Huaya (Melicoccus bijugatus) seed flour

as a new source of starch: physicochemical,

morphological, thermal and functional
characterization

Víctor M. Moo-Huchin, Dulce M. Ac-

Chim, Yasser A. Chim-Chi, Carlos
R. Ríos-Soberanis, Gonzalo Ramos,
Hernani T. Yee-Madeira, et al.
Journal of Food Measurement and
Characterization

ISSN 2193-4126

Food Measure
DOI 10.1007/s11694-020-00573-3

1 23
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 Author's personal copy
Journal of Food Measurement and Characterization
https://doi.org/10.1007/s11694-020-00573-3

ORIGINAL PAPER

Huaya (Melicoccus bijugatus) seed flour as a new source of starch:

physicochemical, morphological, thermal and functional
characterization
Víctor M. Moo‑Huchin1   · Dulce M. Ac‑Chim2 · Yasser A. Chim‑Chi2   · Carlos R. Ríos‑Soberanis3   ·
Gonzalo Ramos4 · Hernani T. Yee‑Madeira5   · Alejandro Ortiz‑Fernández2 · Raciel J. Estrada‑León2   ·
Emilio Pérez‑Pacheco2 

Received: 14 May 2020 / Accepted: 21 July 2020

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
The huaya seed, considered as fruit waste, represents 40% of the total weight of this tropical fruit that is highly consumed
in the Yucatan Peninsula, Mexico. Starch isolated from the fruit seed flour has becoming an alternative to the use of
non-conventional sources starches. The present study aims to isolate starch from huaya seed flour and characterize it by
its physicochemical, structural, thermal and functional properties. The starch yield was 39.38% containing low levels of
protein, lipids, ash and fiber. Starch was characterized by exhibiting an average size of 14.54 µm presenting an oval shape
without cracks, high amylose content and high content of resistant starch (RS). Additional analysis shown that starch pos-
sesses a more ordered microstructure (C-type starch and ratio 1047/1022 cm−1 and 1022/995 cm−1), high decomposition
and gelatinization temperatures, and perhaps these results explain the low swelling capacity of the granule and its low rate
of enzymatic hydrolysis. According to the results, this starch owns the potential to be used in non-transparent foods that
require high temperatures (such as baking), for the design of products with a lower glycemic index and lower caloric value,
and additionally, for the manufacture of bioplastic films for applications as wrappings in food industry.

Keywords  Starch · Physicochemical properties · Tropical fruit · Melicoccus bijugatus · Huaya

Introduction

* Emilio Pérez‑Pacheco Starch is stored as semi-crystalline granules in cereal grains,

eperez@itescam.edu.mx
1
Tecnológico Nacional de México, Campus Instituto
Tecnológico de Mérida, km 5 Mérida‑Progreso,
C.P. 97118 Mérida, Yucatán, Mexico
2 tant in both food (thickener, stabilizer, emulsifier, water
Tecnológico Nacional de México, Campus Instituto
Tecnológico Superior de Calkiní, Cuerpo Académico
Bioprocesos, Av. Ah‑Canul, C.P. 24900 Calkiní, Campeche,
Mexico
3
Centro de Investigación Científica de Yucatán, A.C,
Unidad de Materiales, Calle 43, No. 130 x 32 y 34, Colonia
Chuburná de Hidalgo, C.P 97205 Mérida, Yucatán, Mexico
4
Instituto Politécnico Nacional, Centro de Investigación en
Ciencia Aplicada y Tecnología Avanzada, Unidad Querétaro,
Cerro Blanco 141, Col. Cimatario, 76090 Querétaro, Mexico
5
Instituto Politécnico Nacional, Escuela Superior de Física y
Matemáticas, Av. Instituto Politécnico Nacional, S/N, San
Pedro Zacatenco, Gustavo A. Madero, C.P. 02200 Mexico,
México D.F., Mexico
roots, tubers, stem-piths, leaves, seeds, fruits and pollen.
Starch structure consists of linear amylose and branched
amylopectin fractions. These fractions are responsible for its
crystalline and amorphous properties. Starch is very impor-
retention and gelling agent) and non food industries (paper
manufacture, laundering agent, plywood industries, plastic
manufacturing and pharmaceutical industry).
Nowadays, new starch sources with different physico-
chemical properties are required. New industrial applications
require specific properties, such as high viscosity and trans-
parent gels [1] and these properties depend on their origin:
corn, wheat, potatoes and rice. Corn is commonly used for
starch isolation in the United States, however, in Mexico,
corn is the basis of food for most of the population. Isolation
starch from Mexican corn is impractical, because national
production is insufficient to meet human consumption needs

