International Journal of Food Science and Technology 2022 1

Original article
Annealing processing technique divergently affects starch
crystallinity characteristic related to resistant starch content: a
literature review and meta-analysis

Maria Putri Anugerah,1* Didah Nur Faridah,1,2 Frendy Ahmad Afandi,3 Dase Hunaefi1 &
Anuraga Jayanegara4
1 Departement of Food Science and Technology Faculty of Agricultural Technology, IPB University, Bogor 16880, Indonesia
2 Department of Food Technology, Faculty of Agricultural Technology, Southeast Asian Food and Agricultural Science Technology
(SEAFAST) Center, Bogor Agricultural University, Bogor 16880, Indonesia
3 Deputy Ministry for Food and Agribusiness, Coordinating Ministry for Economic Affairs Republic of Indonesia, Jakarta 10710, Indonesia
4 Department of Nutrition and Feed Technology, Faculty of Animal Science, IPB University, Bogor 16680, Indonesia

(Received 5 November 2021; Accepted in revised form 28 January 2022)

Summary Annealing is one of starch physical modification techniques that can increase resistant starch (RS) con-
tent. However, the effect of annealing on RS varies based on botanical sources. Starch crystal type and
relative crystallinity are two important characteristics of starch. This study aimed to compare the influ-
ence of annealing on starch crystallinity and different starch crystal type (A, B and C) and investigate its
effect on RS content by literature meta-analysis study. The literature review was carried out using 22
research articles and 101 data points. The result showed relative crystallinity increase from the highest
changes to the lowest, respectively; C-type crystal (18.98%) followed by B (13.60%) and A-type crystal
(8.17%). Meta-analysis study results showed a significant increase of RS content on starches with A-type
crystal (SMD = 6.46; 95% CI: 3.22–9.70; P < 0.01) and a positive relationship between the degree of
crystallinity and RS content after annealing modification.
Keywords Allomorphs, annealing, crystallinity, meta-analysis, starch modification.

amylopectin (Alcázar-Alay & Meireles, 2015). Varia-

Introduction
Starch is found abundantly as a stored carbohydrate
source in various parts of plants such as roots, tubers,
trunks, fruits and seeds. Starch granules characteristics
are diverse including in shape and size based on the
plant origins. Zea mays has compound, polyhedral, 5–
15 µm granules, while Solanum tuberosum has simple,
ovoidal, 2–15 µm starch granules (Gismondi et al.,
2019). Starch granule is basically comprised of two
glucose polymer structures called amylose and amylo-
pectin. Amylose is a long chain structure consisting of
D-glucose monomers bound together by α-(1,4) glyco-
sidic bonds while amylopectin is a highly branched
structure with additional α-(1,6) glycosidic bonds that
connect the glucose residues (Vamadevan et al., 2013).
The ratio of amylose to amylopectin is varied depend-
ing on the botanical origins. Potatoes were reported to
contain 29.3% amylose while waxy sorghum was
observed to have 3.5:96.5 ratio of amylose to
*Correspondent: E-mail: didah_nf@apps.ipb.ac.id
tion of amylose ratio in one type of plant is also com-
mon for example on normal wheat (26.2% amylose
content) and waxy wheat (6.4% amylose content) in
Su et al. (2020); high amylose rice (32%), medium
amylose rice (23%) and low amylose rice (7%) in
(Dias et al., 2010); and high amylose maize (85%) and
normal maize (23%) in Wang et al. (2014). Besides the
starch granules types and amylose to amylopectin
ratio, starches are also different in terms of crystal
types (Sarko & Wu, 1978; Jane, 2006; Pérez & Bertoft,
2010; Bertoft, 2017).
These starch differences influence starch properties
when heated or processed. Starch characteristics and
physicochemical properties play important role in
starch application in the food industry. One important
application of starch depends on its resistant starch
(RS) content (Fuentes-Zaragoza et al., 2010). RS is
used in the food industry for various purposes such as
to increase fiber content in breakfast cereal, noodles
or pasta, to provide prebiotic, and to improve bakery
products quality (Brown, 2004; Ashwar et al., 2016).

