Accepted Manuscript

Physicochemical properties and starch digestibility of whole grain sorghums,

millet, quinoa and amaranth flours, as affected by starch and non-starch con-
stituents

Sathaporn Srichuwong, Delphine Curti, Sean Austin, Roberto King, Lisa

Lamothe, Hugo Gloria-Hernandez

PII: S0308-8146(17)30589-7
DOI: http://dx.doi.org/10.1016/j.foodchem.2017.04.019
Reference: FOCH 20889

To appear in: Food Chemistry

Received Date: 17 November 2016

Revised Date: 16 March 2017
Accepted Date: 4 April 2017

Please cite this article as: Srichuwong, S., Curti, D., Austin, S., King, R., Lamothe, L., Gloria-Hernandez, H.,
Physicochemical properties and starch digestibility of whole grain sorghums, millet, quinoa and amaranth flours,
as affected by starch and non-starch constituents, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.
2017.04.019

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Title:
Physicochemical properties and starch digestibility of whole grain sorghums,
millet, quinoa and amaranth flours, as affected by starch and non-starch
constituents

Runner-up title:
Characterization of whole grain flours and starches from sorghums, pearl millet,
quinoa and amaranth

Authors:
Sathaporn Srichuwonga, Delphine Curti, Sean Austin, Roberto King, Lisa Lamothe*
& Hugo Gloria-Hernandezb

Nestlé Research Center, PO Box 44, Vers-chez-les-Blanc, CH-1026 Lausanne,

Switzerland

*
Corresponding author:
Tel./fax:+41 217859162
E-mail address: lisa.lamothe@rdls.nestle.com

a
Present address: ICL Food Specialties, Dr.-Albert-Reimann-Strasse 2, 68526,
Ladenburg, Germany
b
Present address: ALINFO Ltda., Food Research and Development, Edmundo
Eluchans 1250, Viña del Mar, Chile

1
Abstract

Minor grains such as sorghum, millet, quinoa and amaranth can be

alternatives to wheat and corn as ingredients for whole grain and gluten-free
products. In this study, influences of starch structures and other grain constituents on
physicochemical properties and starch digestibility of whole flours made from these
grains were investigated. Starches were classified into two groups according to their
amylopectin branch chain-length: (i) quinoa, amaranth, wheat (shorter chains); and
(ii) sorghum, millet, corn (longer chains). Such amylopectin features and amylose
content contributed to the differences in thermal and pasting properties as well as
starch digestibility of the flours. Non-starch constituents had additional impacts;
proteins delayed starch gelatinization and pasting, especially in sorghum flours, and
high levels of soluble fibre retarded starch retrogradation in wheat, quinoa and
amaranth flours. Enzymatic hydrolysis of starch was restricted by the presence of
associated protein matrix and enzyme inhibitors, but accelerated by endogenous
amylolytic enzymes.

Keywords: cereals; pseudocereals; starch; whole grain; physicochemical property

Highlights
• Chemical composition and amino acid profile of whole grain flours were compared
• Physicochemical properties and enzymatic hydrolysis of starch varied with grain
type
• Starch molecular features were the major factors affecting thermal, pasting and
enzymatic hydrolysis properties
• Additional effects of non-starch constituents on whole grain characteristics are
dependent on botanical source.

2
Introduction
Worldwide consumption of products containing whole grain has grown
continuously due to increasing awareness of their health-promoting benefits
(Marquart, Jones, Cohen & Poutanen, 2007; Mintel, 2011: Schaffer-Lequart et
al., 2015). Whole grain flour is more nutritious than refined flour because higher
contents of dietary fibres, micronutrients and phytochemicals from bran and germ
fractions are retained. Currently, common cereals such as wheat and corn are used
as ingredients for production of most whole grain products. Gluten protein fraction in
wheat, however, cannot be tolerated by persons suffering from celiac disease, and
although corn is considered to be safe for celiac patients, it lacks some essential
nutrients (Hager, Wolter, Jacob, Zannini & Arendt, 2012).
Recently, interest in the development for whole grain and gluten-free products
has been growing, and the lesser known grains such as sorghum, millet, quinoa and
amaranth are postulated as promising alternatives for this purpose (Alvarez-Jubete,
Arendt & Gallagher 2010; Taylor & Emmambux, 2008). Research has shown that
these minor grains are rich in several phytochemicals that exhibit antioxidant and
free-radical scavenging activity (Taylor, Belton, Beta & Doudu, 2014). For
centuries, these grains are important staple food crops in Africa, Asia and South
America. In these regions, whole flours made from these grains are also used as
substrates for producing a wide variety of fermented foods and beverages (Hammes,
Brandt, Francis, Rosenheim, Seitter & Vogelmann, 2005; Taylor & Emmambux,
2008). Sorghum and millet are well adapted to drought and harsh climate in the arid
areas where other crops grow poorly. Pseudocereals such as quinoa and amaranth
are known for their superior protein quality containing a high level of lysine that is
limited in cereals. The United Nations recognizes quinoa as the alternative food
source in fighting against hunger and food insecurity (FAO 2013). In modern food
processing, these grains can be used either as a basic raw material or as admixtures
which improve texture, sensory attributes and nutritional value.
Although these minor grains are viewed as promising ingredients for whole
grain-based products, their current utilization in modern food industry is still limited by
availability, price, insufficient product development and research efforts. Due to the
fact that these minor grains differ in several important respects from each other and
from the common cereals, a sound fundamental knowledge on their grain
characteristics and technological properties is required to promote their industrial

