3 Biotech (2022) 12:62

https://doi.org/10.1007/s13205-022-03128-z

PROTOCOLS AND METHODS

Development and validation of rapid and cost‑effective protocol

for estimation of amylose and amylopectin in maize kernels
Shashidhar Bayappa Reddappa1 · Rashmi Chhabra1 · Zahirul Alam Talukder1 · Vignesh Muthusamy1 ·
Rajkumar Uttamrao Zunjare1 · Firoz Hossain1

Received: 12 April 2021 / Accepted: 23 January 2022 / Published online: 7 February 2022
© King Abdulaziz City for Science and Technology 2022

Abstract
Maize possesses wide variation in amylose and amylopectin which assumes significance as a part of both food-chain and
different industrial applications. Estimation of amylose and amylopectin in maize kernels is important for developing suit-
able hybrids. The existing protocols for estimation of amylose and amylopectin in maize are elaborate and lengthy, and
involve high cost. Here, we developed a rapid and cost-effective method for estimation of amylose and amylopectin in maize
kernels. 10% toluene and 80% ethanol were used for removal of proteins (~ 10%) and lipids (~ 4%) from maize flour. The
over-estimation of amylose was minimized using NaOH with KI to stop free KI to bind with amylopectin. Standards were
improved by mixing amylose and amylopectin in different concentrations (0–100%), rather than using amylose or amylopectin
alone. Standard curve generated regression equation of y = 90.436x + 0.8535 with R2 = 0.9989. Two types of samples viz.,
(1) protein, amylose and amylopectin (2) amylose and amylopectin, showed that starch fractions were highly comparable to
expected values with correlation coefficient (r) of 0.9998 and mean standard deviation of 0.54. The protocol successfully
estimated wide range of amylose (2.79–50.04%) and amylopectin (59.96–97.21%) among diverse maize inbreds including
amylose extender1 (ae1) and waxy1 (wx1) mutants. Present protocol required 75% less time and 92.5% less cost compared
to existing protocols. The newly developed method would be highly useful in developing maize hybrids high in amylose or
amylopectin. This is the first report of rapid and cost-effective protocol for estimation of starch fractions in maize kernels.

Keywords Amylopectin · Amylose · Kernels · Maize · Protocol · Starch

Introduction

Starch is a major energy reserve in higher plants and most

abundant carbohydrates next to cellulose (Zhong et al. 2021).
Starch is generally synthesized in a specialized plastid called
* Firoz Hossain amyloplasts with composition of amylose and amylopec-
fh_gpb@yahoo.com tin (Hossain et al. 2019). Amylose is a linear homopolymer
Shashidhar Bayappa Reddappa of glucopyranose units linked by α-(1,4) linkage, whereas
shashintrsachin@gmail.com amylopectin is a branched homopolymer of glucopyranose
Rashmi Chhabra with α-(1,4) and α-(1,6) linkage (Lin et al. 2019). The ratio
reshu0428@rediffmail.com of amylose and amylopectin plays an important role in
Zahirul Alam Talukder determining the structure of starch granule. Maize starch
zahirbari@yahoo.com is composed of an average of ~ 25% of amylose and ~ 75%
Vignesh Muthusamy of amylopectin (Hasjim and Jane 2009; Chung et al. 2009).
pmvignesh@yahoo.co.in; vignesh@iari.res.in High amylose maize starch has wide application in mak-
Rajkumar Uttamrao Zunjare ing of candies, gums, adhesives and biodegradable plastics
raj_gpb@yahoo.com (Zhang et al. 2018). Amylose also contributes to Resistant
Starch (RS) which takes > 120 min to digest in the human
1
Division of Genetics, ICAR-Indian Agricultural Research gut (Englyst et al. 1992). ‘Diabetes mellitus-type2’ has been
Institute, New Delhi 110012, India