doi:10.1111/ijfs.15628
© 2022 Institute of Food, Science and Technology (IFSTTF).
2 Annealing: crystallinity and resistant starch M. P. Anugerah et al.
RS is the type of starch that is not hydrolysed by
digestive enzymes thus is not absorbed in the small
intestine (Leszczynski, 2004). Various efforts have
been done to increase RS content in food such as
adding it as ingredients or modifying starch. Starch
modification by annealing is one of the techniques
widely studied to increase RS content. Annealing is
one kind of starch physical modification technique
that applies heat to starch suspense in water. Moisture
content for annealing was divided into two types:
intermediate (40–55%) and excess water (>60%) (Tes-
ter & Debon, 2000). Zavareze & Dias (2011) stated
that the process temperature should be above the glass
transition and beneath the gelatinisation temperature.
The combination of excess water and heat changes the
starch structure into a rubbery state that is more
mobile which led to the reorganisation of starch amy-
lopectin and amylose into a more compact structure.
This compact structure forms sterical hindrance
against hydrolysis enzyme thus increasing RS content
(Tester & Debon, 2000). The technique increases the
level of RS through physical modification without the
addition of any chemical reagent thus deemed as clean
and green technology (Park & Kim, 2021). Annealing
does not destroy starch granules thus researchers
attempted to study structural changes in starch orga-
nisation during hydrothermal treatment through this
modification technique.
The changes in starch structure during annealing,
especially changes of RS content related to different
carbohydrate source has not been fully understood.
Starch crystallinity is one of the factors affecting RS
content during annealing (Ashwar et al., 2016) among
other factors such as plant genetics, amylopectin chain
distribution and interactions with starch protein and
lipid. Faridah et al. (2021) have reported the effect of
annealing parameters such as incubation time, temper-
ature and water ration as well as amylose content on
the changes of RS due to annealing. The objective of
this study was to investigate the effect of annealing on
relative crystallinity degree related to RS content based
on the type of starch allomorphs using a literature
review and meta-analysis.
Crystalline type or allomorphs refers to amylopectin
double helices arrangement. According to Hizukuri
(1985), Imberty et al. (1991) and Bertoft et al. (2008)
starch crystal types are divided into three types: A-
type (closely packed, dense and less water), B-type
(less compact and more hydrated) and C-type (mix of
A- and B-type). Allomorphs type is unique to botani-
cal origins and can be recognised by analysis starch X-
ray diffraction pattern. The results of this study were
expected to discover the annealing effect on RS
changes based on starch crystallinity type and crystal-
linity changes thus providing insight into the nature of
starch crystalline behaviour during annealing.
International Journal of Food Science and Technology 2022
Materials and methods
A meta-analysis study and literature review were car-
ried out by following the procedures described in
Afandi et al. (2021). Meta-analysis study was following
several steps, that is, articles screening, data extraction,
meta-analysis and interpretation. Materials used were
research articles related to annealing starch modifica-
tion published from 2000 to 2021 indexed by Scopus,
search engine, and reference manager.
The screening of research articles
Two meta-analyses were carried out to investigate the
effect of starch modification by annealing on the con-
tent of RS based on the type of crystal type and the
relative crystallinity changes. The literature review was
performed to collect data on the effect of annealing on
the relative crystallinity of starch. The materials
needed for the meta-analyses and literature review
were research articles on starch modified by annealing
which investigate the RS changes due to the modifica-
tion and provide data of the starch crystallinity. Inclu-
sion and exclusion criteria were determined for more
effective screening efforts. The inclusion criteria used
in this study were all articles published between 2000
and 2021, indexed by Scopus, published in English,
using raw starch as a sample and applying annealing
technique for the starch modification. The exclusion
criteria applied were all studies that used pre-treated
starch and a combination of dual starch modification
techniques, articles published as a book, literature
review and meta-analysis. The screening was done by
gathering the research articles from various biblio-
graphic databases which are Google Scholar, Wiley
Online Library, Inderscience, Microsoft Academic, Sci-
ence Direct, Pubmed NCBI, Research Gate and
Cochrane Library.
Preliminary screening for meta-analysis of the effect
of annealing on RS based on crystallinity type used
the combination of ‘ANNEAL*’ AND OR ‘RESIS-
TANT STARCH’, with the removal of all words
related to exclusion criteria such as -DEBRANCH, -
ULTRASOUND, -HEAT MOISTURE TREAT-
MENT with the utilisation of Boolean operators such
as AND and OR and removal of exclusion criteria.
Elimination and selection steps of the research articles
were recorded on Preferred Reporting Items for Sys-
tematic Reviews and Meta-analyses as presented in
Fig. 1. The articles gathered from the preliminary
screening were then selected based on title and
abstracts, downloaded and organised in Mendeley ref-
erence manager. The second step of the selection was
to eliminate all the articles with the same title to
remove duplication. All articles selected after the
removal of duplication were to be reviewed thoroughly
© 2022 Institute of Food, Science and Technology (IFSTTF).