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 Author's personal copy
[2]. Consequently, alternative starch sources are needed from
outside the standard range of cereal grains and tubers. The
study of non-conventional sources of starch has increased
to extend the variety of industrial use. Starches from green
banana (Musa paradisiaca), mafafa (Xanthosoma robus-
tum) and ramon (Brosimum alicastrum), have shown dif-
ferent physicochemical, structural and functional proper-
ties [1–3]. However, there are new sources that have not yet
been studied, such as huaya seeds (Melicoccus bijugatus).
Huaya fruit (also known as Mamoncillo fruit) is a globose
drupe, ovoid, which grows in compact clusters, as found in
southeast of Mexico. Peel is strong, rough, green and have
been shown to modulate of acetylcholinesterase activity
[4]. Seeds occupy the most of the surface in the fruit, but
are considered as huaya fruit waste, however, it can contain
significant amounts of starch that can be better explored.
These seeds are covered by a salmon-colored mesocarp and
when fully ripe, the edible pulp is pleasantly acid-sweet. The
sweet and astringent fruit pulp is usually consumed fresh and
occasionally made into jelly, pies, or cold drinks [5].
Huaya fruit production and consumption are important
economic activities for the rural areas of the southeast of
Mexico, specifically, the Yucatan Peninsula. According to
Moo-Huchin et al. [6], huaya fruit exhibits 38.7% yield in
edible portion, 83.67% moisture, 13.9°Brix, 1.87% acidity,
Fig. 1  a Micrograph of native huaya starch. b Average size of huaya granules. c Huaya seed
13
V. M. Moo‑Huchin et al.
is abundant in dietary fiber, vitamin C, phenolic compounds,
flavonoids, carotenoids and antioxidant activity. However,
the isolation and characterization of starch obtained from the
huaya seeds has not been reported. The use of new non-con-
ventional starch obtained from the huaya seeds may increase
the desired properties of value added food products. There-
fore, in the present study, the aim was to characterize the
physicochemical, structural, thermal and functional proper-
ties of starch isolated from huaya seed flour. The informa-
tion obtained will help to define the effective applications of
starch from huaya seed flour in industrial sectors.
Material and methods
Materials
Huaya fruits were collected in the northern region of Campe-
che State, located on the Yucatan Peninsula, Mexico during
July 2019 (Fig. 1c). At the moment of harvesting, the fruits
presented an average weight of 11.70 ± 0.90 g, average diam-
eter of 26.70 ± 0.71 mm and °Brix/acidity ratio = 7.40 ± 0.02.
Fruits without blemishes or signals of damage were selected
to obtain the seeds by removing the peel and edible pulp by
hand. Huaya seeds were dried in a convection oven (Shell Lab
 Author's personal copy
Huaya (Melicoccus bijugatus) seed flour as a new source of starch: physicochemical,…
1350FX-10) at 40 °C for 72 h and then, they were stored in a
desiccator. Seeds were milled in a commercial blender in 10 s
time intervals and sifted through 100-mesh screen to produce
flour. This flour was stored in hermetically sealed glass con-
tainers until further use.
Starch isolation
Native starch (NS) was extracted from huaya seed flour. A dis-
persion containing sodium bisulfite (Sigma-Aldrich, 243973)
and sodium hydroxide (Sigma-Aldrich, S5881) was used. The
suspension was sifted, washed, and centrifuged until polysac-
charide was obtained [3]. After isolation, NS was dried in a
vacuum furnace for 12 h using 1.33 Pa at 40 °C to avoid starch
damage and sifted through a 100-mesh screen. Finally, the
isolated starch was stored in hermetically sealed glass contain-
ers. The amount of recovered starch was calculated according
to Eq. 1:
(WIF) (PS)
RS = × 100
WF
where WIF denote the weight of the isolated fraction (%
dw), PS denote the purity of starch, WF denote the weight
of the huaya seed flour (% dw) and RS denote the percentage
of recovery starch.
Proximal chemical‑composition
Proximal chemical composition of raw flour and NS obtained
from huaya was determined in triplicate, according to AOAC
methods for moisture (925.10), ash (923.03), proteins
(920.87) and lipids (920.39) [7]. Crude fiber content was