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identified as the most widespread lifestyle disease among
humans affecting 366 million people worldwide. This fig-
ure is expected to rise to a level of 552 million by 2030
(Sami et al. 2017). Amylose content shows positive correla-
tion with RS and total dietary fiber (TDF) (Xia et al. 2018).
RS significantly lowers down the glycaemic index (GI) in
humans (Behall and Hallfrisch 2002). The GI of traditional
maize is 81, while the GI of high amylose maize reduces the
GI to 44 (Ai and Jane 2016). Thus, high amylose maize is
ideal food for the diabetic people. Apart from hypoglycaemic
effect, high amylose maize also shows hypocholesterolemic
effect by reducing cholesterol accumulation which further
helps in reduction of bile-stone formations (Hashimoto et al.
2006; Malhotra 1968). On the other hand, maize with high
amylopectin also called ‘waxy maize’ is a popular choice of
food in South-East Asian countries (Xiaoyang et al. 2017).
Waxy maize is consumed as ‘green corn’, especially during
breakfast, and also popular as vegetable (Devi et al. 2017).
Amylopectin possesses the property of high viscosity and is
easily digested in human gut (Lu and Lu 2012). Amylopectin
also serves as a popular ingredient in textile, adhesive and
paper industries (Bao et al. 2012).
Amylose and amylopectin generally vary from 7 to 35%
and 75 to 95%, respectively in maize (Li et al. 2018; Qi
et al. 2020). However, Zhang et al (2020), while analyzing
a set of maize inbreds reported amylose up to 67%, while
waxy maize can have amylopectin up to 100% (Zhou et al.
2016). Precise estimation of amylose and amylopectin from
the maize grain sample assumes great significance for the
identification of suitable donor in the breeding program
followed by development of high amylose or amylopectin
maize. Various methods including colorimetric, potentio-
metric or amperometric titration techniques are available for
determining of amylose and amylopectin (Bates et al. 1943;
Williams et al. 1958). Colorimetric estimation is the widely
and most frequently used technique, which is based on the
ability of amylose to form complex with iodine to give blue
color. However, it has been reported that even amylopectin
can form complex with iodine and absorb minute quantity
of light which interfere in amylose estimation (Davis et al.
1994). Amylose or amylopectin in most of the cereals is
estimated either by extracting starch and then estimating or
directly using the milled powder. In the first case, extrac-
tion of starch is tedious and requires lot of time, whereas in
the second case due to the presence of proteins and lipids
the estimated value of amylose is affected. Cereals like rice
grains has nearly 90% of starch which makes needless to
extract starch hence can be directly ground and used for esti-
mation. However, in crop such as maize which is composed
of 10% protein and 4% lipid with 70% of starch, it becomes
necessary to remove protein and lipid prior to estimation of
amylose or amylopectin. ‘Megazyme’ amylose kit based on
the specific precipitation of amylopectin by concanavalin-A
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3 Biotech (2022) 12:62
lectin has emerged as a popular choice in lab analysis (Yun
and Matheson 1990; Gibson et al. 1997). However, the avail-
able protocols for estimation of amylose and amylopectin
are time consuming and involves cumbersome methodology
and costly chemicals (Zhu et al. 2008; Megazyme 2018).
The present study was, therefore, undertaken to develop and
validate a rapid, simple and cost-effective protocol that can
be used for estimation of amylose and amylopectin in maize
kernels.
Materials and methods
Preparation of standard curve
Amylose and amylopectin standards from maize with purity
of > 99% from Sigma Life Sciences were used in prepara-
tion of standard curve. Standard curve was prepared by
mixing relative proportion of amylose and amylopectin
standards. For example, 60% amylose standard included
40% of amylopectin. Standard curve was plotted using
amylose:amylopectin ratio of 100:0, 80:20, 60:40, 50:50,
40:60, 30:70, 25:75, 20:80, 10:100 and 0:100. Three rep-
licates were used for accurate measurement of absorbance.
Preparation of standard sets for validation
Amylose and amylopectin standards from maize with purity
of > 99% were used in preparation of starch standard mix-
tures. Bovine serum albumin protein (HiMedia Laboratories
Pvt. Ltd.) with purity of > 98% was used. For testing the
effectiveness of estimation method, two types of test samples
were prepared with different contents of (1) protein (ranging
from 5 to 15%), amylose and amylopectin (set 1–set 3), and
(2) amylose and amylopectin (set 4–set 11) (Table 1). The
analysis was carried out in four biological replicates.
Preparation of diverse maize samples for validation
Further, eight diverse maize inbreds of exotic and indige-
nous origin were used for validation. Of these, PMI-ae1-145,
UMI-1200, BML-6, PMI-LAMY-1 and PMI-wx1-301 were
developed by different breeding centers in India, while three
lines (CML-580, CML-313 and CML-247) were developed
by International Maize and Wheat Improvement Center
(CIMMYT) Mexico. Among the inbreds, PMI-ae1-145 pos-
sessed mutant amylose extender1 (ae1) gene, while PMI-
wx1-301 had the recessive waxy1 (wx1) allele. Rest of the
inbreds possessed the wild-type Ae1 and Wx1 alleles. The
experiment was undertaken with four biological replications.
These inbreds with varying amylose and amylopectin were
purposefully selected based on preliminary analysis in our
laboratory.
3 Biotech (2022) 12:62 Page 3 of 8 62

Table 1  Details of sets of Sample Std. starch mixture Protein lost (%)a Expected Estimated SD Relative
standards used for validation (Amy + Amp + Protein) amylose (%) amylose (%) SD Amy
(%) (%)