Figure 1 PRISMA flowchart of the research
articles for the meta-analysis of the effect of
annealing on resistant starch based on starch
crystal type.
to check whether they contained all the information
needed for the analysis or not. Research articles with
incomplete data (the average and standard deviation
of the RS content before and after annealing, the aver-
age and standard deviation of relative crystallinity
before and after annealing, number of replication and
crystal type) were then eliminated to obtain research
articles with complete data that will be further
extracted for the meta-analysis.
Data extraction and grouping
The articles that had been gathered from preliminary
screening were then organised in one folder and coded.
The data from each article were extracted into an Excel
file. The information extricated from the research arti-
cles were authors’ name, publishing year, publisher,
Scopus indexed, country, sample, starch crystalline type,
annealing process, number of replications, average and
standard deviation RS content (percentage of RS) and
relative crystallinity of native starch and annealed
starch. There were 22 research articles selected for the
meta-analysis of the effect of annealing on RS based on
starch crystal type which resulted in 74 data points. A
data point is a combination data set of different sam-
ples, annealing process parameters and percentage of
RS or relative crystallinity degree. Therefore, it is possi-
ble to extract more than one data point from one
research article. Data sets then were grouped based on
the crystalline type of the carbohydrate source (type A,
B or C). Aside from the two meta-analysis studies, a lit-
erature review was also carried out from the articles
© 2022 Institute of Food, Science and Technology (IFSTTF).
Annealing: crystallinity and resistant starch M. P. Anugerah et al. 3
collected from the previous efforts which contained the
data of relative crystallinity changes after the annealing
starch modification.
Meta-analysis and subgroup analysis
Each data point that have been extracted consisted of
key data of the native (V) and modified starch (A) for
meta-analysis such as number of replication (NV and
NA), the average of RS (XV and XA), the standard
deviation of RS (sV and sA Þ. The value of pooled SD,
correction factors and variance were calculated first
following formulas 1, 2 and 4. The value of Hedge’s d
(SMD) and standard error (Sd) were calculated follow-
ing formulas 3 and 5 and inputted to meta-essentials
tools developed by Afandi et al. (2021). The value of
% weight was determined using formulas 7 and 8. The
heterogeneity of the data was determined using formu-
las 9 and 10. The output of meta-essentials tools is in
form of forest plots that display the influence of each
parameter analysed.
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
  
NA  1 ðsA Þ2 þ NV  1 ðsV Þ2
S ðpooled SDÞ ¼  A  (1)
N þ NV  2
3
J ðcorrection factorÞ ¼ 1    N   (2)
4 N þ NA  2  1
XA  XV
d ðeffect sizeÞ ¼ J (3)
S
International Journal of Food Science and Technology 2022
4 Annealing: crystallinity and resistant starch M. P. Anugerah et al.