3
use. Starch is present as a major component in most cereal and pseudocereal
grains. It plays critical roles on technological properties of cereal flours through its
physicochemical transformation and interaction with other ingredients. For example,
viscosity of starch is one of the key sensory drivers for cereal beverages and
porridges. In extruded cereals and snacks, starch constitutes a continuous
amorphous phase, and other grain components such as proteins and fibres impact
the continuity of the starch phase. In pasta, noodles and baked goods, starch
granules represent the discontinuous phase because they are distributed within a
continuous gluten network (Robin & Palzer, 2015). In gluten-free product, starch
plays important role in providing structure, texture and stability to final products. In
cereal fermentation, the enzymatic conversion of starch plays a key role in
determining primary fermentation substrates (Hammes et al., 2005). In addition to
starch, technological properties of whole grain flour are also affected by flour
preparation, dietary fibres, lipids, micronutrients and phytochemicals.
Improving sensorial and textural properties of whole grain products is a major
challenge for the food industry (Robin & Palzer, 2015; Schaffer-Lequart et al.,
2015). Increased knowledge of structure-function relationship is essential to
overcome this challenge, especially for minor cereals and pseudocereals of which
less is known. Previous studies mainly focused on chemical composition and direct
use of these grains in food formulation to evaluate changes in nutritional and textural
properties of final products (Alvarez-Jubete et al., 2010; Hager et al., 2012).
Research that aims to understand roles of starch structures and other grain
constituents on physicochemical properties of whole grain flours is scarce. In this
study, chemical composition, structural features of isolated starch, thermal and
pasting properties and starch digestibility were determined for whole grain flours
made from sorghum, millet, amaranth, quinoa, in comparison with those of wheat and
corn. The characteristics of whole grain flours are discussed in relation to starch
structures, and non-starch constituents.

2. MATERIALS AND METHODS

2.1 Materials
Grains of white and red varieties of sorghum (Sorghum bicolor), millet
(Pennisetum glaucum) and corn (Zea Mays L) were obtained from Nestlé Research
and Development Center in Abidjan, Ivory Coast. Amaranth (Amaranthus caudatus)

4
and quinoa (Chenopodium quinoa) grains were purchased from Mokamo
Agroexportación, Lima, Peru. According to the supplier, saponin was previously
removed from quinoa grains by washing with water. Wheat grains (Triticum aestivum)
were obtained from Grands Moulins de Cossonay (Cossonay, Switzerland). Grains
were manually cleaned from impurities, and broken grains were removed. The grains
were stored at 4°C and used within 30 days of receipt. Whole grain flour (WGF) was
prepared freshly on the day of analysis by dry-milling the grains through a 0.5 mm
screen using a sample mill (Cyclotec-1093, Tecator, Sweden). Average moisture
contents (% of fresh weight) of WGFs were determined using Halogen moisture
analyzer (Mettler Toledo HR73, Volketswil, Switzerland) at 160°C.
Porcine pancreas α-amylase (E.C. 3.2.1.1) (A6255 type I-A approx. 1050
units/mg protein in saline solution, 29 mg proteins/ml), pepsin from gastric porcine
mucosa (P7000) and protease type XIV isolated from Streptomyces griseus (P5147)
were purchased from Sigma-Aldrich Chemie GmbH (Buchs SG
Switzerland). Amyloglucosidase from Aspergillus niger (EC 3.2.1.3., 3300 units/ml on
soluble starch, stabilised liquid in 50% (v/v) glycerol, 0.02% sodium azide) and
isoamylase from Pseudomonas sp. (EC 3.2.1.68, 500 U/ml in ammonium sulphate
suspension, 0.02% sodium azide) were purchased from Megazyme International
Ireland Ltd. (Wicklow, Ireland). Glucose assay kit (Wako Pure Chemical Industries,
Ltd, Osaka, Japan) based on enzymatic method (Mutarotase-GOD) was purchased
from IGZ Instruments AG (Zürich, Switzerland). All chemicals were reagent grade
and obtained from Sigma–Aldrich Chemie GmbH (Buchs SG
Switzerland).

2.2 Grain composition

Total starch, damaged starch and phytic acid contents of WGF were
determined using enzymatic kits (K-TSTA 07/11 with KOH procedure, K-SDAM 07/11,
K-PHYT 12/12, respectively, Megazyme Ltd.). Protein content was calculated from a
sum of total amino acids determined by ion-exchange liquid chromatography
following the AACC 07-01 and 07-11 methods. Total fat content was determined
using a Soxhlet extraction. Total, soluble and insoluble dietary fibres were
determined according to the AOAC 991.43 and AOAC 2011.25 methods. The AOAC
991.43 employs the use of thermo-stable α-amylase at 95-100°C, pH 8.2. Under this

5
method, some parts of resistant starch and non-digestible oligosaccharides (NDO)
are excluded. In the AOAC 2011.25, starch is hydrolyzed by pancreatic α-amylase at
37°C, pH 6.0 to avoid underestimation of resistant starch, whereas quantification of
NDO are performed using HPLC (McCleary, Sloane, Draga & Lazewska, 2012).
Total phenolic content was determined by Folin-Ciocalteau assay using catechin as a
standard (Chiremba, Taylor, Rooney, & Beta, 2012). Ash content was determined
as a weight loss by high temperature incineration (580°C). For all assays,
determinations were made in duplicate.

