retrogradation of starch will be promoted.
The effects of salt ions on the retrogradation of starch can be
explained using the Hofmeister series [79], which classifies ions into
Chaotropes and Kosmotropes based on their ability to change the
hydrogen bonding network of water [80]. Chaotropes are defined as
“water structure breakers”, and can destabilize the structure of macro­
molecules and cause a salting-in effect. Kosmotropes are defined as
“water structure makers”, and are strongly hydrated, can stabilize
macromolecules, and create a salting-out effect [81]. Although the
Hofmeister series is mainly used to explain the effect of salts on protein
denaturation, it has a similar regularity when it is applied to explain the
effect of ions on the behavior of starch [82]. Wang et al. [79] reported
that Kosmotropes can promote hydrogen bond links among starch
molecules, and thus promote the association of starch chains, while
Chaotropes have the opposite effect on the behavior of starch chains. Fu
and BeMiller [83] found that Chaotropic ions can reduce the
retrogradation of starch, while Kosmotropic ions can promote the
retrogradation of starch. The promoting or retarding effect of ions on
retrogradation can influence both native maize starch and hydrox­
ypropylated maize starch.
6. Retrogradation conditions for retrograded starch
Retrogradation of starch can last for a long time when starchy food is
exposed to different environments, especially when the starchy foods are
processed, stored, and transferred. It is acknowledged that the retro­
gradation of starch is influenced by several environmental factors, such
as retrogradation temperature, retrogradation time, retrogradation
pattern, and the changing rate of temperature. Recent studies have taken
different factors into account to investigate the effect of environmental
conditions on the retrogradation of starch, with some typical examples
listed in Table 3. With respect to the effect of retrogradation time on the
retrogradation of starch, it is unanimously believed that retrogradation
can be divided into two stages, i.e., short- and long-term retrogradation
[21]. Short-term retrogradation can be attributed to amylose, while

Table 3
Influences of retrogradation conditions on the retrograded starch.
Factors Additive Starch source Retrograded conditions Results References

Temperature, Waxy potato starch S/W = 1:2 g/mL; T = 4 C, 25 C, and cycles

◦ ◦
Temperature-cycled of 4/25 C is best for
◦
[85]
storage time. of 4/25 ◦ C (24 h each); ST = 1, 3, 5, 7 days. retrogradation, follow by 25 ◦ C, and then 4 ◦ C.
RS content (25.66 to 37.33%).
Temperature. Liquid eggs; Wheat flour. Wheat flour account for 29.5% of cake total Retrogradation is more significant at 20 ◦ C [87]
anhydrous milk weight; T = 4 ◦ C, 20 ◦ C; ST = 17 days; than at 4 ◦ C.
fat; sugar.
Temperature; Waxy wheat starch S/W = 1:1 g/mL; T = 4 ◦ C, 25 ◦ C, and cycles (1) Continuous- treatment is better than [88]
retrogradation of 4/25 ◦ C (time interval of 36 h); ST = 6 intermittent- treatment for starch to
pattern. days for continuous retrogradation twice 3 retrogradate. (2) retrogradation at 25 ◦ C was
days for intermittent retrogradation. greatest, followed by 4/25 ◦ C, and then 4 ◦ C.
RS content (24.03 to 54.12%).
Storage time; Rice 800 g rice: 1040 g water; 4 ◦ C for 0, 1, 3, 7, Rapid cooling rate can retard the [89]
cooling rate, 11 and 14 days, and 18 ◦ C for 0, 1, 2, 3,4, 5, retrogradation of starch.
temperature 6 and 7 months. Cooling rate = 0.09, 0.26,
0.33, 0.53, 1.45 ◦ C/min.
Storage time; Mixing waxy starch and S/W = 3:7; T = 4 ± 1 ◦ C; ST = 24, 48, and (1) Relative crystallinity increases with the [90]
cooling rate non-waxy starch at 72 h; cooling rates = 1, 3, 5, and 9 ◦ C/min. increase of amylose content and the increase of
weight ratios of 100:0, storage time. (2) Cooling rate affects the
75:25, 50:50, 25:75, and retrogradation but not showed regularity.
0:100.

S/W, starch: water; T, temperature; ST, storage time.