NV þ NA d2 crystallinity after annealing modification. The highest
Vd ðVarianceÞ ¼ þ    (4) average relative crystallinity increase of 18.98% was
NV NA 2 NV þ NA observed on C-type crystal starches on 31 out of 35
pffiffiffiffiffiffi data points. This number is followed by 13.60% aver-
Sd ¼ Vd (5) age %RC increase on B-type crystal starches on 12
out of 15 data points and 8.17% average RC increase
on A-type crystal starches on 35 out of 51 data points.
∑ni¼1 wi di
d þ ðcumulative effect sizeÞ ¼ (6) Increases of %RC were observed in moisture level
∑ni¼1 wi above 67–80% and decreases of %RC were observed
on annealing in moisture level of 67% and above
1 90%.
Wd ¼ (7) Based on this study, A-type crystal showed the low-
Vd
est changes of relative crystallinity due to annealing
Wd which might be related to its structure. Structurally,
% weight ¼ (8) A-type crystal consists of 8 water molecules each
∑Wd
monoclonal unit packing of amylopectin double helices
! (Fig. 2; Jane et al., 1997). This structure is compact
ð∑Wd:dÞ2 and stable. According to several studies A-type crystal
Q ¼ ∑Wd:d  2
(9)
∑Wd showed more short-chain fractions (Hizukuri, 1985), a
lower degree of amylose and amylopectin intermin-
gling, more stable (Knutson, 1990), higher crystallites
Q  df density and branch points scattered in crystalline (Jane
I2 ¼ x100 ; df ¼ n  1 (10)
Q et al., 1997). Research by Gérard et al. (2000), Witt et
al. (2012), Gaenssle et al. (2021) showed that A-type
The output of meta-analyses and sub-group analyses crystal has shorter amylopectin branches (DP 6-12)
is the forest plot. It displays the effect size in a form of compared to B-type crystal. These structural character-
Hedges’s d (SMD), confidence intervals (95% CI) and istics support A-type crystal stability during annealing
weights of each data point and subgroup. Positive physical modification. The increase of relative crystal-
SMD value indicated an increase of the parameter linity in starches with A-type crystal was due to the
analysed. These changes in considered significant if the melting of short amylopectin chain (DP 6-12) to form
confidence interval does not intersect the null value or stronger crystalline (Chung et al., 2009), rearrange-
centreline of the forest plot. ment of amylopectin chain in amorphous and crystal-
line lamellae (Babu et al., 2019; Sudheesh et al., 2019)
Results and discussion and crystal perfection (Zeng et al., 2015).
The change of B-type relative crystallinity was
The effect of annealing on relative crystallinity higher than those of A-type crystal. It indicated that
B-type crystal is more susceptible to changes by
Polymorph crystal type of native starch does not annealing compared to A-type crystal. B-type crystal
change upon annealing treatment as reported on consists of 36 water molecules in each hexagonal pack-
starch with A-type crystal such as rice by Dias et al. ing with most of its branch points clustered in amor-
(2010), De La Rosa-Millán (2017), wheat by R. F. phous regions and is comprised of longer double
Tester et al.(1998), Lan et al. (2008), Su et al. (2020) helices (Jane et al., 1997). A higher number of water
and corn by Rocha et al. (2012), Wang et al. (2014); molecules lead to a more hydrated structure which will
starch with B-type crystal such as potato by Stute be easier to plasticised during annealing. It is called
(1992), Rocha et al. (2011), Xu et al. (2018a,b) and ‘hydrated’ structure because there are more water mol-
starch with C-type crystal such as mung bean by Zou ecules in B-type crystal as it has an empty lattice filled
et al. (2019), red adzuki bean by Xu et al. (2018a,b) with water inside the double helix hexagonal packing
and pea by Wang et al. (2013). However, it affected (Kong et al., 2014; Pinto et al., 2020). Thus, making it
starch relative crystallinity. A literature review on more susceptible to a physical modification such as
starch relative crystallinity (%RC) changes after annealing. These results are in accordance with studies
annealing was shown on Table 1. One hundred and by Chi et al. (2019) which reported that the B-type
one data points were extracted from 22 research crystal of potato starch was easier to move or more
papers covering 51 data points of A-type crystal, 15 mobile due to hydration during annealing. Xu et al.
data points of B-type crystal and 35 data points of C- (2018a,b) stated that this increase was due to crystal-
type crystal starches. The majority of data points in all line perfection and crystallites rearrangement by vibra-
crystal type starches showed an increase of relative tional movements.

International Journal of Food Science and Technology 2022 © 2022 Institute of Food, Science and Technology (IFSTTF).
 Annealing: crystallinity and resistant starch M. P. Anugerah et al. 5

Table 1 Systematic review on relative crystallinity changes upon annealing based on crystal type of starch

%RC
Relative
Crystal Native Annealed crystallinity
No. Research article Year Sample type MC (%) T (°C) t (h) XV XA changes %