determined by acid–alkali digestion [8], total carbohydrate
as nitrogen-free extract (NFE) was calculated by difference,
additionally, pH was determined using a Metrohm 827 poten-
tiometer in dispersion at 1% (w/v, in distilled water) at room
temperature.
Color determination
The color of NS was measured in triplicate using a Hunter
Lab MiniScan EZ colorimeter. The color was expressed
in the CIELAB space as L* (lightness, from 0 = black to
100 = white), a* (+a = redness, −a = greenness) and b*
(+b = yellowness, −b = blueness) and the final result was
expressed as hue angle (h) with Eq. 2 and chromaticity C*
(intensity of the tone) was calculated with Eq. 3.
b∗
h = tan−1
a∗
√
C ∗= (a ∗)2 + (b ∗)2
Apparent amylose content
Amylose content was determined according to Ratnayake
et al. [9]. This analysis consists of solubilizing the huaya
starch in dimethyl-sulfoxide after which it is exposed in an
iodine solution. The apparent amylose content was deter-
mined from a standard curve prepared, using mixtures of
pure potato amylose and amylopectin solutions (over the
range 0–100% amylose) and expressed as percentage. The
sum of amylose and amylopectin corresponds to 100% of
starch. Amylopectin content was calculated by the difference
at 100% of amylose content using the colorimetric method
of Morrison, Laignelet [10].
Total starch
Total starch was determined using the starch assay kit
(Sigma-Aldrich, STA20), which is based on the hydrolysis
of starch to glucose catalyzed by α-amylase and amyloglu-
(1) cosidase. Glucose is oxidized to gluconic acid and hydrogen
peroxide by glucose oxidase. Hydrogen peroxide reacts with
ο-dianisidine in the presence of peroxidase to form a colored
product. Oxidized ο-dianisidine reacts with sulfuric acid to
form a stable colored product. The intensity of the pink color
measured at 540 nm is proportional to the original glucose
concentration. The analyses were performed according to
the instructions supplied with the kits.
Scanning electron microscopy (SEM) and particle
size
The morphological characteristics of NS were observed
using a SEM. Starch samples were mounted on a metallic
slide and the examination was performed with a scanning
electron microscopy JEOL JSM 6360 LV electron probe
microanalyzer at 15 kV in low vacuum. Subsequently, the
starch was suspended in an appropriate volume of distilled
water and placed in a Beckman Coulter LS100Q laser dif-
fraction particle size analyzer.
Differential scanning calorimetry (DSC)
Starch gelatinization was measured using a DSC-6 (Perki-
neElmer Corp., Norwalk, CT) according to Estrada-León
et  al. [11]. Approximately, 1  mg of huaya starch was
weighed into an aluminum sample pan. Water (3 µl) was
added by using a microsyringe to obtain a starch:water
ratio of 1:3 (w/w) in the DSC pans, which were sealed and
left at room temperature for 1 h. Pans were heated from
(2)
25 to 110 °C (temperature increased at a rate of 10 °C/
min). Sample chamber was flushed with nitrogen to avoid
(3)
moisture condensation. An empty aluminum pan was used
as the reference. The onset (­ To), peak (­ Tp) and conclusion
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temperatures ­(Tc) were recorded. The enthalpy change of the
thermal transition (ΔHgel) was estimated by integrating the
area between the thermogram and a base line under the peak,
and was expressed as Joules per gram dry weight of starch.
Fourier transform infrared (FTIR) spectra
The FTIR spectra of NS was determined by using FTIR
Spectrophotometer (Thermo Nicolet, Nexus 670-FTIR, UK).
The scanning range of measurement was 400–4000 cm−1.
Samples were prepared by grinding the solid powder starch
with 200 mg potassium bromide (KBr) powder (1:100) and
then pressing the mixture into a tablet for the IR analysis.
X‑ray diffractometry and relative crystallinity
The X-ray diffraction patterns of amylose (Sigma-Aldrich,
A0512, USA), amylopectin (Sigma-Aldrich, 10120, USA)
and huaya isolated starch were studied. For the analysis, a
Bruker D5005 wide-angle X-ray diffractometer equipped
with a copper source operating at 35 kV and 15 mA produc-
­ uKα radiation with a wavelength of 1.54 Å and from
ing a C
5° to 35° on a 2θ scale with a step size of 0.02° was used.