Set-1 23.75 + 71.25 + 5.00 5.42 25 25.01 ± 0.70 2.79

Set-2 22.5 + 67.5 + 10.00 11.4 25 24.62 ± 0.51 2.09
Set-3 21.25 + 63.75 + 15.00 14.34 25 25.84 ± 0.55 2.14
Mean 25.15 ± 0.58 2.34
SE 0.23 – –
CD (5%) 0.54 – –
Set-4 90 + 10 + 0 – 90 92.44 ± 1.05 1.13
Set-5 75 + 25 + 0 – 75 76.56 ± 0.59 0.77
Set-6 60 + 40 + 0 – 60 60.44 ± 0.45 0.75
Set-7 50 + 50 + 0 – 50 50.51 ± 0.62 1.22
Set-8 40 + 60 + 0 – 40 40.51 ± 0.15 0.38
Set-9 30 + 70 + 0 – 30 30.18 ± 0.42 1.39
Set-10 20 + 80 + 0 – 20 21.00 ± 0.65 3.10
Set-11 10 + 90 + 0 – 10 10.16 ± 0.28 2.76
Mean 47.73 ± 0.52 1.44
SE 0.40 – –
CD (5%) 0.85 – –
a
Protein lost was calculated as the amount of weight lost after toluene treatment
Std standard, Amy amylose, Amp amylopectin, SD standard deviation, SE standard error, CD critical differ-
ence

Equipments and reagents
Seed miller with filter of diameter < 0.2 mm, vortex, centri-
fuge, heating water bath, spectrophotometer and tubes (2 ml
and 50 ml) were required. Further, the protocol also required
80% ethanol (stored at either 4°C or room temperature more
than a year), 0.1 M sodium chloride solution (0.1 M NaCl)
containing 10% toluene (can be stored for one month in 4°C
and care should be taken from direct sunlight exposure and
kept away from heat), 1 M sodium hydroxide (1 M NaOH)
(can be stored at room temperature for more than a month),
1 N acetic acid (1 N-AA) (can be stored at 4°C or room
temperature and is extremely stable) and potassium iodide
solution (KI, 0.26 g of Iodine in 10 ml of potassium iodide
solution containing 2.6 g of KI) (should be freshly prepared
before use and kept out of light). All the chemicals and rea-
gents used in the protocol were purchased from HiMedia
Laboratories Pvt. Ltd.
Amylose estimation protocol
Seed powder was obtained by milling 4–5 completely
dried maize seeds having moisture content 10–12% with
a diameter of < 0.2 mm. Approximately 100 mg of seed
powder was transferred into 2 ml tubes. Sample was
treated with 80% ethanol of 500 µl and vortexed for a short
period of time (2 min). The sample tubes were centrifuged
for 5 min at 10,000 rpm and supernatant was separated. It
was repeated two times. The pellet obtained was further
treated with 500 µl of 0.1 M NaCl solution containing
10% toluene and vortexed. The tubes were centrifuged
for 5 min at 10,000 rpm and supernatant was discarded.
It was repeated until the supernatant was clear of white
milky layer. Obtained residue was again washed with 80%
ethanol and dried completely in incubator at 80 °C for 4 h.
The residue, thus, obtained is starch with < 5% impurity of
fiber or thick outer covering of kernel.
Starch residues of 25 mg was carefully measured and
transferred into 50 ml tubes. It was solubilized with 2.5 ml
1 M NaOH and was kept in hot boiling water bath for
at least 15 min with intermittent shaking. Samples were
removed from water bath, cooled at room temperature and
volume was adjusted to 25 ml using double-distilled water.
An aliquot of 1.25 ml was transferred to new 50 ml tube
and treated with 150 µl 1 N acetic acid, 100 µl 1 M NaOH
and 500 µl KI solution and made up to 25 ml using double-
distilled water. The solution was incubated at room tem-
perature for at least 20 min to develop color and absorb-
ance was measured at 620 nm. Amylose was expressed as
percentage by calculating the proportion of amylose over
25 mg starch residues taken in the final step. The proce-
dure has been represented as a flow chart in Fig. 1.