1 Wang et al. (2014) 2014 Normal maize A 67 45 24 34.60 33.50 −1.10 −3.179
2 Wang et al. (2014) 2014 Waxy maize 42.3 42.8 0.50 1.182
3 Wang et al. (2014) 2014 Waxy maize 72 42.3 41.60 −0.70 −1.655
4 Wang et al. (2014) 2014 Normal maize 34.60 34.20 −0.40 −1.156
5 Babu et al. (2019) 2018 Foxtail millet 50 24 42.24 46.89 4.65 11.01
6 Bian & Chung (2016) 2016 Brown rice 70 50 24 31.30 31.40 0.10 0.319
7 Lan et al. (2008) 2008 Wheat 11132 75 42.6 72 28.60 29.80 1.2 4.196
8 Lan et al. (2008) 2008 Wheat CDC 48.3 72 30.50 30.90 0.4 1.311
9 Lan et al. (2008) 2008 Wheat 99WAX27 49.6 72 42.80 45.30 2.5 5.841
10 Waduge et al. (2006) 2006 Barley HB 364 50 72 39.8 41.9 2.1 5.276
11 Trinh & Le (2017) 2017 Tapioca 12 38.88 45.67 6.79 17.46
12 Trinh & Le (2017) 2017 Tapioca 48 38.88 50.9 12.02 30.92
13 Waduge et al. (2006) 2006 Barley CDC Fibar 72 44.3 46.6 2.3 5.192
14 Waduge et al. (2006) 2006 Barley CDC McGwire 37 41.7 4.7 12.7
15 Trinh & Le (2017) 2017 Tapioca 38.88 57.21 18.33 47.15
16 Su et al. (2020) 2020 Waxy wheat 80 50 12 24.92 25.37 0.45 1.806
17 Su et al. (2020) 2020 Normal starch 21.19 22.04 0.85 4.011
18 4 Chi et al. 2019 Potato starch 24 25.50 25.30 −0.20 −0.784
19 Su et al. (2020) 2020 Waxy wheat 24.92 25.77 0.85 3.411
20 Su et al. (2020) 2020 Normal starch 21.19 22.06 0.87 4.106
21 Su et al. (2020) 2020 Waxy wheat 24.92 25.90 0.98 3.933
22 Su et al. (2020) 2020 Normal starch 21.19 22.21 1.02 4.814
23 Liu et al. (2016) 2016 Sorghum 30.10 32.60 2.5 8.306
24 Liu et al. (2016) 2016 Maize starch 29.70 32.20 2.5 8.418
25 Liu et al. (2016) 2016 Tartary buckwheat 32.20 36.80 4.6 14.29
26 Su et al. (2020) 2020 Normal starch 48 21.19 22.26 1.07 5.05
27 Su et al. (2020) 2020 Waxy wheat 24.92 26.02 1.1 4.414
28 Su et al. (2020) 2020 Normal starch 21.19 22.40 1.21 5.71
29 Su et al. (2020) 2020 Waxy wheat A 80 50 48 24.92 26.41 1.49 5.979
30 Su et al. (2020) 2020 Waxy wheat 72 24.92 26.40 1.48 5.939
31 Su et al. (2020) 2020 Normal starch 21.19 22.73 1.54 7.268
32 Su et al. (2020) 2020 Normal starch 21.19 22.81 1.62 7.645
33 Su et al. (2020) 2020 Waxy wheat 24.92 26.80 1.88 7.544
34 Su et al. (2020) 2020 Normal starch 96 21.19 22.60 1.41 6.654
35 Su et al. (2020) 2020 Normal starch 21.19 22.65 1.46 6.89
36 Su et al. (2020) 2020 Waxy wheat 24.92 26.83 1.91 7.665
37 Su et al. (2020) 2020 Waxy wheat 24.92 27.23 2.31 9.27
38 Dias et al. (2010) 2010 High wmylose Rice 90 45 16 49.1 47.6 −1.5 −3.055
39 Dias et al. (2010) 2010 Low amylose Rice 37.7 37 −0.7 −1.857
40 Dias et al. (2010) 2010 Medium amylose rice 38.5 38.4 −0.1 −0.26
41 Dias et al. (2010) 2010 High amylose rice 50 16 49.1 47.4 −1.7 −3.462
42 Dias et al. (2010) 2010 Low rmylose rice 37.7 36.1 −1.6 −4.244
43 Dias et al. (2010) 2010 Medium amylose rice 38.5 38.1 −0.4 −1.039
44 De La Rosa-Millán (2017) 2017 Corn 55 12 28.43 21.26 −7.17 −25.22
45 De La Rosa-Millán (2017) 2017 Rice 12 25.19 19.17 −6.02 −23.9
46 Dias et al. (2010) 2010 High amylose rice 49.1 39.5 −9.6 −19.55
47 Dias et al. (2010) 2010 Low amylose rice 37.7 33.5 −4.2 −11.14
48 Dias et al. (2010) 2010 Medium amylose rice 38.5 36.4 −2.1 −5.455
49 Rocha et al. (2011) 2011 Cassava 95 56 24 43.1 37 −6.1 −14.15
50 Rocha et al. (2012) 2012 Waxy corn 62 24 41.4 44.3 2.9 7.005
51 Rocha et al. (2012) 2012 Normal corn A 63 24 32.6 33.7 1.1 3.374
52 Wang et al. (2014) 2014 High amylose maize B 67 45 24 23.40 23.20 −0.20 −0.855
53 Wang et al. (2014) 2014 High amylose maize 72 23.40 22.70 −0.70 −2.991
54 Xu et al. (2018) 2018 Potato starch 75 55 12 19.68 20.92 1.24 6.301

© 2022 Institute of Food, Science and Technology (IFSTTF). International Journal of Food Science and Technology 2022
6 Annealing: crystallinity and resistant starch M. P. Anugerah et al.

