364

DOI 10.1002/star.201100008

Starch/Starke 2011, 63, 364372

RESEARCH ARTICLE

Isolation and characterization of Mexican jackfruit

(Artocarpus heterophyllus L) seeds starch in two
mature stages
Deyanira L. Madrigal-Aldana1, Beatriz Tovar-Gomez1, Miguel Mata-Montes de Oca1,
Sonia G. Sayago-Ayerdi1, Felipe Gutierrez-Meraz2 and Luis A. Bello-Perez2
1

Laboratorio Integral de Investigacion en Alimentos, Division de Estudios de Posgrado, Instituto Tecnologico de Tepic,
Tepic, Nayarit Mexico
2
Centro de Desarrollo de Productos Bioticos (CEPROBI) del Instituto Politecnico Nacional, Yautepec, Morelos, Mexico

Starch was isolated from jackfruit (Artocarpus heterophyllus L) seeds grown in Mexico at
different stages of fruit maturity and ripeness. Seeds represent about 815% from the fruit
that can weigh around 236 kg. Proximate composition of seeds showed a high protein
content (ca. 22%). Starch yield was 14% with a purity of 81% in both ripeness stages and AM
content was lower (12.27%) than other non-common starch sources. The starch granules in
physiological mature (PM) and consumption ripeness (CR) jackfruit showed birefringence
with diverse shapes such as semi-oval or bell shapes. The size of starch granules for PM
ranged between 3 and 9.5 mm and for CR between 3 and 12 mm. A-type XRD pattern was
similar to cereal starches. PM starch had higher peak viscosity than CR, but CR did not show
breakdown; both starches presented setback during cooling. Thermal properties of gelatinization and retrogradation in PM and CR starches were similar. Characterization performed
on this non-common starch showed that it could be an alternative to use in food systems.

Received: January 10, 2011

Revised: February 21, 2011
Accepted: February 22, 2011

Keywords:
Granule size / Jackfruit / Molecular characteristics / Non-common starch sources / Thermal properties

Introduction

Abbreviations: CR, consumption ripeness; DSC, differential

scanning calorimeter; PM, physiological mature; RVA, rapid visco
analyzer; WRC, water retention capacity.

spread to other tropical countries of America such as

Mexico, where the annual production is around 6800 ton
(SIACON, 2009, http://www.siap.gob.mx). Jackfruits are
extremely large, up to 90 cm and their weight can range
from 2 to 36 kg [4]. The edible part of this fruit is the pulp,
while the seeds are from 2 to 4 cm length and one fruit may
contain from 100 until 500 seeds, which represent 815%
of the fruit weight [4, 5]. The seeds usually are consumed
roasted, boiled, steamed, and are eaten as a snack. Fresh
seeds cannot be kept for a long time. Additionally, obtaining
seed our can be an alternative as an ingredient for food
products [6]. The literature on jackfruit seeds is rather
scarce; biochemical differences between varieties, starch
content, and some physicochemical properties have been
studied [4, 7]. An elevated starch content in jackfruits
seeds has been reported [6], although, these values are
varied [4, 6, 8]. Starch content in jackfruits seeds, as in
other cultivars, depends of their structural composition,

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Jackfruit is the name of the fruit from a jacktree (Artocarpus

heterophyllus L) that belongs to the Moraceae family. It is an
exotic fruit native to Asiatic countries, mainly Thailand,
Malaysia, and India [1, 2]. Currently, jackfruit is being cultivated in Burma and Thailand. In America it had been introduced to Brazil in the 19th century [3]. Recently, it has

Correspondence: Professor Luis A. Bello-Perez, Centro de

Desarrollo de Productos Bioticos (CEPROBI) del Instituto
Politecnico Nacional, Yautepec, Morelos, Mexico.
E-mail: labellop@ipn.mx
Fax: 52-735-3941896

Starch/Starke 2011, 63, 364372

growing conditions, harvesting periods, and climates
[9, 10]. Jackfruit is a climacteric fruit and is harvested
when physiological mature (PM), and it is consumed
when the fruit reaches the proper consumption ripeness
stage. In this period, diverse physiological changes are
produced that can modify the chemical characteristics of
the components present in the pulp and seeds. The interest in the characterization of starch from non-common
sources such as jackfruit harvest in a tropical zone of
Mexico is relevant because of the potential useful in
food systems. The aim of this work was the isolation
and characterization of starch from jackfruits seeds produced in a tropical zone in Mexico.