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62 Page 4 of 8 3 Biotech (2022) 12:62

Fig. 1  Flow chart for the proce-

dure for estimation of amylose

Statistical analysis 60:40, 50:50, 40:60, 30:70, 25:75, 20:80, 10:100 and 0:100)
was prepared, which generated regression equation of
Mean, standard error (SE) and critical difference (CD) were y = 90.436x + 0.8535 with R2 value of 0.9989 (Fig. 2).
analyzed using Windostat v8.0. Standard deviation (SD)
between the replicated and relative standard deviation were Validation of developed protocol
calculated using MS-Office Excel-2019.
Estimation of amylose and amylopectin in different sets
of standard samples
Estimation of amylopectin
Two types of test samples were prepared with different
Amylopectin was estimated by subtracting the value of
mixtures of (1) protein, amylose and amylopectin (set 1–set
amylose from the total 25 mg starch residues taken for final
3) (2) amylose and amylopectin (set 4–set 11) for validat-
analysis.
ing the protocol. All the eleven sets showed amylose and
amylopectin concentrations comparable to expected values
Comparison with existing protocol with correlation coefficient of 0.9998 and mean standard
deviation was 0.54. Repeated analysis for the standard starch
The newly developed protocol for the estimation of amylose
in maize grains were also compared with (1) dual-wave-
length method (Hovenkamp-Hermelink et al. 1988; Zhu
et al. 2008; Zeng et al. 2012) and (2) GOPOD reagent and
concanavalin-A-based Megazyme kit (© Megazyme 2018)
for its efficacy besides time and cost analysis.

Results

Preparation of standard curve

Standard curve with different concentrations of stand-

ard amylose and amylopectin in triplicates (100:0, 80:20,
Fig. 2  Standard curve for estimation of amylose

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3 Biotech (2022) 12:62 Page 5 of 8 62

samples yielded repeatability with relative standard devia-
tion of 1.68% (Table 1).
Estimation of amylose and amylopectin in diverse maize
samples
it was found that values of the amylose estimated in the
protocol developed here were similar to the values meas-
ured through Megazyme’s kit, while the values of amylose
using dual-wavelength method were quite lower than both
protocols.

Eight diverse maize genotypes were employed for estimation

of amylose and amylopectin in maize kernels. The amylose
ranged from 2.79% in waxy line (PMI-wx1-3) to 50.04%
(PMI-ae1-145) with mean standard deviation of 0.62.
While, amylopectin varied from 49.96% (PMI-ae1-145)
to 97.21% (PMI-wx1-301). Other genotypes with different
proportion of amylose and amylopectin included CML-580
(amylose: 35.59%, amylopectin: 64.41%), CML-313 (amyl-
ose: 29.77%, amylopectin: 70.23%), UMI-1200 (amylose:
24.18%, amylopectin: 75.82%), BML-6 (amylose: 21.69%,
amylopectin: 78.31%), CML-247 (amylose: 19.74%, amy-
lopectin: 8026%) and PMI-LAMY-1 (amylose: 12.13%,
amylopectin: 87.87%). Repeated analysis of these maize
samples yielded repeatability with relative standard devia-
tion of 4.08% (Table 2).
Comparison with existing protocols for amylose
and amylopectin
The time taken for completion of this developed protocol
for estimation of amylose and amylopectin starting with the
defattening of flour up to final estimation was approximately
8 h for 100 samples, whereas the Megazyme’s kit took much
longer time up to 24 h for 24 samples (© Magazyme 2018).
The protocol reported here required a cost of US$ 0.25 per
sample compared to US$ 3.20 per sample using Megazyme’s
kit (© Megazyme 2018) (Table 3). On the other hand, dual-
wavelength method involved a cost of US$ 0.49 per sample,
with a requirement of 4.3 h for 100 samples. Interestingly,
Discussion
Amylose possesses linear helical configuration which makes
a stable compact structure resistant to digestive enzymes,
thereby lowering the GI (Wee and Henry 2020). The GI
of traditional maize is 81, while the GI of high amylose
maize is reduced to 44 (Ai and Jane 2016). Similarly, white
rice and brown rice have a GI of 78 and 65, respectively.
Whereas, high amylose rice possesses low GI of 39 (Rohman
et al. 2014; Wee and Henry 2020). The present investigation
was targeted to develop a rapid, simple and cost-effective
protocol for estimating amylose and amylopectin in maize
kernels. Different methods were tested with standards to
get reproducible and robust results, viz., Iodine-DMSO
colorimetric method in combination with calcium chloride
(Knutson and Grove, 1994), potassium iodide method (Jain
et al. 2012) and Megazyme kit for estimation of amylose
and amylopectin (© Megazyme 2018). Moreover, various
reports were carefully studied for different steps starting
from defatting and deproteinization of maize flour. Zhu et al.
(2008) followed aqueous leaching process given by Mua and
Jackson (1998) to get purified amylose fractions. Jane et al.
(1992) employed general procedure given by Schoch (1942)
to fractionate starch of normal corn, potato and normal rice
which takes approximately 40 h for fractionation into amyl-
ose and amylopectin. Since protein is present in maize ker-
nels in significant amount (~ 10%), deproteinization of maize
flour is an essentiality.