The relative crystallinity of the starch granules was calcu-
lated by using Eq. 4.
Relative crystallinity (%)
Crystalline area
= × 100
Amorphous area + Crystalline area (4)
Swelling power and solubility
The procedure of Rafiq et al. [12] was used for determin-
ing swelling power (SP) and solubility (IS) of huaya starch.
These analyses were measured over a temperature range
between 60 and 90 °C. Starch slurry (1 g/100 mL, starch
dw) was placed in centrifuge tubes and heated in water
bath at different temperatures like 60, 70, 80 and 90 °C for
30 min with regular vortexing after every 5 min. The tubes
were centrifuged after cooling at 2500 rpm for 15 min so
the supernatant was decanted in pre-weighed petri dish and
evaporated at 105 °C for 12 h, finally, dried supernatant were
weighed to calculated the IS. The sediment after centrifuga-
tion was weighed for swelling power estimation (SP). SP and
IS were calculated with Eqs. 5 and 6, respectively:
WDS(g)
IS(%) = × 100 (5)
SW(g)
WS(g)
SP(g∕g) = (6)
SW(g) − WSS(g)
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V. M. Moo‑Huchin et al.
where IS denote the percentage of solubility, WDS denote the
weight of dried supernatant, SW denote the sample weight,
SP denote the swelling power, WS denote the weight of wet
sediment and WSS denote weight of solid soluble.
Starch gel clarity
Starch gel clarity was measured using the method of Perera,
Hoover [13]. 1% aqueous suspension of huaya starch was
heated within a water bath at 90 °C for 1 h at constant stir-
ring. Then, the starch paste obtained was cooled for 1 h at
30 °C. The samples were stored for 6 days at 4 °C and the
percentage of transmittance (%T) was determined every 24 h
(after homogenization) at 640 nm using the spectrophotom-
eter UV–Vis Perkin Elmer Lambda 11 against a water blank.
In vitro digestibility
The in vitro digestion of huaya starch was carried out fol-
lowing the methodology proposed by Bello-Perez et al. [14].
The digestible fractions were estimated by the Englyst’s
method [15]. The starch classification based on its digest-
ibility was: RDS as the starch that was hydrolyzed within
20 min of incubation, RS as the starch not hydrolyzed with
120 min, and SDS as the starch digested during the period
between 20 and 120 min.
Statistical analysis
All the experiments were performed at least in triplicate and
results are presented as mean ± SD (standard deviation). Dif-
ferences among means from each determination (proximal
chemical composition, swelling power, solubility, starch gel
clarity and in vitro digestibility) were evaluated by one-way
analysis of variance (ANOVA) by Tukey Test (P ≤ 0.05),
using the statistical package Statgraphics Plus software, ver-
sion 2.1 (Manugistic, Inc., Rockville, MD, USA).
Results and discussion
Chemical proximate analysis
The proximal chemical composition of raw flour and NS
are summarized in Table 1. Chemical composition of huaya
starch is significantly different (P ≤ 0.05) from raw flour.
Huaya starch exhibited lower (P ≤ 0.05) amount of ash,
lipid, fiber and protein as compared to raw flour, which is
due to the isolation starch process. Lower amount of chemi-
cal compounds indicate the purity of isolated starch. The
moisture content of the raw flour (7.39 ± 0.16%) and the
huaya starch (8.59 ± 0.02%) are within the accepted values
 Author's personal copy
Huaya (Melicoccus bijugatus) seed flour as a new source of starch: physicochemical,…
Table 1  Proximal chemical composition of raw flour and native starch from huaya seed flour (% dw)
Sample Moisture Ash (%) Lipids (%)
(%)
Raw flour 7.39 ± 0.16a 2.99 ± 0.09b 1.70 ± 0.08b
Huaya starch 8.59 ± 0.02b 0.24 ± 0.02a 0.69 ± 0.01a
Values are expressed as mean ± standard deviation (n = 3); Values within a column with different superscripts are significantly different at
P ≤ 0.05
Dw dry weight, nd no detected
for dry powdered products (< 15%). In addition, the mois-
ture percentage of huaya starch is lower than the suggested
values (< 20%) for other starches obtained from conventional
sources such as potatoes [16].
The ash content in the huaya starch is consistent with that
reported for yam starch (Dioscorea alata) by Zhu [17], with
a value of 0.24 ± 0.02%. Similarly, values above 0.5% are