2.3 Isolation of starch

Starch was isolated from the grains according to the alkaline-protease wet
milling procedure (Song & Jane, 2000). Residual proteins were removed by
suspending starch pellet in 0.1 M NaCI solution containing 12% v/v toluene under
mechanical stirring for 1 h, and allowed starch to settle. This step was repeated until
the toluene layer was clear. The resulting starch pellet was washed three times with
deionized water, twice with absolute ethanol and dried in a convection oven at 35°C
for 36 h. Isolated starch was stored in a desiccator at room temperature. Starch purity
was > 95% dry basis determined using the total starch kit.

2.4 Scanning electron microscopy

Isolated starches and starch granules embedded in cereal endosperms and
pseudocereal perisperms were observed under a scanning electron microscope
(SEM). For the latter, grains were frozen in liquid nitrogen and fractured along the
transverse axis using frozen hammer or blade. Samples were mounted on aluminum
stubs using a conductive carbon tape. The samples were sputter-coated with a 5 nm
layer of gold using a sputter coater SCD-05 (Leica Microsystems, Wetzlar). The
ultrastructure was visualized in the Quanta 200F FEG microscope (FEI company,
Eindhoven) operated at 8-10kV in low vacuum mode using a secondary electron
detector.

2.5 Amylose content and amylopectin branch chain-length distribution

Amylose content of isolated starch was determined by the lectin concanavalin
A (ConA) method using the assay kit (K-AMYL 07/11, Megazyme Ltd.). Molar-based

6
branch chain-length distribution of amylopectin was determined on isoamylase-
treated starch using fluorophore-assisted capillary electrophoresis (FACE) according
to Srichuwong, Gutesa, Blanco, Duvick, Gardner & Jane (2010).

2.6 Thermal properties

Gelatinization and retrogradation properties were determined using differential
scanning calorimetry (DSC) (Mettler DSC 820, Mettler Instrument, Volketswil,
Switzerland) equipped with a sample robot (TSO801RO). The instrument was
calibrated with indium (28.5 J/g; 156.0°C) and n-decane (202.5 J/g; −29.9°C).
Isolated starch or WGF was weighed in an aluminum pan and added with distilled
water to obtain 33% w/w dry starch equivalent. The pan was sealed and allowed to
stand at room temperature for 1 h before determination. The scanning temperature
range and heating rate were performed at 5 – 110°C and 5°C/min, respectively. An
empty sealed pan was used as a reference. Onset (To), peak (Tp), conclusion (Tc)
temperatures (°C), and enthalpy change (∆H, J/g dry starch) of endothermic
transition were recorded. After the first run, the pan was stored at 4°C for 6 days to
allow starch retrogradation. Endothermic transition of retrograded starch was
determined using the same procedure. Each sample was measured in triplicate.

2.7 Pasting properties

Pasting properties of isolated starch and WGF were determined using Rapid
Viscoanalyzer (RVA model 3D; Newport Scientific Ltd, Sydney, Australia). Isolated
starch or WGF was weighed in a canister and added with distilled water to obtain 8%
w/v, dry starch equivalent (total weight of 28.0 g). The pasting profile was monitored
following the temperature profile: heating from 37 to 95°C at 8.3°C/min (after an
equilibration time of 1 min at 37°C), a holding period at 95°C for 10 min, cooling from
95 to 37°C at 8.3°C /min and a holding phase at 37°C for 20 min. The constant
rotating speed of the paddle was 160 rpm. Influence of disulfide-linked proteins was
evaluated by replacing distilled water with 100mM sodium metabisulfite (SMB)
solution.

2.8 Enzymatic hydrolysis of starch

7
 Isolated starch or WGF containing 100 mg starch (dry basis) was weighed into
50-ml Oak Ridge polypropylene copolymer tubes, and mixed thoroughly with 3.5 ml
distilled water. Protein digestion procedure was modified from Englyst, Hudson,
Cole & Cummings (1999). The starch suspension was kept at 37°C for 5 min and
added with 1.5 ml of pepsin-HCI solution (1.35 % w/w pepsin, 0.05 M HCI, pH 2.0),
and the mixture was incubated at 37°C for 30 min on a magnetic stirrer. The pH was
brought up to ∼6.0 by adding 3.0 ml pH 6.4 maleate buffer (0.1 M, 10 mM CaCl2). To
start starch digestion, 2 ml of enzyme solution (0.1 M maleate buffer pH 6.0, 10 mM
CaCl2) containing 110 units of porcine pancreas α-amylase and 33 units of
amyloglucosidase was added. Starch digestion was performed at 37°C on a
magnetic stirring. This buffer condition for hydrolysis of starch was performed
according to the condition used for determining resistant starch (AOAC 2002.02)
(McCleary et al., 2012). Aliquots of 0.5 ml were taken at selected time intervals, and
immediately added to 1.5 ml cold ethanol solution (90% v/v). The mixture was kept in
an ice bath for 10 min, and then centrifuged (3000×g, 10 min) to separate the
supernatant. The supernatant collected was analyzed for glucose content using the
assay kit. The glucose released from starch hydrolysis was obtained by subtracting
the resulting glucose obtained from the tested sample with values obtained from
blanks (a tube without sample and a tube without enzyme). The resulting glucose
was multiplied by a factor of 0.9 to convert glucose concentration into starch, and
reported as percentage of total starch content (dry basis). The test was also
performed without pepsin in order to investigate the influence of WGF-associated
proteins.