Table 2  Estimation of amylose and amylopectin in diverse maize inbreds

Inbreds Amylose (%) Amylopectin (%) SD Amylose (%) Amylopectin (%) SD Amylose (%) Amylopectin (%) SD
Present study Dual wavelength Megazyme

PMI-ae1-145 50.04 49.96 ± 0.28 47.93 52.07 ± 0.60 49.96 50.04 ± 1.01
CML-580 35.59 64.41 ± 1.08 33.19 66.81 ± 0.46 35.46 64.54 ± 0.43
CML-313 29.77 70.23 ± 0.62 28.23 71.77 ± 0.55 29.55 70.45 ± 0.74
UMI-1200 24.18 75.82 ± 0.53 22.18 77.82 ± 0.67 24.85 75.15 ± 0.72
BML-6 21.69 78.31 ± 0.59 20.57 79.43 ± 0.83 22.44 77.56 ± 0.60
CML-247 19.74 80.26 ± 1.00 18.54 81.46 ± 0.49 19.68 80.32 ± 0.74
PMI-LAMY-1 12.13 87.87 ± 0.47 11.65 88.35 ± 0.77 12.55 87.45 ± 0.49
PMI-wx1-301 2.79 97.21 ± 0.36 2.15 97.85 ± 0.19 2.79 97.21 ± 0.25
Mean 24.49 75.50 ± 0.62 23.05 76.94 ± 0.58 24.66 75.34 ± 0.62
SE 0.50 0.50 0.50 0.50 0.49 0.49
CD (5%) 4.74 4.74 4.74 4.74 4.74 4.74

SD standard deviation, SE standard error, CD critical difference

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Table 3  Cost and time analysis of the developed protocol

Cost analysis
Present study Dual wavelength Megazyme

Chemicals Cost in INR (100 samples) Cost in INR (100 samples) Cost in INR (24 samples)
Ethanol 1155.00 2310.00 -
Sodium chloride 0.75 – -
Toluene 21.45 – -
Sodium hydroxide 6.00 12.00 -
Glacial acetic acid 14.11 28.20 -
Potassium iodide 362.15 724.30 -
Iodine 27.00 54.00 -
Amylopectin standard 24.10 48.20 -
Amylose standard 250.00 500.00 -
Total cost 1860.56 3676.70 5760.00
(INR 18.60 per sample) (INR 36.77 per sample) (INR 240.00 per sample)
US$ 25.36 US$ 48.99 US$ 76.74
(US$ 0.25 per sample) (US$ 0.49 per sample) (US$ 3.20 per sample)
Time analysis
Preparation of reagents 0.3 h 0.3 h 2h
Procedure 7.3 h 4.0 h 22 h
Total time 7.6 h (~ 8 h) 4.3 h 24 h

1 US$ = INR 75.05

Baker et al. (1979) used various alcohols and solvents in
different concentrations in order to remove oil from grain
sample. Here, we optimized 80% ethanol for defattening of
maize flour. While, deproteinization of maize flour has been
standardized with 0.1 M NaCl with 10% toluene. This step
of removal of proteins has not been reported in many of
the amylose estimating protocols. Toluene is more effec-
tive with the use of NaCl, as the solubility of proteins is
increased by using low concentration (0.1 M NaCl) of salts
which increases the interaction between the protein and the
solvent (salting-in). On the other hand, toluene being an
organic solvent denatures protein and forms a milky layer
which is ultimately discarded. Repetition of this step aids
in removal of protein. As organic solvents denature proteins
by disrupting hydrophobic interaction between non-polar
side chains of proteins, toluene was also suggested to be
a potential solvent for protein denaturation (Asakura et al.
1978). This step was optimized and repeated thrice in case of
maize flour until clear toluene phase was obtained. The clar-
ity of the toluene phase depends upon the content of protein,
therefore, the repetitions of treatment with 0.1 M NaCl with
10% toluene would vary depending upon the protein content
in other cereals.
After the removal of fats and proteins from the maize flour,
amylose was estimated using spectroscopic method. Amylo-
pectin forms complex with iodine which shows absorption
at 620 nm thereby interfering with the precise estimation
of amylose (Gibson et al. 1997; Jain et al. 2012). In earlier
reports, no step was involved to avoid the formation of this
complex. In our study, we suggested to add NaOH along with
KI, which initiates a reaction with iodine complexed with amy-
lopectin, resulting in a clear solution with no absorption at
620 nm. The remaining NaOH in the solution was neutralized
by acetic acid.
Preciseness of the developed protocol
For accuracy and precision, preparation of standards was also
improvised by mixing amylose and amylopectin in different
concentrations, rather than using amylose alone. By employ-
ing this strategy, interference due to absorbance by amylo-
pectin was further minimized to a greater extent. In terms of
accuracy, as mentioned in Megazyme’s kit protocol, repeated
analyses of a set of samples yielded repeatability (within
laboratory) with relative standard deviations of < 5% for pure
starch and ~ 10% for cereal flours (© Magazyme 2018). In our
protocol, the repeated analysis for the standard starch samples
yielded repeatability with relative standard deviation of 1.68%
and it was 4.08% for maize samples. This depicts the robust-
ness and reproducible nature of the newly developed protocol.
Variation in amylose and amylopectin in maize
genotypes
Wide variation was observed among the eight genotypes.
These inbreds were deliberately selected based on our

