MRS Advances

https://doi.org/10.1557/s43580-021-00134-w

ORIGINAL PAPER

Starch from Ramon seed (Brosimum alicastrum) obtained by two

extraction methods
Soledad Cecilia Pech‑Cohuo1   · Jovani Hernandez‑Colula2 · Nancy G. Gonzalez‑Canche3 · Ivan Salgado‑Transito3 ·
Jorge Uribe‑Calderon4 · Jose Manuel Cervantes‑Uc4 · Juan Carlos Cuevas‑Bernardino5 · Teresa Ayora‑Talavera1 ·
Neith Pacheco1

Received: 21 August 2021 / Accepted: 17 September 2021

© The Author(s), under exclusive licence to The Materials Research Society 2021

Abstract 
Brosimum alicastrum is a native tree widely distributed in the Yucatan peninsula where is called Ramon. Some studies have
reported that Ramon seeds contain high starch content, recently used in developing novel and sustainable biomaterials.
This work aimed to evaluate the effect of the extractive solution on the starch isolation Ramon seed flour; for that, distilled
water (S1) and NaOH solution (S2) were used. The Ramon starch yield was 28.0 ± 1.4% and 31.9 ± 1.7% for S1 and S2. The
morphology of starches was observed with scanning electronic microscopy, the functional groups were determined through
Fourier-transform Infrared Spectroscopy and crystallinity was calculated by X-ray diffraction analysis. The results indicate
that both types of starch presented spherical morphology, similar functional groups and crystallinity values, suggesting that
both extraction methods are suitable. The starches isolated exhibited similar thermal behavior assessed by differential scan-
ning calorimetry and thermogravimetric analysis.

Introduction bijugatus) [2] and parotta (Enterolobium cyclocarpum) [3],

Currently, starch is widely used in multiple applications such
as the food industry and materials development [1]. Many
recent studies report starch isolations for material production
from non-conventional sources, such as huaya (Melicoccus
* Soledad Cecilia Pech‑Cohuo
spech_al@ciatej.edu.mx
1
Centro de Investigación y Asistencia en Tecnología y
Diseño del Estado de Jalisco, Subsede Sureste, Parque
Científico Tecnológico de Yucatán, Km 5.5 Carretera Sierra
Papacal‑Chuburná Puerto, 97302 Mérida, Yucatán, Mexico
2
Universidad Interserrana del Estado de Puebla, Chilchotla,
Av. Miguel Hidalgo s/n, 75070 Chilchotla, Puebla, Mexico
3
Centro de Investigaciones en Óptica, A.C., Unidad
Aguascalientes, Prol. Constitución 607 Fracc. Reserva Loma
Bonita, 20200 Aguascalientes, Aguascalientes, Mexico
4 and for soil conservation [5]. A more recent application to
Centro de Investigación Científica de Yucatán, Unidad
de Materiales, Calle 43 No. 130, Chuburná de Hidalgo,
97302 Mérida, Yucatán, Mexico
5
CONACyT‑Centro de Investigación y Asistencia en
Tecnología y Diseño del Estado de Jalisco A.C., Subsede
Sureste, Parque Científico Tecnológico de Yucatán, Km 5.5
Carretera Sierra Papacal‑Chuburná Puerto, 97302 Mérida,
Yucatán, Mexico
which do not compete with human consumption and can be
used as raw material for industrial processes [1, 3]. Besides,
this polysaccharide can be obtained from Ramon seeds, a
tree available in southeastern Mexico [4]. In Mexico, B.
alicastrum is known by more than 50 common names, of
which Ramon, ojite, ojoche, and capomo are the most com-
mon [5]. The Ramon tree can reach a height of 40 m and
diameter of more than 1 m [5], and each tree produce 95.5 kg
of seeds per year [6]. This plant species is considered an
underutilized and undervalued species because it is not com-
mercially available like maize or beans, and only indigenous
communities with knowledge of its users take advantage
[7]. B. alicastrum is one of the tropical trees of which all
its parts can be used and has broad potential as food (fruits
and seeds), forage (leaves, fruits, and seeds), medicine, tim-
ber products, beverages, ornamental, like shade (for coffee
and cocoa plantations), to reduce the impact of strong winds
produce biomaterials such as thermoplastic material [7] and
adhesives [8]. Studies on the obtaining and characterization
of starch indicate that Ramon seeds possesses 92.57% of
this polysaccharide with amylose content of 25.36% [9]; in
this respect, just one extraction method has been reported
using sodium bisulfite (SBS) solution as extraction solvent