2.9 Data analysis

Linear regression analysis and analysis of variance (ANOVA) by Tukey’s
comparison test (P < 0.05) were conducted using Minitab 17 (Minitab Inc.,
Pennsylvania, USA).

3. Results and Discussion

3.1. Composition of WGF
Table 1 shows that sorghum, millet and corn WGFs contained higher starch
contents (72.2–73.8%) than those of quinoa, amaranth and wheat (66.3 – 68.1%, P <

8
0.05). Damaged starch contents ranged from 4.7-11.7% in which the highest level
was shown for amaranth (17.7%). Physical damage to starch was attributed to the
dry-milling process employed for WGF preparation. Starch damage increases rate of
water absorption and enzyme susceptibility (Robin & Palzer, 2015; Srichuwong et
al., 2010). As shown in Table 1, protein contents of amaranth and wheat WGFs were
comparable (13.2-13.3%) and higher than other grains (9.6-11.4%, P < 0.05). Data
on relative percentages of amino acids confirmed that amaranth and quinoa
contained higher proportions of lysine (6.8 - 6.9%) than cereals (2.2 – 3.1%)
(supplementary data, Table S1). High levels of albumins and globulins would be
responsible for their high lysine content (Fairbanks, Burgener, Robison, Andersen
& Ballon, 1990). Amaranth WGF also showed the highest fat content which may
cause potential shelf-life and quality issues in food products.
As listed in Table 1, dietary fibres obtained from the AOAC 2011.25 method
were higher than those from the AOAC 991.43. This would be due to a higher
recovery of resistant starch and NDO as discussed in the section 2.2. Based on the
AOAC 2011.25, wheat WGF contained higher TDF (14.5%) than other grains (11.5 –
8.4%) (P < 0.05). The SDF contents of 4.1, 3.9 and 3.7% were shown for wheat,
quinoa and amaranth WGFs, respectively, accounting for 28.3, 41.1 and 32.6% of
their TDF contents, respectively. Smaller percentages of SDF in TDF were found in
other cereals (15.0 – 19.4%). Lamothe and co-workers (2015) demonstrated that
IDF and SDF from quinoa and amaranth were mainly pectic polysaccharides and
xyloglucans, unlike those of cereals which were mostly arabinoxylans. Phenolic
contents of red sorghum, millet and quinoa (133.6 – 203.5 mg/100g) were higher
than other grains (92.4 – 110.1 mg/100g) (P < 0.05). The highest levels of ash and
phytic acid contents were shown for amaranth. Our results on chemical composition
of whole flours from the selected grains were comparable to those reported
previously (Alvarez-Jubete et al., 2010; Hager et al., 2012; Kaur, Singh & Rana,
2010).

3.2. Starch structural features

Cellular nature featuring the starch granules embedded in storage protein
matrix was observed in cereal endosperms under SEM (supplementary data, Fig.
S1). Such feature was not clearly observed for pseudocereal perisperms. In sorghum,
millet and corn, starch granules were tightly packed in corneous endosperm

9
contributing to the polygonal granule, while floury endosperm regions contained
starch granules which were loosely-packed and more spherical. Sorghum
endosperms featured denser packing of starch granules within the protein matrices,
which might be responsible for the indentations left by protein bodies, noticeable on
some polygonal starch granules (supplementary data, Fig. S2). Sorghum and corn
starch granules had 5 - 35 µm diameter, which were larger than millet starch with 2 -
15 µm. Wheat starch comprised large, lenticular granules (10 – 40 µm) and small,
spherical granules (2 - 8 µm). Quinoa starch granules were polygonal with 0.3 - 1.7
µm diameter, occurred as single, spherical entities and oblong aggregates (10 - 20
µm). Amaranth starch granules were also polygonal with 0.5 - 2.5 µm diameter.
Figure S2 illustrates surface pores on some granules of sorghum, millet, wheat and
corn starches. The pores were not observed on quinoa and amaranth granules.
Amylopectin is a highly branched glucan polymer and a main component of
starch granule. It is characterized by α(1-4)-linked D-glucose with different branch
chain length connected by α(1-6) linkages, organized into cluster structures. These
branch chains form double helical structures, a basis of semi-crystalline nature of
starch granules. Therefore, amylopectin branch chain-length distribution determines
physicochemical properties and enzyme susceptibility of starch granules (Jane et al.,
1999; Srichuwong & Jane 2007; Srichuwong, Sunarti, Mishima, Isono &
Hisamatsu, 2005). Figure 1 demonstrates that two distinguished distribution
patterns of amylopectin branch chain-length could be classified. The first group
comprising wheat, quinoa and amaranth starches was characterized by a larger
proportional amount of short chains with degree of polymerization (DP) of 6-12
(Table S2) and a distinguished shoulder around DP 18-20. A second group
comprising corn, sorghums and millet starches had smaller proportional amounts of
branch chains DP 6-12 (Table S2) without the distinguished shoulder on their
distribution patterns. Jane and co-workers (1999) suggested that branch chains with
DP 18-20 may represent the proximal length of crystalline regions for most starches,
therefore a relatively high ratio of shorter chains (e.g. DP 6-12) might indicate inferior
crystalline structures. Our findings pointed out that amaranth, quinoa, and wheat
starch granules would comprise inferior crystalline structure to those of sorghums,
millet and corn. Amylose is fundamentally a linear molecule of α(1-4)-linked D-
glucose chains and a minor component in starch granules. Table 1 shows that