Materials and methods

2.1 Sample preparation of jackfruit seeds

Jackfruits in two ripeness stage, PM and consumption
ripeness (CR) were collected in an orchard located in
Compostela, Nayarit, Mexico. The fruits were exposed
at ambient temperature for 30 min, washed with tap water,
peeled off, and the seeds were manually separated from
the pulp and the mucilage peel of the seeds was removed
manually. Part of peeled seeds were cut into 1 cm slices
and ground using a commercial grinder (Mapisa
Internacional S.A. de C.V., Mexico, D.F.), these ground
samples were used in chemical composition analyses.
The remaining peeled seeds was stored at
48C for further
starch isolation.

2.2 Chemical composition

Moisture content was determined by gravimetric heating
(130  28C for 2 h) using a 23 g sample. Ash, protein,
fat, and crude ber were analyzed according to
AOAC methods 923.03, 920.87, 920.85, and 945.18,
respectively [11].

Mexican jackfruit starch

365

pressed through a sieve to eliminate seed bers. The

resulting milky suspension was allowed to settle at
458C and rewashed with distilled water to eliminated
soluble sugars. It was then centrifuged at 4500 g at
208C during 30 min and supernatant was discarded.
The precipitate was re-suspended in 80% ethanol and
dried at 458C for 48 h until a 13% of moisture content.
Later, starch powder and residue were quantied and
starch recovery was determined.

2.4 Starch content

Total starch content was determined by the method of Goni
et al. [13]. In brief, a 50 mg sample was dispersed in 2 mol/L
KOH to hydrolyze all the starch (30 min) and subsequently
incubated (608C, 45 min, pH 4.75) with amyloglucosidase
(Roche No. 102 857, Roche Diagnostics, Indianapolis, IN,
USA); glucose content was then determined using the
glucose oxidase/peroxidase (GOD/PAD) assay (SERAPAK1 Plus, Bayer de Mexico, SA de CV). Total starch
content was calculated as glucose (mg)  0.9; potato
starch was used as a reference.

2.5 AM content
The AAM content was determined by the test of Hoover
and Ratnayake [14]. A sample of isolated starch (20 mg,
db) was dissolved in 90% DMSO (8 mL) in 10 mL screwcap reaction vials. The contents were vigorously mixed for
20 min and then heated in a water bath (with intermittent
shaking) at 858C for 15 min. The vials were then cooled to
ambient temperature, and the contents diluted with water
to 25 mL in a volumetric ask. The diluted solution
(1.0 mL) was mixed with water (40 mL) and 5 mL of
I2/KI solution (0.0025M I2 and 0.0065M KI) and then
adjusted to a nal volume of 50 mL. The contents were
allowed to stand for 15 min at ambient temperature and the
absorbance at 600 nm was measured.

2.6 Light microscopy

2.3 Starch isolation
The stored seeds (
48C) were exposed at ambient
temperature during 30 min and were treated using a
method previously reported [12]. Briey, the spermoderms
was removed in jackfruit seeds using lye peeling. A 0.5% of
NaOH solution was used to soak jackfruits seeds during
30 min and after rinsed with distilled water. White cotyledon obtained were soaked in 0.5% NaHSO3 for 30 min,
and rewashed with distilled water three additional times.
PM and CR samples were blended separately with
1000 mL of 0.5% NaHSO3 solution during 4 min. Starch
was isolated using the method proposed by Bobbio et al.
[4]. Ground cotyledons were made into a slurry and
2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

A polarized light microscope (Eclipse 80i, Nikon, Japan)

was employed with 40x objective lenses equipped with a
digital camera (Digital Imaging Head, DC330 camera MTI,
Japan). Dry native starch was sprinkled on a slide and a
cover was added.

2.7 SEM
For SEM, samples were xed to a double glue conductive
copper tape, which was covered with a layer of coal of
20 nm thickness. It was deposited under a vacuum using
an evaporator in a JEOL JSMP 100 (Japan) electron
microscope. Later on, samples were covered in the ionizer
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D. L. Madrigal-Aldana et al.

metals JEOL with a 50 nm thickness gold layer. A lm piece

was mounted on aluminum stubs using a double-sided
tape and then coated with a gold layer (4050 nm), allowing surface and cross-section visualization. All samples
were examined using an accelerating voltage of 5 kV.