170
Q. Chang et al.
long-term retrogradation is mainly conducted by amylopectin [5]. In
general, the degree of retrogradation increases over time, but sometimes
the degree of retrogradation remains unchanged.
Temperature is another factor that influences the retrogradation of
starch. The degree and rate of retrogradation are different at different
temperatures. It has been found that 4 ◦ C can promote the formation of a
crystalline nucleus, but the temperature is not suitable for the growth of
the crystal, while a temperature around room temperature (25 ◦ C) is
disadvantageous to the formation of a crystalline nucleus but is good for
the growth of starch. A temperature cycle promotes the formation of
slowly digested starch because more imperfect crystals are formed under
such conditions [71,84]. Xie, Hu, Jin, Xu, and Chen [85] found that
retrogradation at 4/25 ◦ C intervals retrogradation could boost the for­
mation of slowly digested starch, because starch granules at these
temperatures were prone to form imperfect crystals. However, it was
suggested by Zhou, Baik, Wang, and Lim [86] that the imperfect crystals
that form at 4 ◦ C are prone to melt at 30 ◦ C, and thus lower the total
enthalpy of melting. This can be regarded as a parameter influencing the
degree of retrogradation. At the beginning of the retrogradation process,
the crystalline nucleus is small and unstable. Crystals at this stage are
vulnerable to melting. After retrogradation continues, the crystal grows
to a larger size, and its internal structure is rearranged toward a more
ordered pattern. Additionally, Hesso et al. [87] and Hu et al. [88] found
that compared with retrogradation at 4 ◦ C, either 20 or 25 ◦ C are better
temperatures. The reason that influenced the high level of RS was
mainly the crystallite stability according to the condition of
temperature.
The rate of temperature change also can affect the retrogradation of
starch. Yu, Ma, and Sun [89] studied the influence of the cooling rate on
the retrogradation of starch as well as the final texture of the retrograded
starch. It was found that the degree of retrogradation of starch decreased
if the cooling rate was too quick. This was because a quick cooling rate
restricted the time available for the starch to rearrange. Moreover, after
being frozen at a temperature below 0 ◦ C, it is difficult for starch chains
to undergo further rearrangement, even when given sufficient time. In
terms of the effect of the cooling rate on the retrogradation of starch,
similar results were also obtained by Jiamjariyatam, Kongpensook, and
Pradipasena [90].
7. Conclusion
Starch is one of the most common ingredients in food, and its
retrogradation can significantly influence the quality of starchy food.
However, despite the natural properties of starch, the retrogradation of
starch is also inevitably affected by other components in food (e.g.,
water, lipids, proteins, carbohydrates, and salt ions) and retrogradation
conditions. A diverse range of retrogradation results have been reported
from various studies, due to the complexities of the starch gel system and
environmental conditions. The internal factors (amylose, amylopectin,
other food ingredients) play a significant role in the formation of the
ordered structure of retrograded starch, while the external factors
(processing conditions) have a direct impact on its structural rear­
rangement, and the creation of suitable conditions enables food com­
ponents to affect the rearrangement of starch. In general, it is reasonable
to conclude that water plays a critical role in the retrogradation of
starch, because it can not only directly affect the gelatinization and
retrogradation of starch, but also serves as a bridge to deliver the in­
fluence of other components that influence retrogradation. This paper
provides a basic reference for future studies of starch retrogradation.
CRediT authorship contribution statement
Qing Chang: Conceptualization, Formal analysis, Roles/Writing -
original draft. Baodong Zheng: Project administration, Supervision. Yi
Zhang: Project administration, Resources, Software, Supervision.
Hongliang Zeng: Project administration, Visualization; Writing - review
171
International Journal of Biological Macromolecules 186 (2021) 163–173
& editing, Supervision.
Declaration of competing interest
Authors declare there is no conflict of interest.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (grant numbers 31701552 and 31972076), the Natural Science
Foundation for Distinguished Young Scholars of Fujian Province (grant
number 2019J06012), the Program for Leading Talent in Fujian Pro­
vincial University (grant number 660160190) and Program for New
Century Excellent Talents in Fujian Province University (grant number
KLA18058A).
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