10
amylose contents of 24.0 - 25.8% were observed for millet, sorghums, corn, and
wheat starches. Quinoa starch contained 8.4% amylose, whereas amylose was
almost absent in amaranth starch (1.2%). The result supported the mostly
amylopectin nature of amaranth (Kaur et al., 2010). In this study, differences in
starch structural features were shown among starches from different grain sources.
However, it is noted that the variations in starch structures are also reported within
different varieties of sorghum (Beta & Corke, 2001), wheat (Singh, Singh, Isono &
Noda, 2010), quinoa (Lindeboom, Chang, Falk & Tyler, 2005) and amaranth (Sing,
Kaur, Kaur, Isono, Ichihashi & Noda, 2014).

3.3. Gelatinization properties

Endothermic transition corresponding to the loss of double-helices upon starch
gelatinization was analyzed by DSC, and the effects of non-starch components on
gelatinization of starch in WGFs were evaluated. Previous study showed that gluten
from wheat demonstrated negligible or no denaturation peak in DSC (Hoseney,
Zelenak, & Lai, 1986). Little data is available for the thermal property of native
proteins from the minor grains used in this study. Therefore, we believed that the
endothermic transitions observed in WGFs were mainly from starch gelatinization
event.
Table 2 shows that gelatinization temperatures of starch in WGFs shifted to
higher ranges in comparison to those of their isolated starch counterparts, especially
for the Tp and Tc values. The shifting effect was more pronounced in corn, red
sorghum and white sorghum WGFs. Previous DSC study on rye and wheat flours
suggested that water separated into two phases: a protein aggregate phase and a
starch phase, resulting in a shift of starch gelatinization temperature to a higher range
(Wannerberger & Eliasson, 1993). In addition to proteins, dietary fibres might also
contribute to the shifting effect by competing with starch for hydration. Therefore, the
variation in temperature shifting shown among different grains might be attributed to
several factors including the characteristics of proteins and dietary fibres, and a
presence of other hydrophilic/hydrophobic compounds. The ∆H values of starch in
WGFs were smaller than those of isolated starches, which were attributed to a partial
loss of crystallinity during mechanical milling.
Both isolated starches and WGFs from white sorghum, red sorghum, millet
and corn required higher gelatinization temperatures than those of wheat, amaranth

11
and quinoa (P < 0.05, Table 2). The results supported the inferior crystalline
structures of wheat, amaranth and quinoa starches. Linear regression analysis
revealed significant relationships between amylopectin features and DSC parameters.
For isolated starches, relative proportion of short chains DP 6-12 had negative
correlations with To (R2 = 0.89, P = 0.002), Tp (R2 = 0.84, P = 0.004) and Tc (R2 =
0.70, P = 0.018). The longer chains DP 13-24 showed positive correlations with To
(R2 = 0.82, P = 0.005), Tp (R2 = 0.80, P = 0.007) and Tc (R2 = 0.72, P = 0.016).
Significant relationship with ∆H values, however, was not observed. Similar trends
were also observed for WGFs. Figure 2 illustrates the plots of correlation coefficient
(R) values between proportion of respective chain-length and Tp and ∆H values.
Results showed similar plot patterns between isolated starches (Fig. 2A) and WGFs
(Fig. 2B), implying that amylopectin features had a dominant role in determining the
gelatinization property of starch in WGF, although the gelatinization process could be
delayed by the associated proteins and/or dietary fibres. The results agreed well with
the previous study on starches isolated from different botanical sources
(Srichuwong et al., 2005), and starches isolated from large varieties of wheat
(Singh et al., 2010) and amaranth (Sing et al., 2014).

3.4. Retrogradation properties

Short-term developments of gel structure and hardness of cooked starches
are attributed to retrogradation of amylose, while retrogradation of amylopectin
involving re-association of the branch chains occurs at a much slower rate
(Srichuwong & Jane 2007). Long-term retrogradation determines changes in
textural properties and shelf-life stability of many starchy foods. The endothermic
event of dissociation of the retrograded orders was analyzed by DSC. The Tp and ∆H
values describe the perfectness and total quantity of re-associated double helical
orders, respectively. For isolated starches, the Tp (47.9 – 49.2°C) of retrograded
quinoa, amaranth and wheat starches were lower than those of millet and sorghums
(51.8– 52.2°C) (P < 0.05, Table 2). The ∆H values of retrograded sorghums, millet
and corn (6.9-8.3 J/g) starches were also larger than those of wheat (5.9 J/g), quinoa
(4.2 J/g) and amaranth (0.4 J7g) (P < 0.05). The result indicated that amaranth starch
was the most resistant to retrogradation.
For isolated starches, short amylopectin branch chains (DP 6-12) showed
negative correlations with Tp (R2 = 0.96, P = 0.000) and Tc (R2 = 0.91, P = 0.001) of
12
retrograded isolated starches. The longer chains (DP 13-24) had positive correlations
with Tp (R2 = 0.83, P = 0.004) and Tc (R2 = 0.71, P = 0.017). Unlike the gelatinization
event, the ∆H values were also negatively and positively correlated with amylopectin
branch chains (DP 6-12) (R2 = 0.73, P = 0.014) and DP 13-24 (R2 = 0.75, P = 0.012),
respectively, and also positively correlated with amylose content (R2 = 0.87, P =
0.002). The findings suggested that amylose/amylopectin ratio and amylopectin
branch chain-length contributed to both perfectness and quantity of retrograded
starches. Amylose-lipid complexes restrict granular swelling and dispersion; therefore
the starch molecules are more closely located within swollen granules, increasing
opportunities for starch retrogradation (Jane et al., 1999; Srichuwong, Isono, Jian,
Mishima & Hisamatsu, 2012).
The ∆H values of retrograded starch in sorghum, millet, wheat, and corn
retrograded WGFs were 9 – 28% lower than those of the isolated starches.
Interestingly, a large reduction in ∆H values was shown for amaranth, wheat and
quinoa WGFs, i.e. 55, 78 and 72% reduction, respectively (Table 2). The plot
patterns of R values between proportion of respective amylopectin chain-length and
DSC parameters revealed the differences between of WGFs (Fig. 2D) and isolated
starches (Fig. 2C). The results suggested that non-starch constituents also affected
degree of starch retrogradation. A large body of studies has shown that wheat water-
soluble fibres and some hydrocolloids can retard long-term retrogradation of
amylopectin, and this inhibition effect is concentration dependent (BeMiller, 2011). In
this study, the weight ratios of SDF to total starch of wheat, quinoa and amaranth
WGFs (0.056 – 0.060) were about two times higher than those of other grains (0.021
- 0.026). Accordingly, the low retrogradation tendency of starches in wheat, quinoa
and amaranth WGFs would be resulted from their short amylopectin branch chains
and high SDF contents.