considered as an indication of deficiencies in the extraction
process. In another result, lipids contained in huaya starch
have been reduced by 60%, compared to raw flour. Lipids are
part of the minor components of commercial starches and
are found in starch granules conforming organic complexes
with the amylose. It is known that lipids modify the proper-
ties of starch [18].
Crude fiber was also reduced by 85% compared to raw
flour. This is another indication of the efficiency of the
extraction process. This value is lower than that reported by
Witono et al. [19], for Canna edulis starch, which reports
1.4% content of fiber. Proteins were not detected in the
native starch. It has been reported that protein content is
dependent on the source of starch and the extraction proce-
dure [18]. The use of native huaya starch is suggested for the
production of high glucose syrup, since Maillard reactions
could not occur. For syrup production, the maximum protein
limit is 0.4% [20].
The proximal composition of huaya starch reported in
this work is similar to that reported for starches that were
isolated from other seeds sources such as ramón (B. alicas-
trum) and parota (Enterolobium cyclocarpum) [3, 11, 21].
However, the dissimilarities between such seeds are mainly
due to the vegetable source and the isolation processes used.
Morphology of starch granules
Morphological characteristics of the huaya starch were
observed by SEM (Fig. 1a).
Huaya starch granules (size ranged from 4.00 ± 0.10 to
28.00 ± 0.20 µm in Fig. 1b) are oval in shape and the sur-
faces of all granules appeared smooth and showed no evi-
dence of fissures or ruptures, comparable to the parota (E.
cyclocarpum) and Ramón (B. alicastrum) starch granules
[11], which were also starches extracted under the same
Crude fiber (%) Proteins (%) Nitrogen-free extract (%)
1.70 ± 0.07b 0.54 ± 0.02 85.68 ± 2.58a
0.26 ± 0.02a nd 90.22 ± 0.52b
conditions. Smooth surfaces with no cracks suggest purity
in starch extraction, but morphological characteristics may
vary according to the cultivar, plant growth, environmental
conditions and methods of extraction and purification [22].
Differences in starch granule size is attributed to the biologi-
cal source, since starch biosynthesis induces natural vari-
ability in amylose and amylopectin molecules, which may be
responsible for granular diversity [23]. The average granule
diameter was 14.54 ± 0.95 µm, in comparison smaller than
the potato starch granules with 27 µm [24]. According to
Lindeboom et al. [23], starch granules are classified into
three groups: large (> 25 µm), medium (10–25 µm) and very
small (< 5 µm), so the granules of the huaya starch belong
to medium size group (Fig. 1b). Starch granule size and
size distribution have been shown to affect physicochemi-
cal properties such as solubility, enzyme susceptibility and
crystallinity [25].
Physicochemical characteristics
The physicochemical characteristics of the huaya starch are
shown in Table 2. The purity of the extracted starch was
98.01 ± 0.50% with a 39.38 ± 1.00% yield. These results are
comparable to those reported by Estrada-León et al. [11], for
an E. cyclocarpum starch extracted under similar conditions
to those settled in this study. The amylose content present
(39.13 ± 0.35%) classifies the starch as normal type, accord-
ing to [26, 27]. It is noteworthy that the amylose content is
comparable than that reported for Jackfruit seed starch [28,
29], who report 32.14% and it is proposed as a thickener
and stabilizer in chilli sauce. Consequently, the amylopectin
content will be similar than those reported by these authors.
The amylose/amylopectin ratio provides information about
the functional properties of starch: amylose conforms and
confers stability to gels while amylopectin contributes to
viscosity. Based on the results obtained (amylose/amylopec-
tin = 0.64 ± 0.01), this starch would be feasible to be used in
products that require high viscosity as a thickening agent,
since it displays a low retrogradation tendency [30].
The measured pH was 6.70 ± 0.10, which indicates a neu-
tral pH suspension. This value is similar to that reported by
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V. M. Moo‑Huchin et al.