2.8 XRD
The XRD was obtained from a D/max 2500 X-ray diffractometer (Tokyo, Japan), a conventional X-ray tube set to
40 kV and 200 mA. The X-ray source was Cu Ka radiation.
Data were collected from 2u of 5 to 358 (u being the angle of
diffraction) with a step width of 0.028 and step time of 0.4 s,
scanning speed of 88/min, divergence slit width of 0.2 mm,
scatter slit width of 0.6 mm, receiving slit width of 0.2 mm,
at room temperature. Starch samples were dried at 508C to
constant moisture (10%) in a vacuum oven, then 50 mg
samples were added into the slide for packing prior to
X-ray scanning. Starches were equilibrated at 508C to
constant moisture (10%) prior to analysis.
The crystallinity percentage (%C) was determined from
the diffractogram by calculation of the area corresponding
to the crystalline peaks (Ap; from the difference between
the area under the curve and the area of the amorphous
halo), the total area under the curve (At), and the instrumental noise (N) according to the following equation [15].
%C

Ap
At
N

(1)

The amorphous halo was determined with the amorphous component of starch obtained with an extraction
procedure reported elsewhere [16].

temperature; peak, breakdown, nal, and setback

viscosity. All measurements were replicated three times.

2.11 Starch gelatinization

Gelatinization parameters were measured using a differential scanning calorimeter (DSC TA Instruments), equipped
with a thermal analysis data station (TA Instruments,
Newcastle, DE) and following the procedure of ParedesLopez et al. [19]. The sample of starch (2 mg dwb) was
weighed directly in an aluminum DSC pan (TA series
900796.901) of 20 mL capacity. Deionized water was added
with the help of a Hamilton micro syringe to achieve a starchwater suspension containing 70% water. Pans were hermetically sealed, and allowed to stand for 1 h at room temperature for even distribution of water before DSC measurements. Indium and empty aluminum pan were used as
reference to calibrate the DSC. Sample pans were heated
at a rate of 108C/min from 20 to 1208C. Onset temperature
(To), peak temperature (Tp), conclusion temperature (Tc),
and enthalpy of gelatinization (DH) was calculated automatically. DH was calculated on starch dry basis.

2.12 Starch retrogradation

After gelatinization test the samples was stored at 48C for
7 days before re-scanning. Temperature range and heating
rate were 251208C and 108C/min, respectively. An empty
pan was used as reference for all measurements.
Retrogradation enthalpy (DH) was calculated automatically.

2.13 Statistical analysis

2.9 Water retention capacity and solubility

Water retention capacity (WRC) and solubility were
measured according to Nunez-Santiago et al. [17] method.
The reported values are the mean of triplicate
measurements.

2.10 Pasting profile

Pasting properties of starches were evaluated with a Rapid
Visco Analyzer (RVA) (RVA-4, Newport Scientic, Sydney,
Australia) [18]. Starch (2.24 g, 8% moisture db) was
weighed directly in the aluminum RVA sample canister,
and distilled water was added to a total constant sample
weight of 28 g. The slurry was manually homogenized
using the plastic paddle to avoid lump formation before
the RVA run. A programmed heating and cooling cycle was
set for 23 min where the samples were held at 308C for
1 min, heated to 958C in 7.5 min, further held at 958C for
5 min before cooling to 508C within 7.5 min, and holding at
508C for 2 min. Parameters recorded were pasting
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The means (at least three replicates) and SDs were determined for all samples. A statistical software (SAS version
9.0) was used to evaluate by ANOVA the differences in
mean values based on data collected from replications of
each measurement. Statistically signicant differences
( p<0.05) were evaluated using the LSD multiple comparison procedure.