3.5. Pasting properties

Amylopectin provides basic molecular structure for granular swelling and
pasting during cooking. Starch with inferior crystalline structure, i.e. amylopectin with
shorter branch chain-length, may display low pasting peak viscosity and large
viscosity breakdown because of its lower interaction in holding granule integrity
(Srichuwong & Jane, 2007). In cereal starches, amylose limits swelling properties
by decreasing amylopectin concentration and/or forming helical complexes with lipid

13
molecules, especially phospholipids. These insoluble complexes often lead to a high
pasting temperature, low viscosity breakdown and the development of an opaque
starch paste (Srichuwong & Jane, 2007). In our previous study, the endothermic
events of amylose-lipid complexes were observed for some minor cereals and
pseudocereals (Robin, Théoduloz, & Srichuwong, 2015).
Figure 3 shows that, among the isolated starches, quinoa had the highest
peak viscosity (2860 cp), followed by sorghums (2240 – 2400 cp), amaranth (2000
cp), corn ~ millet (1850 - 1891 cp), and wheat (1319 cp). Peak viscosity and peak
time had a weak parabolic relationship with amylose content (supplementary data,
Fig. S3A). The amount of amylose-lipid complexes formed during starch pasting may
play a critical role in the resistance of swollen granules against heat and shear force.
In amaranth starches, the inhibitory effects of amylose-lipid complexes would be
absent or negligible because of the low amylose content (1.2%). This would allow
starch granules to swell more freely, resulting in low pasting temperature and high
sensitivity to shear-thinning. For sorghum, corn, millet and wheat starches containing
22.9 – 25.8% of amylose, the impact of amylose-lipid complexes would be more
significant, resulting in restricted granular swelling, lower peak viscosity and smaller
breakdown. For quinoa starch having 8.2% of amylose, the swollen granules might
be stabilized by a moderate effect of amylose-lipid complexes. This characteristic
together with its very small granule size might be responsible for the highest viscosity
and smallest breakdown observed for quinoa starch. For WGFs, a similar parabolic
trend was also found between RVA parameters and amylose contents
(supplementary data, Fig. S3B). The parabolic relationship was also reported
between amylose content and expansion volume of extruded corn starches
(Chinnawasmy & Hanna, 1988). This might also explain our previous finding on the
lower expansion property of extruded amaranth WGF compared to those of sorghum
and quinoa (Robin et al., 2015).
As shown in Figure 3, sorghum WGFs were characterized by smaller peak
and breakdown viscosities compared to its isolated starch. Hamaker and co-
workers (1987) reported that disulfide-bonds between oligomeric proteins formed
during wet-cooking of sorghum flours may reduce degree of starch swelling. The
disulfide-bond formation can be prevented or cleaved by reducing agents such as
sodium metabisulfite (SMB). Figure 3 shows that peak and breakdown viscosities of
white and red sorghum WGFs were increased with SMB treatment, supporting the

14
inhibitory effect of disulfide-linked proteins. Peak viscosities of quinoa and amaranth
WGFs were not affected by SMB addition. Their WGFs had lower peak viscosities
than the isolated starches. This might be attributed to an activation of grain-
associated endogenous amylolytic enzymes, which have been reported in several
studies (Hager, Mäkinen & Arendt, 2014; Kaur et al., 2010; Lorenz & Nyanzi,
1989). Our preliminary study also showed higher activities of endogenous amylolytic
enzymes in WGFs of quinoa and amaranth (unpublished data). The presence of
additional amylolytic activity from endogenous enzymes might affect the
quantification of damaged starch for these grains in which the assay was also
performed enzymatically (K-SDAM 07/11, Megazyme Ltd.). Unlike others, wheat
WGF displayed a slightly lower pasting temperature and higher peak viscosity than
its isolated starch. It was likely that the damaged starch granules in wheat WGF
could directly increase hydration rate to granule interior, bypassing the extensive
inhibitory effect of amylose-lipid complexes which was dominant in isolated wheat
starch.