Table 2  Physicochemical characteristics of the huaya starch

Parameter Native starch

Starch yield (% dw) 39.38 ± 1.00

Total starch (% dw) 98.01 ± 0.50
RDS (%) 17.96 ± 1.80
SDS (%) 21.24 ± 1.05
RS (%) 59.59 ± 0.56
Amylose (%) 39.13 ± 0.35
Amylopectin (%) 60.87 ± 0.36
Amylose/amylopectin ratio 0.64 ± 0.01
pH 6.70 ± 0.10
L* 83.41 ± 0.50
a* 2.40 ± 0.03
b* 8.70 ± 0.02
Hue angle 74.70 ± 0.11
Chromaticity (C*) 9.10 ± 0.01
Values are expressed as mean ± standard deviation (n = 3). RDS (rap-
idly digestible starch), SDS (slowly digestible starch) and RS (resist-
ant starch)
dw dry weight
Estrada-León et al. [11], where E. cyclocarpum starch iso-
lated using three different methods (alkaline steeping, water
steeping and acid steeping) showed pH values between 5.7
and 6.5. Color parameter L* (luminosity) (L* = 83.41 ± 0.50
is less than 100; which indicates that huaya starch is not
completely white. Based on the values of a* (2.40 ± 0.03),
b* (8.70 ± 0.02), C* (9.10 ± 0.01) and the hue angle
(74.70 ± 0.11), confirm that the huaya starch extracted is
light salmon colored.
The in vitro starch hydrolysis profile is presented in Fig. 2
and the content of RDS, SDS and RS summarized in the
Table 2. The RDS, SDS and RS contents of huaya starch
were 17.96 ± 1.80%, 21.24 ± 1.05% and 59.59 ± 0.56%,
respectively; thus, this starch is slightly susceptible to enzy-
matic hydrolysis (as shown in the hydrolysis curve). The sum
of the RDS and SDS is defined as digestible starch; which
was approximately 39.20 ± 1.41%. The starch hydrolysis rate
showed a significant increase (P ≤ 0.05) with increasing time
and the final hydrolysis was determined to be 46.00 ± 1.81%
(Fig. 2). The high RS content of huaya starch is comparable
to that reported for kithul palm starch (49.02%) [31]. The
high RS content of huaya starch is beneficial to management
of diseases, such as diabetes, whereas high levels of SDS and
RS did not produce hyperglycemia [32], since RS is a frac-
tion of the starch that is able to resist digestion and remains
intact throughout the gastrointestinal tract.
Starch digestibility is influenced by a combination of fac-
tors, such as starch source, granule size, amylose content
and crystal structure [33]. α-amylase hydrolysis of starch
is known to be inversely related to amylose content and
Fig. 2  In vitro hydrolysis of native huaya starch. Values within each
time with different letters are significantly different at P ≤ 0.05
amylose content was positively correlated with RS content
[32, 34]. More ordered crystalline structure leads to a lower
rate of enzymatic hydrolysis, which is due to the strong
interaction between neighboring helices [35]. The average
granular size of starch, the high content of amylose and RS,
and probably, along with an ordered crystalline structure
partially explain the low susceptibility of huaya starch to
enzymatic hydrolysis.
SP and IS
It is known that as the temperature (above the gelatinization
temperature) of a starch paste increases, there is an increase
in the swelling rate and a large portion of carbohydrates
is leached from the starch granule. The SP and IS patterns
(at different temperatures) of huaya starch are depicted in
Fig. 3. The SP of huaya starch showed a significant increase
(P ≤ 0.05) at different temperatures. The values reported
for SP at different temperatures demonstrated that huaya
starch resists swelling between 60 and 70 °C. This can be
explained by the high gelatinization temperature exhibited
(81.45 ± 0.15 °C). Meanwhile, between 80 and 90 °C the
starch granules swell owing to the breakdown of intermo-
lecular hydrogen bonds in amorphous areas, which allows an
irreversible and progressive absorption of water. The SP val-
ues of huaya starch (between 0.40 ± 0.01 and 6.00 ± 0.20 g/g)
are comparable to that reported for rice starch (between 4
and 12 g/g) with high amylose content (32%) [33]. Grace,
Henry [36] indicated that the high amylose content (59.74%)
restricted the swelling capacity of corn starch. Therefore, the
high amylose content of huaya starch (39.13 ± 0.35%), the
average size of the granule (14.54 ± 0.95 µm) and the exist-
ence of strong interactions between the molecules present
inside the starch granule probably explain the low swelling