Results and discussion

3.1 Proximate composition

The chemical composition of ground jackfruits seeds is
shown in Table 1. The stage of ripening inuenced the
moisture content, PM seeds had higher moisture content
than CR seeds. This elevated moisture content could lead
to increasing microbiological growth [20], and it is necessary to further process by starch isolation or drying to
produce a our. Jackfruits seeds showed a low lipid conwww.starch-journal.com

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Mexican jackfruit starch

367

Table 1. Chemical composition of jackfruit seeds in PM and CR stagesa)

Composition (%)
Moisture
PM
CR

Ash

62.66  0.25
54.06  0.85b

3.61  0.11
3.14  0.22a

Proteinb)

Lipid
a

0.81  0.01
0.70  0.01b
a

Crude fibre

22.41  0.26
21.99  0.51a
a

8.93  0.33a
7.32  0.14b

a) Values are mean  SD, n 3; values with similar letters in the same column do not differ signicantly ( p<0.05).
b) N  6.25.
tent, this value was similar those reported in other Jackfruit
studies [4, 6, 21]. The protein content was similar those
reported by Bobbio et al. [4]. However, protein content has
been found to change depending on the variety and the
ripening stage of the fruit [68, 22]. The differences can
also be attributed to diverse varieties, ripening stage, type
of soil, and environmental conditions [23]. The high protein
content suggests that it should be used in a bioavailability
study in further investigations, as an alternative for seed
consumption.

3.2 Starch yield, total starch, and AM content

The moisture content of starch isolated from jackfruit
seeds was signicantly different ( p<0.05) for each ripening stage. Physiological stage is correlated to water content in fruits. The yield of starch isolated was similar than
that reported by Bobbio et al. [4] (Table 2).
Values in total starch content in jackfruit seeds or pulp
were scare and did not indicate the ripeness stage in the
fruit. Starch yield was higher in CR than PM seeds; this
difference is attributed to the lower moisture content in the
former sample. However, other physiological events proceed in the seeds where starch accumulates during pulp
ripening. There is a reverse relationship between the
starch content of the two tissues. When the fruit is ripe,
starch is converted to monosaccharides decreasing the
starch content [24]. Tulyathan et al. [6] reported in jackfruit
seeds, without endosperm, similar values as those
obtained in the present study.
Difference in AM content was obtained in both starches
(CR and PM), with lower AM content in PM than in CR. This
pattern can be caused by continued starch biosynthesis

during fruit storage, where AM synthesis occurs within the

matrix formed by the synthesis of AP [25], and the synthesis of AM decreased due to that the amount of maltooligosaccharides is minor [26]. AM content determined in
CR and PM starches is considered low and can be considered important in the retrogradation of these jackfruit
seed starches. Regarding AM content, it was lower than
other non-conventional starch sources such as tubers of
Dioscorea bulbfera with 29.37% [27] and arracacha with
17.46% (Arracacia xanthorriza) [28]. Results in AM content
for Artocarpus genus vary extensibility. In Brasil Bobbio
et al. [4] reported 28% in AM content, while Tulyathan et al.
[6] obtained 32% in fruit from Bangkok and Thailand.
However, within the same country South Thailand,
Tondang [21] obtained values in AM from 24.4%. These
samples variations can be ascribed from differences in
source, growing environment, harvesting season, and
ripeness of fruit. Additionally, measurement variations
can depend on the analytical method employed [10, 21].

3.3 Microscopy studies

Micrographs in jackfruit seeds starch granules viewed under
light microscopy and polarized light are shown in Fig. 1. The
size of starch granules from PM seeds was smaller than CR
seeds. This pattern is caused by continued starch biosynthesis during fruit storage, with a concomitant increase in the
chain length of AP and consequently, granule size [29]. The
Maltese cross was viewed under polarized light in both
starches, indicating the arrange of starch components
(AM and AP), and that these were not disorganized during
starch isolation procedure. The crosses were symmetrical
and sharp in the center of the granules and it was equally

Table 2. Starch yield, total starch and AM content in physiologic mature (PM) and ripeness (CR) stages of jackfruit seeds
flour (%)a)
Sample

Moisture

PM
CR

7.26  0.33
6.49  0.23b
a

Starch yieldb)

Total starch

9.30
14.07

73.34  0.70
81.16  0.33b

AM content
a

8.69  0.36a
11.46  0.41b

a) Values are mean  SD, n 3; values with similar letters in the same column do not differ signicantly ( p<0.05).
b) It was calculated in wet basis of jackfruit seeds our.
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D. L. Madrigal-Aldana et al.