3.6 Enzymatic hydrolysis of starch

In cereal fermentation, type of grain plays a key role in determining quantity
and quality of carbohydrates as primary fermentation substrates, nitrogen sources
and other growth factors for microorganism. A conversion of starch to fermentable
sugars is resulted from actions of grain-associated endogenous, exogenous and/or
extracellular amylases of lactobacilli (Hammes et al., 2005; Gänzle & Follador,
2012).
In this study, influences of grain components on enzymatic hydrolysis of starch
to glucose were evaluated. Figure 4 illustrates the starch hydrolysis (%) of isolated
starches and WGFs. Without pepsin pre-incubation step (Fig. 4A), at 240 min,
amaranth and quinoa starches were more readily hydrolyzed to glucose (90%) than
wheat (79.5%) and other starches (60-67%). The results supported that inferior
crystalline structures of quinoa, amaranth and wheat starches were more susceptible
to enzyme catalytic activities (Srichuwong et al., 2005). Amylose content, granule
size and a presence of less organized zones of starch granule e.g. surface pores
may also influence rate of starch hydrolysis to some extent (Srichuwong & Jane,
2007). Figure 4B shows that inclusion of pepsin pre-incubation did not largely modify
digestion patterns among isolated starches. A slight increase in hydrolysis extent,

15
observed for some starches might be attributed to the removal of surface proteins by
pepsin (Bhattarai, Dhital & Gidley, 2016).
For WGFs, other factors including physically damaged starch granules,
proteins, lipids, endogenous enzymes, inhibitors, and phytochemicals may have
additional impact on enzyme-substrate accessibility and available enzyme activities.
As shown in Figure 4C and D, the digestion patterns of WGFs differed from those of
isolated starches. Without pepsin pre-incubation (Fig. 4C), quinoa and amaranth
WGFs were more rapidly hydrolyzed to glucose than their isolated starches (Fig. 4A),
achieving > 90% within 120 min. This might be due to the additional activity of
endogenous amylolytic enzymes included in these grains. With pepsin pre-incubation
(Fig. 4D), their hydrolysis extents were reduced to similar levels shown for the
isolated starches (Fig. 4B), indicating that the endogenous enzymes were
deactivated by pepsin. Without pepsin pre-incubation, sorghum WGFs had lower
starch digestibility (48-52% at 240 min) than their isolated starches (60-62% at 240
min). Figure 4D shows that starch digestibilities of millet and sorghum WGFs were
increased after pepsin treatment. This would be due to the actions of pepsin in
breaking down the dense protein matrix associated with starch granules, allowing
more enzyme accessibility to starch substrates.
Corn WGF showed higher starch digestibility than its isolated starch, and its
digestibility was not largely modified by pepsin. This would be attributed to the loosely
adhesion of storage proteins to starch granules as observed in the SEM (Fig. S1). As
shown in Fig. 4C, without pepsin pre-incubation, starch in wheat WGF was highly
resistant to enzymes in which only 16% was hydrolyzed at 240 min. Inclusion of
pepsin, however, drastically increased the hydrolysis extent to 79% (Fig 4D). The
limited enzymatic hydrolysis of starch in wheat WGF might be attributed to the
presence of proteinaceous amylolytic inhibitors which were widely reported in refined
and whole wheat flours (Oneda, Lee & Inouye, 2004; Silano 1978). These proteins
showed inhibitory effects on several α-amylases including porcine pancreas α-
amylases at optimal pH 5.3 - 6.0 and 30 - 37°C. Pepsin incubation and heat
treatment deactivated their inhibitory activities. Another study suggested that a
physical binding of enzyme on surface of wheat gluten may reduce available enzyme
activity for starch digestion (Bhattarai et al., 2016). It is noted that hydrolysis of
protein matrix by pepsin or other protease may release phytochemicals, such as
phenolic compounds and phytic acid, from the grain matrix. At certain concentrations,
16
these compounds could inhibit α-amylase activity and reduce rate of starch digestion
to some extent (Deshpande & Cheryan 1984; Thompson & Yoon, 1984).
Consequently, the enzymatic hydrolysis of starch in WGF was resulted from
interplays of several factors including starch structures, protein matrices, endogenous
enzymes, enzyme inhibitors and phytochemicals. Influences of these factors are
grain-specific and would be further varied with degree of grain comminution,
additional proteolytic enzymes and heat treatment.

4. Conclusions
Minor cereals and pseudocereals are considered as emerging food ingredients
for several food applications. Whole grain flours made from these grains have
different chemical composition, physicochemical properties and starch digestibility.
The present study showed that the starch structural features contributed largely to
physicochemical properties of each grain type. The additional effects of non-starch
components were also grain-specific. Protein and soluble fibre could delay
gelatinization and retrogradation of starch in WGF, respectively. Pasting property and
starch digestibility of WGF were influenced by several factors including associated
proteins, endogenous enzymes and proteinaceous amylolytic inhibitor. This study
provides fundamental understanding on chemical and physicochemical properties of
whole grain flours from sorghum, millet, amaranth and quinoa.

Acknowledgement

The authors would like to thank Martine Rouvet and Bertrand Schmitt for their
great support on scanning electron microscopy, Charlotte Gancel and Christine
Théoduloz for analytical support.