13
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Huaya (Melicoccus bijugatus) seed flour as a new source of starch: physicochemical,…
Fig. 3  a Solubility and b swelling power of isolated huaya starch at different temperature. Values within each temperature with different letters
are significantly different at P ≤ 0.05
Fig. 4  DSC thermogram of gelatinization of native huaya starch
capacity. As for the solubility, it increase significantly
(P ≤ 0.05) according to the temperature directly, because
the swollen granules of the huaya starch allow the removal
of amylose from them. IS values of huaya starch are compa-
rable to that reported for kithul starch [31].
Differential scanning calorimetry analysis (DSC)
DSC was used to study starch gelatinization which involved
disruption of the native structure of the huaya starch. The
gelatinization temperatures ­(To, ­Tp and ­Tc) and enthalpy
(∆H) of huaya starch are shown in Fig. 4. This process
starts at 78.15 ± 4.50 °C (­ To) (melting temperature of the
weak-structured crystal) through the phase transition where
water acts as a plasticizer. When temperature rises, the starch
granules suffers fragmentation until finally the amorphous
part (amylose) is fully solubilized, meanwhile the starch
crystalline zone is maintained in the aqueous solution. The
­Tp was observed at 81.45 ± 0.15 °C. At this point, the highest
values of heat absorption are recorded and the starch tran-
sits to a gummy state due to the rupture of its granules. At
the end of the process, the temperature was 85.42 ± 4.25 °C
­(Tc) (melting temperature of the strong-structured crystal).
­To, ­Tp and T
­ c values of huaya starch were comparable than
those reported by Sudheesh et al. [31] for kithul starch (a
starch rich in amylose that gelatinizes at high tempera-
tures). High gelatinization temperatures indicate that a high
amount of energy is required for gelatinization of huaya
starch, and that it may be influenced by its low swelling
capacity, high amylose content, medium size, higher pro-
portion of crystalline region by the interaction of starch
chains and internal arrangement of starch fractions within
the granule. The ∆H value calculated by DSC is related to
the dissociation of double helices and it is related to the
conversion of the starch suspension to viscous paste. This
∆H value (16.63 ± 0.66 J/g) for huaya starch was similar to
that reported for common buckwheat starch [37] which is
typified as a high gelatinization temperature starch. There-
fore, the high value of ∆H indicates that huaya starch has a
well-organized microstructure and more energy is required
to gelatinize.
Fourier‑transform infrared (FTIR) spectroscopy
FTIR analysis of huaya starch provides information on the
arrangement of functional organic groups and their interac-
tions in the molecule. Since the IR beam only penetrates
the first few micrometers (about 2 µm) into the granule, it
implies that the IR spectra are representative of the out-
side of the starch granules [38]. Figure 5 depicts the FTIR
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 Author's personal copy
Fig. 5  FTIR spectra of native huaya starch
spectra of the huaya starch. The broad band at 3332 cm−1
corresponds to the vibrations of the free hydroxyl groups.
Therefore, there is little inter and intramolecular interaction
of these functional groups, due to its size (OH– molecular
weight) [39]. The band at 2927 cm−1 is due to CH stretching
associated with the ring methane hydrogen atoms. Water
absorbed by starch is observed in the band at 1637 cm−1. In
the characteristic region of the starch, the major peaks cor-
responding to the vibrations of the C–O–C glucose bonds
(1144, 1075 and 1009 cm−1) and 851 cm−1 corresponding
to pyranose are observed [40].
The absorbance band at 1047 cm−1 was sensitive to the
crystalline structure and 1022  cm−1 was referred to the
amorphous starch [34]. The relationship 1047/1022 cm−1
(0.90 ± 0.01) (reflect the degree of order) reported for huaya
starch is similar to that reported for C-type chesnut starch,
considered as a starch with a high degree of short-range
molecular order [41], which indicates that huaya starch
contained higher amount of crystallites and these results
are consistent with data observed in the X-ray diffraction
pattern.
The intensity ratio at 1022/995  cm −1 (0.85 ± 0.03)
(degree of the double helix) reported for huaya starch
is similar to that reported for starches in lotus rhizome
(between 0.80 and 0.83), suggesting that huaya starch has
a higher number of double helix structure just as reported
for starches from rhizomes of two lotus cultivars [38]. The
lower number of double helix structure resulted in lower
△H [42], consequently, the greater number of double
helix structure would explain the high value of △H for the
huaya starch (16.63 ± 0.66 J/g) and rhizome starch (values
between 11.2 and 13.3 J/g) [38].
13
V. M. Moo‑Huchin et al.
X‑ray diffraction
X-ray diffraction provides an elucidation of the long-range
molecular order, typically termed as crystalline, which is
due to ordered arrays of double helices formed by the amylo-
pectin side chains [38]. Figure 6 shows the X-ray diffraction
patterns of amylopectin from corn, amylose from potato, and
isolated huaya starch. The prominent peaks located around
15°, 17° and 23° are characteristic of a starch consisting
mainly of amylopectin and this result coincides with the
reported values for the amylose and amylopectin content of
huaya starch. Huaya starch gave rise to an X-ray pattern that
corresponds to a mixture between type A and B polymorphs,
also called Type C. The main difference between A- and