Starch/Starke 2011, 63, 364372

Figure 1. Micrographs of starch granules isolated from jackfruit seeds in two ripeness stages. (a) Light microscopy, ripeness;
(b) polarized-light microscopy, ripeness; (c) light microscopy, physiologic mature; (d) polarized-light microscopy, physiologic
mature.

Figure 2. Scanning electron micrographs of starch isolated from jackfruit seeds in physiologic mature (a), (c); ripeness (b),
(d) stages.
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Mexican jackfruit starch

369

divided into four segments. This indicates an intact structure

in the starch of jackfruits seeds.
The scanning electron micrographs are shown in Fig. 2.
Jackfruit seeds starch presented granules with spherical or
dome-shaped and split, similar shapes were reported in
other starch fruit (mango) [30]. Jackfruit seeds starch had
small granule size (between 3 and 12 mm), and smaller
starch granules were observed in PM than in CR samples.
This pattern is in agreement with the light microscopy
study and AM content results obtained for jackfruit seeds
starch. The small granule size of jackfruit seed starch can
be important for diverse applications such as encapsulation and water retention. Granule size for jackfruit starch
ranged between 1 and 18 mm [4, 12, 21], suggesting that
the variations can be due to agronomic trials, ripeness
stage of the fruit, and climatic conditions.

3.4 XRD
The XRD pattern of PM and CR of jackfruit seeds starches
is shown in Fig. 3. Both samples presented a A-type XRD
pattern, similar to that determined in jackfruit seeds starch
[6] and cereal starches [31, 32]. Slight differences in the
crystallinity percentage were found; CR jackfruit seeds
starch had higher crystallinity level (28%) than PM seeds
starch (26.3%). The XRD pattern and the crystallinity level
(long range order) give information of the arrangement of
AP chains in double helices. This is important in properties
such as starch digestibility [33] and retrogradation [34],
because starches with A-type XRD pattern were more
easily digestible than B-type and the former present high
amount of slowly digestible starch [33].

3.5 Solubility and water retention capacity

Water retention capacity and solubility results of the two
starch samples (PM and CR) are shown in Fig. 4.

Figure 3. X-ray diffractograms of jackfruit starch isolated

from seeds in PM and ripeness (CR) stages.
2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 4. (a) Water retention capacity and (b) solubility

(S) at different temperatures of starch isolated from jackfruit seeds in PM and CR ripeness stages.
Regarding the rst parameter measured, the PM sample
presented higher WRC than CR in a temperature range of
60708C (Fig. 4a). Thereafter at 80908C, statistically
differences ( p>0.05) in WRC were found. This property
in native starches is increased constantly with an increase
in temperature. Solubility was increased as well at elevate
temperature (Fig. 4b). The increase in solubility at higher
temperatures is associated with leaching of starch chains,

Figure 5. RVA pasting profiles of starch isolated from PM

and CR stages jackfruit seeds flour.
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D. L. Madrigal-Aldana et al.

Table 3. RVA parameters of starch isolated from PM and ripeness (CR) stages jackfruit seeds floura)
Temperature (8C)
Pasting
PM
CR

Viscosity (mPa s)
Peak

79.90  1.2
82.50  0.8b
a

Breakdown

321.92  3.6
221.53  5.2b

Final

55.50  2.2
1.25  0.1b

Setback

372.42  5.5
293.90  6.3b

106.00  3.1a
73.62  2.9b

a) Values are mean  SD, n 3. Values with similar letters in the same column do not differ signicantly ( p<0.05).

3.7 Thermal analyses in jackfruit seeds starch

AM, and the external long chains of AP, toward the continuous phase. At higher temperatures, WRC was higher
due to the increase in the mobility of water molecules that
diffuse toward starch granules, and they can be retain
because of the link formed with the OH groups of glucose
units of starch [35].