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3 Biotech (2022) 12:62
preliminary analysis among a set of ~ 300 lines available with
us. Among the lines, PMI-wx1-301 possessed the recessive
wx1 gene which conferred highest amylopectin. The gene
Wx1 located on chromosome-9 encodes a granule-bound
starch synthase (GBSS-I), which catalyses amylose syn-
thesis from ADP-glucose in the endosperm (Mason-Gamer
et al. 1998). The recessive wx1 allele inhibits the conversion
thereby leading to the increased amylopectin (Hossain et al.
2019). On the other hand, the recessive ae1 gene present in
chromosome-5 codes for starch branching enzyme (sbeIIb)
that enhances amylose to a level of > 50% (Li et al. 2008).
Other inbreds possessed amylose in the range of 10–20%,
20–30% and 30–40%, while amylopectin varied from 60 to
70%, 70 to 80% and 80 to 90%. Wide variation of amylose
and amylopectin has been reported in diverse maize germ-
plasm (Li et al. 2018). This suggests that wide variability of
amylose and amylopectin in maize genotypes including the
recessive wx1 and ae1 genes can be effectively measured
using our newly developed protocol.
Cost and time analysis
The analysis showed that the procedure developed here
takes ~ fourfold less time and ~ 13-fold less cost as com-
pared to Megazyme kit. On the other hand, though dual-
wavelength method required nearly half of the time com-
pared to the newly developed method, its cost is double the
cost of the new protocol. Further, the amylose data generated
in the newly developed method suggested that values were
at par with the Megazyme kit and, thus, was highly reli-
able. On the other hand, amylose content measured through
dual-wavelength method was quite low as compared to the
Megazyme kit as well as the present protocol. The lower
values of amylose obtained in dual-wavelength method are
possibly due to non-separation of starch and protein from
flour sample during the process. In our method developed
here, we included steps to remove proteins and separated
starch from the flour sample which gave the amylose values
highly comparable to widely used Megazyme kit. In any
breeding program, large number of genotypes is required to
be screened for amylose and amylopectin in a shorter time
with less cost. The present protocol is simple as it requires
very basic and less costly equipments. Further, the method
is rapid and cost-effective, besides being robust and repro-
ducible. These features of the protocol suite the need of
any breeding program to select for the promising inbreds
followed by development of amylose- or amylopectin-
rich maize. This rapid and cost-effective protocol can also
be used in estimation of amylose in other cereals such as
rice which possess similar to maize in grain composition
with ~ 70–75% starch, 7–10% protein and 2–3% oil (Chiang
et al. 2005). Due to these similar grain composition, Mega-
zyme’s kit is universally used for estimation of amylose in
Page 7 of 8 62
rice as well (© Megazyme 2018). In rice as well, increase
in amylose proportion leads to lower GI (Wee and Henry
2020), while lowering of the same leads to stickiness while
cooking (Zhang et al. 2021). Thus, analysis of diversity of
amylose in rice accession using this newly developed proto-
col assumes great significance in breeding program.
Conclusion
The present study reported the development of protocol for
rapid estimation of amylose and amylopectin in a time- and
cost-effective manner. The protocol has been validated with
the mixtures containing different amylose and amylopectin
content and diverse set of maize inbreds with varying degree
of starch fractions. The results were robust and reproducible
with standard deviation of 1.68% in standards and ~ 4.08% in
unknown maize flour samples. It required 49–92% less cost
compared to the existing methods. The developed method
would be useful in maize breeding programs targeting on
high amylose and amylopectin. This is the first report of
development and validation of protocol for rapid and cost-
effective method for estimation of amylose and amylopectin
in maize kernels.
Acknowledgements First author thanks Indian Council of Agricultural