B-type is that the former adopts a close-packed arrangement
with water molecules between each double-helical structure,
while the B-type is more open, there being more water mol-
ecules, essentially all of which are located in a central cavity
surrounded by six double helices [38].
The slightly prominent peak observed at 2θ = 5.4°, is not
normally seen in type A starches, the peak at 2θ = 17° was
more prominent than that found at 2θ = 18° and the peak at
2θ = 23° is broad; which confirms the X-ray type C diffrac-
tion pattern of huaya starch. The X-ray type C diffraction
pattern of huaya starch is very similar to that reported for
isolated starches from acorn kernel and lotus rhizome [38,
43, 44]. To the best of our knowledge, this is the first time
that C-type crystalline was observed in the starch isolated
from huaya seed flour. The level of crystallinity calculated
was 31.22 ± 0.20% for huaya starch.
Starches with polymorphism type B and C, have been
reported to resist enzymatic hydrolysis more than polymorph
type A starch; this is because the latter consists of double
helices of branch chains that are more orderly aligned in
the granule, and thus, they are more resistant to enzyme
Fig. 6  Thermogravimetric analysis of native huaya starch
 Author's personal copy
Huaya (Melicoccus bijugatus) seed flour as a new source of starch: physicochemical,…
Fig. 7  X-ray diffractogram of native huaya starch
hydrolysis [45]. This fact explains why huaya starch is not
very susceptible to the hydrolysis of pancreatic α-amylase.
Thermogravimetric analysis (TGA)
Additionally, the thermal behavior of the huaya starch was
investigated with TGA. The thermal behavior of the huaya
starch is described in three stages, according to Fig.  7.
The first stage corresponds to the reduction of the mass at
100 °C, which is due to the loss of water [37]. Such loss cor-
responds to 16.00 ± 0.15% of the weight of the huaya starch.
During second stage, which ranges from 245.01 ± 4.90 to
385.00 ± 14.50 °C, the mass loss is due to the main decom-
position processes of starch macromolecules. This starch has
a decomposition temperature of 312.00 ± 15.60 °C. Temper-
ature of this thermal event is lower compared to that reported
by Pérez-Pacheco et al. [3] for corn starch with 338 °C. At
this stage, the mass of the huaya starch decreases by 85%.
Finally, during third stage, starting from 400 °C, the remain-
ing mass is stabilized by the formation of carbon residues
from amylose and amylopectin.
Starch gel clarity
Analysis of starch pasta clarity (measured as %T) was per-
formed for 6 continuous days (Fig. 8). The clarity of huaya
starch paste decreased significantly (P ≤ 0.05) until day 4
(from 3.54 ± 0.10 to 1.75 ± 0.10 %T). It is noteworthy that
the greatest reduction occurs between days 1 and 2, which
suggests that the dispersion of native starch tends to stabi-
lize. Finally, during days 5 and 6, the transmittance values
remained unchanged (there are no significant differences).
Comparing with other starches such as common buck-
wheat (8.85 %T) and tartary buckwheat (7.56 %T) [46],
huaya starch paste is not very transparent (values between
Fig. 8  Paste clarity of native huaya starch. Values within each storage
day with different letters are significantly different at P ≤ 0.05
1.55 ± 0.09 and 3.54 ± 0.10 %T). Ayucitra [47] indicates
that the clarity of starch pastes is an important property to
consider for the manufacture of some food products. There-
fore, the use of huaya starch in non-transparent foods such as
mayonnaise, meat products, concentrated nectar-type drinks
or bakery products is suggested [48]. The transparency of
the starch paste is affected by many factors, such as amylose
content, granule size, amylose/amylopectin ratio, granule
swelling and granule remnants [49].
The low %T values of huaya starch can be attributed to
the high amylose content, granule size, resistance to swell-
ing and swollen granular remnants with little association
of chains after pasting, which is consistent with Chao et al.
[49].
Conclusions
This paper pretends to provide the first study regarding the
properties of an isolated starch obtained from huaya seed
meal. Obtained information showed that the proximal chemi-
cal composition, yield of huaya starch and physicochemi-
cal, morphological, thermal and functional properties are
comparable to other conventional and non-conventional
source starches. Additionally, Huaya starch is resistant to
enzymatic hydrolysis and its paste is not very transparent.
The results of this research support the idea that the starch
obtained from a by-product (seed) of the huaya fruit pos-
sesses adequate and interesting properties, therefore, can be
an alternative to conventional source starches (i.e. cereals)
in food applications such as ingredient for non-transparent
foods that require high processing temperatures or for the
design of slow-digesting foods. However, recommendations
13
 Author's personal copy
for future research include further studies on rheological,
pasting, molecular weights of starch fractions and chain
lengths properties. Accordingly, this novel starch can be
supplementary investigated to expand its use on a variety
of applications.
Acknowledgements  The authors would like to express their gratitude
to the Tecnológico Nacional de México (TecNM), for the financial
support for the project 5697.19-P. Technical support is acknowledged
to MSc José Rodriguez-Laviada.
Compliance with ethical standards  J.F. Toro-Vazquez, L.A. Bello-Pérez, Carbohydr. Polym. 52(3),
Conflict of interest  The authors have no conflict of interest to declare.
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