Thermal properties in starch are related to the behavior in

an industrial heating process. For this reason the values
obtained in non-common starch sources such as jackfruit
seeds starch in different stage of ripeness can bring useful
information from these samples. The results of the thermal
properties are shown in Table 4. The jackfruit seeds
starches did not show statistical differences ( p<0.05) in
thermal parameters of gelatinization, indicating that the
differences in the granule size and AM content between
both starches did not inuence gelatinization behavior.
Additionally, gelatinization temperatures indicate the structural characteristics of AP. AP with higher amount of short
chains had lower gelatinization temperatures than AP with
higher amount of longer chains [39]. In the same sense, the
enthalpy value reects the loss of the ordered double
helices, indicating that both starches had similar arrange
of the double helices. In jackfruit seeds our and starch
from Thailand, the enthalpy values were lower those
obtained in this study [12]. The AM content reported by
these authors was higher than obtained for our samples,
although Gudmundsson and Eliasson [40] considered that
the enthalpy required for starch gelatinization must be
between 10 and 20 J/g.
Starch retrogradation is a process that occurs during
the storage of gelatinized starch. At 48C, gelatinized starch
molecules were re-associate, but in a less ordered struc-

3.6 Pasting properties

One of the most important functional properties in starch is
viscosity. It is an important factor in the determination of
future applications from diverse extraction sources. The
RVA viscograms and parameters of seeds starches were
shown in Fig. 5 and Table 3. The maximum peak temperature of PM seeds starch was higher than CR seeds starch,
but in the former this parameter was obtained at lower
temperature, showing higher stability of starch granules in
CR seeds starch. Additionally, CR seeds starch showed
very little breakdown value, indicating stability of swollen
starch granules at shear stress. Both jackfruit seeds
starches presented setback that is low compared with
other cereal starches [36] due to the low AM content.
These results were similar than reported before in jackfruit
from Brazil and Thailand [4, 12]. The increase in viscosity
from both samples during the setback phase indicates the
retrogradation of starch or reassociation of the AM molecules during the cooling period after gelatinization to form
a gel network [37, 38].

Table 4. Thermal properties of starches isolated from jackfruit seeds in physiologic mature (PM) and ripeness (CR)
stagesa),b)
Temperature [8C]
Starch
PM
CR

Process
Gelatinization
Retrogradation 7 days
Gelatinization
Retrogradation 7 days

To
72.60
45.75
72.60
44.48

Tp





0.1
0.5b
0.2a
0.1b

77.70
57.04
78.30
55.98

Tc





0.2
0.1b
0.4a
0.6b

85.70
68.93
86.10
69.13






0.3
0.1b
0.5
0.4b

Tc
To

DH (J/g)

13.10
23.18
13.50
24.65

14.40
8.04
13.50
7.19






0.4a
0.4c
0.7a
0.3c

a) Values are mean of three replicates  SE. Means in column not sharing the same letter are signicantly different
( p<0.05).
b) To onset temperature; Tp peak temperature; Te conclusion temperature; DH gelatinization or retrogradation
enthalpy.
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Mexican jackfruit starch

371

[9] Sriroth, K., Santisopasri, C., Petchalanuwat, K.,

Kurotjanawong, K., et al. Cassava starch granule structure-function properties: Inuence of time and conditions
of harvest on four cultivars of cassava starch. Carbohydr.
Polym. 1999, 38, 161170.

ture, resulting in a less stable form than in native granular

state. The generation of such an unstable structure could
be assumed, in view of the lower melting temperature
required for melting this material compared to the native
preparation [41]. Both jackfruit seeds starches showed
similar retrogradation parameters, indicating that structure
of starch components is reorganized at the same level. The
small difference in the AM content between both jackfruit
starches did not inuence the retrogradation properties.
Retrogradation of jackfruit seeds starch can be important
during the utilization of this starch in foods because of the
formation of RS [34].

[10] Bello-Perez, L. A., Agama-Acevedo, E., Sayago-Ayerdi,

S. G., Moreno-Damian, E., Figueroa, D. C., Some structural,
physicochemical and functional studies of banana starches
isolated from two varieties growing in Guerrero, Mexico.
Starch/Starke 2000, 52, 6873.

[13] Goni, I., Garca-Dz, L., Saura-Calixto, F., A starch hydrolysis

procedure to estimate glycemic index. Nutr. Res. 1997, 17,
427437.

Conclusions

The jackfruit seeds showed an elevated protein content.

The small granule size of jackfruit seed starch can be an
alternative for encapsulation. Different starch content,
sizes, and shapes of starch granules were observed in
the different ripening stages. Pasting characteristics of the
starch were inuenced by the ripening stage of the seed,
but no difference was found in the thermal parameters.
Seeds of jackfruit that are now considered as waste can
be used to starch isolation. The characterization of this
non-common starch source could be an alternative to use
in food systems.
The authors have declared no conict of interest.

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