Table 1 (Continued)

%RC
Relative
Crystal Native Annealed crystallinity
No. Research article Year Sample type MC (%) T (°C) t (h) XV XA changes %

55 Xu et al. (2018) 2018 Potato starch 24 19.68 21.79 2.11 10.72

56 Xu et al. (2018) 2018 Potato starch 19.68 21.85 2.17 11.03
57 Xu et al. (2018) 2018 Potato starch 48 19.68 22.74 3.06 15.55
58 Xu et al. (2018) 2018 Potato starch 19.68 23.25 3.57 18.14
59 Xu et al. (2018) 2018 Potato starch 72 19.68 22.95 3.27 16.62
60 Xu et al. (2018) 2018 Potato starch 19.68 23.44 3.76 19.11
61 Xu et al. (2018) 2018 Potato starch 96 19.68 23.55 3.87 19.66
62 Xu et al. (2018) 2018 Potato starch 19.68 23.74 4.06 20.63
63 Chi et al. (2019) 2019 Maize starch 80 50 24 31.20 31.80 0.60 1.923
64 De La Rosa-Millán (2017) 2017 Potato 90 45 12 24.43 19.01 −5.42 −22.19
65 Rocha et al. (2011) 2011 Peruvian carrots 95 54 24 37.1 43.1 6 16.17
66 Rocha et al. (2011) 2011 Potato 59 24 38.3 41.1 2.8 7.311
67 Zhang et al. (2015) 2015 Kudzu C 60 50 24 19.3 20.2 0.9 4.663
68 Zhang et al. (2015) 2015 Kudzu 72 19.3 20.4 1.1 5.699
69 Wang et al. (2013) 2013 Pea (PRL417) 67 45 24 34.60 35.50 0.90 2.601
70 Wang et al. (2013) 2013 Pea (Maki) 36.40 37.40 1.00 2.747
71 Wang et al. (2013) 2013 Pea (PRL95) 34.70 36.00 1.30 3.746
72 Wang et al. (2013) 2013 Pea (PRL417) 72 34.60 35.10 0.50 1.445
73 Wang et al. (2013) 2013 Pea (Maki) 36.40 37.10 0.70 1.923
74 Wang et al. (2013) 2013 Pea (PRL95) 34.70 35.70 1.00 2.882
75 Molavi et al. (2018) 2018 Acorn starch 70 50 72 47.8 46.8 −1 −2.092
76 Zou et al. (2019) 2019 Mung bean 75 50 12 17.12 19.82 2.7 15.77
77 Zou et al. (2019) 2019 Mung bean 24 17.12 20.73 3.61 21.09
78 Zou et al. (2019) 2019 Mung bean 17.12 21.27 4.15 24.24
79 Zou et al. (2019) 2019 Mung bean 48 17.12 21.19 4.07 23.77
80 Zou et al. (2019) 2019 Mung bean 17.12 21.93 4.81 28.1
81 Afolabi et al. (2018) 2018 Bambara groundnut C 75 50 48 36.30 47.04 10.74 29.59
82 Waduge et al. (2006) 2006 Barley CDC McGwire 72 42.3 42.6 0.3 0.709
83 Waduge et al. (2006) 2006 Barley CDC McGwire 40.8 42.1 1.3 3.186
84 Waduge et al. (2006) 2006 Barley CDC McGwire 40 43.3 3.3 8.25
85 Zou et al. (2019) 2019 Mung bean 17.12 22.6 5.48 32.01
86 Zou et al. (2019) 2019 Mung bean 17.12 23.16 6.04 35.28
87 Zou et al. (2019) 2019 Mung bean 96 17.12 23.21 6.09 35.57
88 Zou et al. (2019) 2019 Mung bean 17.12 23.76 6.64 38.79
89 Xu et al. (2018) 2018 Red bean starch 55 12 26.45 32.53 6.08 22.99
90 Xu et al. (2018) 2018 Red bean starch 24 26.45 33.08 6.63 25.07
91 Xu et al. (2018) 2018 Red bean starch 26.45 33.41 6.96 26.31
92 Xu et al. (2018) 2018 Red bean starch 48 26.45 33.79 7.34 27.75
93 Xu et al. (2018) 2018 Red bean starch 26.45 34.50 8.05 30.43
94 Xu et al. (2018) 2018 Red bean starch 72 26.45 34.04 7.59 28.7
95 Xu et al. (2018) 2018 Red bean starch 26.45 34.90 8.45 31.95
96 Xu et al. (2018) 2018 Red bean starch 96 26.45 34.56 8.11 30.66
97 Xu et al. (2018) 2018 Red bean starch 26.45 35.80 9.35 35.35
98 Song et al. (2014) 2014 Sweet potato SPSS 90 55 72 25.50 23.20 −2.30 −9.02
99 Song et al. (2014) 2014 Sweet potato Yulmi (YM) 25.50 23.50 −2.00 −7.843
100 Song et al. (2014) 2014 Sweet potato 29.70 28.50 −1.20 −4.04
Yeonwhangm (YHM)
101 Song et al. (2014) 2014 Sweet potato CSPS 24.90 26.70 1.80 7.229