Research (ICAR), New Delhi for providing the fellowship during the
M.Sc. program. Financial help received from Division of Genetics,
ICAR-IARI, New Delhi, and ICAR-Consortia Research Platform on
‘Molecular breeding for improvement of tolerance to biotic and abiotic
stresses, yield and quality traits in crops - Maize component’ (IARI
Project Code No.: 12-143C) is thankfully acknowledged. We thank
breeders of the national program and CIMMYT, Mexico for sharing
the inbreds.
Author contributions Conduct of experiment: SBR; development of
protocol: RC, SBR; validation of protocol with maize inbreds: ZAT;
development and maintenance of inbreds: VM; Statistical analysis:
RUZ; Manuscript writing: SBR, RC and FH; Design of experiment:
FH.
Declarations
Conflict of interest On behalf of all authors, the corresponding author
states that there is no conflict of interest.
References
Ai Y, Jane JL (2016) Macronutrients in corn and human nutrition.
Compr Rev Food Sci Food Saf 15:581–598
Asakura T, Adachi K, Schwartz E (1978) Stabilizing effect of various
organic solvents on protein. J Biol Chem 253:6423–6425
Baker EC, Mustakas GC, Warner KA (1979) Extraction of defatted
soybean flours and flakes with aqueous alcohols: evaluation of
flavour and selected properties. J Agric Food Chem 27:969–973
13
62 Page 8 of 8
Bao JD, Yao JQ, Zhu JQ, Hu WM, Cai DG, Li Y, Shu QY, Fan LJ
(2012) Identification of glutinous maize landraces and inbred lines
with altered transcription of waxy gene. Mol Breed 30:1707–1714
Bates FL, French D, Rundle RE (1943) Amylose and amylopectin con-
tent of starches determined by their iodine complex formation1. J
Am Chem Soc 65:142–148
Behall K, Hallfrisch JB (2002) Plasma glucose and insulin reduction
after consumption of breads varying in amylose content. Eur J
Clin Nutr 56:913–920
Chiang CM, Yeh FS, Huang LF, Tseng TH, Chung MC, Wang CS,
Lur HS, Shaw JF, Yu SM (2005) Expression of a bi-functional
and thermostable amylopullulanase in transgenic rice seeds leads
to autohydrolysis and altered composition of starch. Mol Breed
15:125–143
Chung HJ, Liu Q, Hoover R (2009) Impact of annealing and heat-
moisture treatment on rapidly digestible, slowly digestible and
resistant starch levels in native and gelatinized corn, pea and lentil
starches. Carbohydr Polym 75:436–447
Davis H, Skrzypek W, Khan A (1994) Iodine binding by amylopectin
and stability of the amylopectin–iodine complex. J Polym Sci Part
A-1 Polym Chem 32:2267–2274
Devi EL, Hossain F, Muthusamy V, Chhabra R, Zunjare RU, Baveja A,
Jaiswal SK, Goswami R, Dosad S (2017) Microsatellite marker-
based characterization of waxy maize inbreds for their utilization
in hybrid breeding. 3 Biotech 7:1–9
Englyst HN, Kingman SM, Cummings JH (1992) Classification and
measurement of nutritionally important starch fractions. Eur J
Clin Nutr 46:S33-50
Gibson TS, Solah VA, McCleary BV (1997) A procedure to meas-
ure amylose in cereal starches and flours with concanavalin A. J
Cereal Sci 25:111–119
Hashimoto N, Ito Y, Han KH, Shimada K (2006) Potato pulps lowered
the serum cholesterol and triglyceride levels in rats. J Nutr Sci
Vitaminol 52:445–450
Hasjim J, Jane JL (2009) Production of resistant starch by extru-
sion cooking of acid modified normal maize starch. J Food Sci
74:C556–C562
Hossain F, Chhabra R, Devi EL, Zunjare RU, Jaiswal SK, Muthusamy
V (2019) Molecular analysis of mutant granule-bound starch syn-
thase-I (waxy1) gene in diverse waxy maize inbreds. 3 Biotech 9:3
Hovenkamp-Hermelink JH, De Vries JN, Adamse P, Jacobsen E,
Witholt B, Feenstra WJ (1988) Rapid estimation of the amylose/
amylopectin ratio in small amounts of tuber and leaf tissue of the
potato. Potato Res 31:241–246
Jain A, Rao SM, Sethi S, Ramesh A, Tiwari S, Mandal SK, Singh NK,
Modi N, Bansal V, Kalaichelvani C (2012) Effect of cooking on
amylose content of rice. Eur J Exp Biol 2:385–388
Jane JL, Chen YY, Lee LF, McPherson AE, Wong KS, Radosavljevic
M, Kasemsuwan T (1992) Effects of amylopectin branch chain
length and amylose content on the gelatinization and pasting prop-
erties of starch. Cereal Chem 76:629–637