17
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 Table 1 Composition of whole grain flours*

Whole grain flour (WGF)

Composition White Red Whe Quin Amaran
sorghum sorghum Millet Corn at oa th
Moisture (g/100g fresh 9.8b, 11.1a,
wt.) 7.3d 7.5d 7.1d 9.3c c b
11.2a
b
Total starch (g/100g 72.6
dm**) 72.2c 73.4a, b 73.8a ,c
68.1d 66.8e 66.3e
a, a
Amylose 24.0 25.2
(g/100g starch dm) 25.8a 24.6 a, b b
22.9b ,b
8.4c 1.2d
c, a,
Damaged starch 7.3 10.6
(g/100g dm) 10.1a, b 8.1b, c 4.7d 8.2b, c d b
11.7a
Total protein (g/100g
dm) 11.4b 9.8c 9.6c 9.8c 13.2a 11.7b 13.3a
Total fat (g/100g dm) 5.0b, c 3.7d 4.1c, d 4.2c, d 2.5e 5.4a, b 6.5a
Dietary fibre (g/100g
dm)***
AOAC 2011.25
9.1a,
c, d b, c b, c b
IDF 6.9 8.1 7.9 10.4a 5.6d 7.7b, c
SDF 1.5b 1.9b 1.9b 1.6b 4.1a 3.9a 3.7a
10.7b
TDF 8.4c 10.0b, c 9.8b, c ,c
14.5a 9.5b, c 11.4b
AOAC 991.43
IDF 5.2c, d 5.7c, d 6.5c 8.0b 9.8a 4.9d 5.5c, d
SDF 0.6b, c 1.0b 0.9b, c 0.3c 1.7a 2.1a 1.8a
TDF 5.8c 6.7b, c 7.4b, c 8.3b 11.4a 7.0b, c 7.3b, c
1.6b,
Ash (g/100g dm) 1.9a, b, c 1.4b, c
1.5b, c
1.2 c c
2.5a, b 2.9a
b, b
0.91 1.12
Phytic acid (g/100g dm) 0.91b, c 1.09b, c c
0.74c ,c
1.44b 2.06a
Polyphenols (mg/100g 177.6 110. 133.6
dm) 95.2e 203.5a b
1d 92.4e c
97.2e

* Values in the same row followed by the same letter are not significantly different (P
< 0.05)
** dm = dry matter basis
*** TDF = total dietary fibres; IDF = insoluble dietary fibres; SDF = soluble dietary
fibres

22
Table 2 Thermal properties of isolated starches and WGFs *, **

gelatinization of starch dissociation of retrograded starch

To Tp Tc ∆H ∆H
Source (°C) (°C) (°C) (J/g) To (°C) Tp (°C) Tc (°C) (J/g)

Isolated
starch
35.7a, b,
a a a b c
White sorghum 71.3 74.9 82.6 11.0 52.2a 70.9a 8.3 a
69.7a,
b
Red sorghum 73.2b 81.2a 11.2b 38.7a 52.0a 69.0b 6.9b
72.9b, 36.4a, b,
Millet 68.6b c
82.7a 10.5b c
51.8a 67.6b 8.1a
Corn 66.4c 71.4 c
80.9a 10.4b 33.9 c
50.3b 65.5c 6.9b
Wheat 54.2d 60.8e 69.6d 8.5c 35.0b, c 49.2b, c 63.5d 5.9c
36.2a, b,
Quinoa 53.9d 60.6e 66.0c 10.3b c
47.9d 61.7d, e 4.2d
Amaranth 55.5d 63.5d 75.8b 12.4a 38.1a, b 48.3c, d 61.1e 0.9e

WGF
78.0a,
White sorghum 71.9b b
90.2a 8.3c, d 38.3a 52.7a 68.6a 6.0b
88.9a,
Red sorghum 74.6a 79.2a b
8.9c 37.7a 52.5a 69.5a 6.3b
78.1a,
Millet 72.7b b
87.4b 10.0b 37.1a 52.7a 66.7b 7.2a
Corn 66.3c 76.7 b
90.2a 8.4c, d 38.0a 52.3a 67.2b 5.3c
Wheat 58.1d 65.6d 75.2d 7.9d 37.7a 51.5b, c 63.1c 1.7d
Quinoa 57.4d 66.0d 72.7e 8.4c, d 34.3b 48.7c 61.8d 1.2d
Amaranth 58.3d 67.9c 79.9c 10.9a 35.4b 50.2b, c 61.1d 0.4e

* To = onset temperature, Tp = peak temperature, Tc = conclusion temperature, and

∆H = enthalpy change
** Values in the same column of each material type (isolated starch or WGF) followed
by the same letter are not significantly different (P < 0.05)

23
Figure(s)
Figure 1

7
Relative molar distribution (%)

5 White sorghum
Red sorghum
4 Millet
Corn
3 Wheat
Quinoa
2 Amaranth

0
0 10 20 30 40 50 60 70

Degree of polymerization (DP)

Correlation coefficient (R) Correlation coefficient (R)
Figure 2
A B

Correlation coefficient (R) Correlation coefficient (R)

C D
 Figure 3

(A) (B) (C) (D)

(E) (F) (G)

Figure 4
Highlights
• Chemical composition and amino acid profile of whole grain flours were compared
• Physicochemical properties and enzymatic hydrolysis of starch varied with grain
type
• Starch molecular features were the major factors affecting thermal, pasting and
enzymatic hydrolysis properties
• Additional effects of non-starch constituents on whole grain characteristics are
dependent on botanical source.

24