MC, moisture content; T, temperature; t, time.

The highest increase of relative crystallinity was type crystal thus possessing both long and short chains.
observed on starches with C-type crystal. This crystal Tester et al.(1998) postulated that the greater defects
type consists of a mix between A-type crystal and B- found in starches, the more changes of starch structure

International Journal of Food Science and Technology 2022 © 2022 Institute of Food, Science and Technology (IFSTTF).

Figure 2 Structure of double helix packing
in A-type crystal (monoclonal unit) and B-
type crystal (hexagonal). Red circles are dou-
ble helices and small blue circles are water
molecules.
will experience upon annealing. In accordance with
these results, Vamadevan et al. (2013) reported that
starch with greater structural disorders (defects) showed
structural improvement due to the alignment of double
helices and ordering of untwisted ends. Similarly, Piecyk
et al. (2018) reported an increase of relative crystallinity
on C-type crystal of pea starch due to the formation of
new hydrogen bonds between double helices which lead
to crystalline perfection.
The effect of annealing on RS content based on the
crystalline type
Seventy-four data points were extracted from 22
research papers. The results of subgroup analyses of RS
changes based on starch allomorph crystal type was dis-
played on Fig. 3. Significant RS increase was observed
on annealing of starches with A-crystal type (n = 43;
SMD = 6.46; 95% CI = 3.22–9.70; P < 0.01). RS
increase was also found in annealing of starch with
B-type (n = 6; SMD = 0.28; 95% CI = −4.44–4.99;
Figure 3 Forest plot of annealing effect on resistant starch subgroup analysis based on allomorph crystal type (square symbol on the right side
of the centre line indicates an increase of resistant starch content).
© 2022 Institute of Food, Science and Technology (IFSTTF).
Annealing: crystallinity and resistant starch M. P. Anugerah et al. 7
P = 0.00). However, this increase was not significant as
indicated by the intersection of confidence interval and
centerline. Annealing on starches with C-type crystal
resulted in insignificant decrease of the RS level (n = 25;
SMD = −0.66; 95% CI = −6.19 to 6.80; P = 0.00).
Although the previous result showed that the high-
est relative crystallinity increase was observed in C-
type crystal, this meta-analysis study showed that
regarding the RS increase A-type crystal displayed the
highest increase after annealing. Even with the low
increase in relative crystallinity, annealing on A-type
crystal melted the ‘weak points’ in branch ends result-
ing in a more compact and perfect crystalline. A smal-
ler number of water molecules in this crystal type
resulted in very low mobility and starch chain dense
packing (Knutson, 1990; Tester & Debon, 2000) sup-
port its resistance to enzyme hydrolysis. RS increase
on A-type crystal starch was also reported by Chung
et al. (2009), Liu et al. (2016a,b), Chi et al. (2019) on
maize starch, Trung et al. (2017) on sweet potato
starches and Faridah et al. (2021) on cereals starches.
International Journal of Food Science and Technology 2022
8 Annealing: crystallinity and resistant starch M. P. Anugerah et al.
The effect of annealing on RS content based on relative
crystallinity changes
Further subgroup analysis was carried out to investi-
gate the effect of annealing on RS level based on rela-
tive crystallinity changes using 47 data points (Fig. 4).
The results showed an insignificant decrease of RS
level on starches with %RC reduction between 0 and
10 (n = 9; SMD = −0.81; 95% CI −6.10 to 4.49;
P = 0.00). Significant increase of RS was observed on
other three groups: %RC rise between 0.10 and 10
(n = 28; SMD = 7.32; 95% CI: 1.96–12.67; P < 0.00),
%RC rise between 20 and 30 (n = 6; SMD = 32.49:
95% CI: 8.52–56.47; P < 0.00), and %RC rise
between 30 and 40 (n = 4; SMD = 53.15 : 95% CI:
25.35–80.96; P < 0.00).
These results showed that there is a positive rela-