Knutson CA, Grove MJ (1994) Rapid method for estimation of amylose
in maize starches. Anal Tech Instrum Cereal Chem 71:469–471
Li L, Jiang H, Campbell M, Blanco M, Jane J (2008) Characterization
of maize amylose-extender (ae) mutant starches: Part I. Relation-
ship between resistant starch contents and molecular structures.
Carbohydr Polym 74:396–404
Li C, Huang Y, Huang R, Wu Y, Wang W (2018) The genetic archi-
tecture of amylose biosynthesis in maize kernel. Plant Biotechnol
J 16:688–695
Lin F, Zhou L, He B, Zhang X, Dai H, Qian Y, Ruan L, Zhao H (2019)
QTL mapping for maize starch content and candidate gene predic-
tion combined with co-expression network analysis. Theor Appl
Genet 132:1931–1941
13
3 Biotech (2022) 12:62
Lu D, Lu W (2012) Effects of protein removal on the physicochemical
properties of waxy maize flours. Starch 64:874–881
Malhotra SL (1968) Epidemiological study of cholelithiasis among rail
road workers in India. Gut 9:290–295
Mason-Gamer RJ, Weil CF, Kellogg EA (1998) Granule-bound starch
synthase: structure, function, and phylogenetic utility. Mol Biol
Evol 15:1658–1673
Megazyme (2018) For the measurement of the amylose/amylopec-
tin contents of starch. Amylose/Amylopectin assay procedure.
K-AMYL 06/18
Mua JP, Jackson DS (1998) Retrogradation and gel textural attributes
of corn starch amylose and amylopectin fractions. J Cereal Sci
27:157–166
Qi X, Wu H, Jiang H, Zhu J, Huang C, Zhang X, Liu C, Cheng B
(2020) Conversion of a normal maize hybrid into a waxy version
using in vivo CRISPR/Cas9 targeted mutation activity. Crop J
8:440–448
Rohman A, Helmiyati S, Hapsari M, Larasati Setyaningrum D (2014)
Rice in health and nutrition. Int Food Res J 21
Sami W, Ansari T, Butt NS, Ab Hamid MR (2017) Effect of diet on
type 2 diabetes mellitus: a review. Int J Health Sci 11:65
Schoch TJ (1942) Fractionation of starch by selective precipitation with
butanol. J Am Chem Soc 64:2957–2961
Wee MS, Henry CJ (2020) Reducing the glycemic impact of carbo-
hydrates on foods and meals: strategies for the food industry and
consumers with special focus on Asia. Compr Rev Food Sci Food
Saf 19:670–702
Williams VR, Wu WT, Tsai HY, Bates HG (1958) Rice starch, varietal
differences in amylose content of rice starch. J Agric Food Chem
6:47–48
Xia J, Zhu D, Wang R, Cui Y, Yan Y (2018) Crop resistant starch and
genetic improvement: a review of recent advances. Theor Appl
Genet 131:2495–2511
Xiaoyang W, Dan C, Yuqing L, Weihua L, Xinming Y, Xiuquan L,
Juan D, Lihui L (2017) Molecular characteristics of two new waxy
mutations in China waxy maize. Mol Breed 37:27
Yun SH, Matheson NK (1990) Estimation of amylose content of
starches after precipitation of amylopectin by concanavalin-A.
Starch 42:302–305
Zhang B, Bai B, Pan Y, Li XM, Cheng JS, Chen HQ (2018) Effects of
pectin with different molecular weight on gelatinization behavior,
textural properties, retrogradation and in vitro digestibility of corn
starch. Food Chem 264:58–63
Zhang XD, Gao XC, Li ZW, Xu LC, Li YB, Zhang RH, Xue JQ, Guo
DW (2020) The effect of amylose on kernel phenotypic char-
acteristics, starch-related gene expression and amylose inherit-
ance in naturally mutated high-amylose maize. J Integr Agric
19:1554–1564
Zhang O, Liang C, Yang B, You H, Xu L, Chen Y, Xiang X (2021)
Effects of starch synthesis-related genes polymorphism on quality
of glutinous rice. Front Plant Sci 12:1587–1596
Zeng FK, Zhao X, Zhou TH, Liu G (2012) Dual-wavelength colori-
metric method for measuring amylose and amylopectin contents
of potato starch. Mod Food Sci Technol 28:119–122
Zhong Y, Qu J, Blennow A, Liu X, Guo D (2021) Expression pattern of
starch biosynthesis genes in relation to the starch molecular struc-
ture in high-amylose maize. J Agric Food Chem 69:2805–2815
Zhou Z, Song L, Zhang X, Li X, Yan N, Xia R, Zhu H, Weng J, Hao
Z, Zhang D, Yong H (2016) Introgression of opaque2 into waxy
maize causes extensive biochemical and proteomic changes in
endosperm. PLoS One 11:
Zhu T, Jackson DS, Wehling RL, Geera B (2008) Comparison of amyl-
ose determination methods and the development of a dual wave-
length iodine binding technique. Cereal Chem 85:51–58