tionship between relative crystallinity and RS which
could be related to:
(i) The increase of amylose – amylose or amylose – amy-
lopectin interaction resulted in crystallites formation
within the amorphous regions. This phenomenon will
hinder the access of water to the glycosidic bonds
and act as steric hindrance to enzyme (Tester &
Debon, 2000; Jayakody et al., 2009).
(ii) Co-crystallisation of amylose with amylopectin side
chain forming double helices contributed to the crys-
tal thickening which resulted in the increase of crystal
stability (Tester & Debon, 2000; Gomand et al.,
2012).
(iii) Re-orientation of double helices in the crystalline
lamellae will increase homogeneity and perfection of
the dense packing of the crystalline (Vermeylen et al.,
2006; Vamadevan et al., 2013).
(iv) The twisting of unordered amylopectin branch ends
by the formation of new hydrogen bonds that
Figure 4 Forest plot of annealing effect on resistant starch subgroup analysis based on relative crystallinity changes (square symbol on the
right side of the centre line indicates an increase of resistant starch content) Attached is the pdf file of the last revision version.
International Journal of Food Science and Technology 2022
lengthen the double helices and improvement in dou-
ble helices parallel packing led to a more compact
structure (Kiseleva et al., 2005; Vamadevan et al.,
2014).
(v) Enhancement of the order and the formation of V-
amylose lipid complex (Waduge et al., 2006; Wang
et al., 2014).
Several studies observed a similar positive relation-
ship between the relative crystallinity and RS increase
upon annealing on Tartary buckwheat starch (Liu
et al., 2016a,b), red adzuki bean starch (Xu et al.,
2018a,b), wheat starch (Su et al., 2020) and waxy
maize starch (Wang et al., 2014). While Song et al.
(2014) reported a decrease of RS and relative crystal-
linity after annealing. It was predicted to be caused by
the removal of some helices and crystallites.
Conclusion
Relative crystallinity and starch crystal type are impor-
tant factors in the changes of starch structure because
of annealing starch modification. The annealing pro-
cess significantly increase the RS content on A-crystal
type starch which probably due to its compact and sta-
ble structure resulted in resistance to enzyme hydroly-
sis. This structure also supports the low relative
crystallinity increase compared to other allomorph
types (B- and C-type crystals). There is a positive rela-
tionship between the relative crystallinity degree of all
crystal types and RS content upon annealing. There-
fore, carbohydrate sources with A-type crystals such
as cereals are suitable for annealing with the purpose
of increasing RS content. These results provide a dee-
per understanding of the response of starch molecules
during annealing from different botanical origins
which is advantageous for starch-based products and
RS industries.
© 2022 Institute of Food, Science and Technology (IFSTTF).

Acknowledgments
The experiment was supported by Ministry of
Research and Technology/National Research and
Innovation Agency as a part of Pendanaan Penelitian
di Perguruan Tinggi Tahun Anggaran 2021 program
grant number B/112/E3/RA.00/2021
Author contributions
Maria Putri Anugerah: Formal analysis (equal); Meth-
odology (equal); Writing – original draft (equal).
Didah Nur Faridah: Conceptualization (equal); Super-
vision (equal); Writing – review & editing (equal).
Frendy Ahmad Afandi: Conceptualization (equal);
Methodology (equal); Supervision (equal); Writing –
review & editing (equal). Dase Hunaefi: Supervision
(equal); Writing – review & editing (equal). Anuraga
Jayanegara: Methodology (equal); Supervision (equal);
Writing – review & editing (equal).
Conflict of interest
The authors declare that there is no conflict of
interest.
Peer review
The peer review history for this article is available at
https://publons.com/publon/10.1111/ijfs.15628.
Data availability
The data of the research were not shared.
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