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[1]. The isolation methods may change the physicochemi-
cal and functional properties of starch to suit its end-use
[3]. Therefore, the objective of the present research was to
determine the morphological and physicochemical proper-
ties of starch granules isolated from B. alicastrum using two
different extraction methods.
Materials and methods
Ramon seed flour (RSF) from Maya Oox (Kishur S.P.R. de
L.R. de C.V., Chochola, Yucatan, Mexico), NaOH from the
Golden Bell Company (Guadalajara, Jalisco, Mexico).
Extraction methods: The first method (S1) [3, 10], RSF
was soaked in distilled water (DW) (500 g/L) for 24 h. the
moist RSF was blended for 2 min using a blender (Oster-
izer Galaxie 4107/869-16 G). The wet milled was screened
again through a US no. 100, 200 and 300 sieves. The filtered
product was centrifuged at 1174 × g for 20 min at 25 °C, and
the resulting supernatant was decanted. The starch granules
were washed with DW and dried at 50 °C for 24 h. The
second method (S2) is an alkaline extraction [10], RSF was
soaked in 500 ml of 1% (w/w) NaOH for 24 h. The moist
RSF was treated such as in the above procedure. The bottom
starch layer was re-slurried and washed with 0.1% (w/v)
NaOH solution, then this was neutralized with 1 M HCl
solution to pH 6.5 and centrifuged. The starch was washed
with DW and dried.
The statistical analysis was carried out using the results
corresponding to two independent essays for yield, which
were expressed as the mean ± standard error and analyzed
by the student’s t-test. Values of P < 0.05 were considered
significant. Minitab 16 software was used for statistical
analysis.
Scanning Electronic Microscopy: The size and shape of
starches were examined using a SEM JEOL JSM 6360 LV
electron probe microanalyzer at 15 kV.
TGA: 5 mg of sample was heated from 25 to 800 °C a
10 °C/min with helium flux at 20 ml/min using a Netzch
STA 449F5 thermal analyzer.
DSC: Starch gelatinization was determined with a Dia-
mond DSC (Perkin Elmer). 1 mg of starch was weighed into
an aluminum sample pan. DW (3 μl) was added to obtain a
starch/water ratio of 1:3 (w/w). The tests conditions were 25
to 110 °C/min at heating ratio 10 °C/min, flux of dry nitro-
gen. The onset (To), peak (Tp) and conclusion (Tc) tempera-
tures were recorded, also the ­Gelinterv (Tc − To). The enthalpy
changes of the thermal transition (ΔHgel) was estimated by
integrating the area between the thermogram and a base line
under the peak [3].
FTIR: This determination was performed using a
Thermo Scientific Model Nicolet 8700 spectrometer in the
13
S. C. Pech‑Cohuo et al.
transmission mode, with 4 ­cm−1 and 100 scans on 2 mg sam-
ple/135 mg KBr tablets [3].
XRD: Samples were analyzed with a Diffractometer
Bruker D8 Advance (CuKα,  λ = 1.5418 Ǻ, 40  keV and
30  mA). The diffraction pattern was determined by the
occurrence of crystallinity peaks in the angular range
(2θ = 5°–50°), with a time and size step of 0.5 s and 0.02°,
respectively. The crystallinity was determined according to
Castaño et al. [11].
Results and discussion
Figure 1 shows the Ramon tree (a) and their seeds (b). In
this work, starch yields of 28.0 ± 1.40% and 31.9 ± 1.71%
were found for S1 and S2, respectively. These values of
yields were like those obtained for Pérez-Pacheco et al. [1]
using SBS as an extraction medium. The statistical analy-
sis showed no differences between S1 and S2 (Supplemen-
tary material). Figure 2 shows micrographs with spheri-
cal particles of RSF, S1, and S2. RSF particles present a
rough surface in comparison to smooth surfaces for S1 and
S2 particles. The average diameter was 14.07 ± 1.46 μm,
7.46 ± 1.64 μm, and 8.07 ± 1.47 μm for RSF, S1, and S2.
Lindeboom, Chang and Tyler [12] categorized the size of
starch granules as large (> 25 μm), medium size (10–25 μm),
small (5–10 μm) and very small (< 5 μm). The size of the
granules found in this study are classified as small. Similar
values of diameter were found for Ramon starch extracted
with SBS [1].
In Fig. 3a, the TGA and DTGA curves of RSF show a
degradation temperature ­(Td) at 279 °C and 23% residual
mass at 700 °C. S1 and S2 presented Td at 321 and 324 °C,
respectively (Table 1). The TGA Curves of the RSF and
starches showed three stages of degradation. The first
stage corresponds to mass reduction for temperatures
below 150 °C associated with loss of water and volatiliza-
tion of light biomass compound [3, 6]. In the second stage
(250 ± 10 ≤ T ≤ 360 ± 10 °C), the loss mass is due to the prin-
cipal process of decomposition for carbohydrates in RSF [6]
and starch macromolecules [3]. Degradation temperatures
of S1 and S2 in the DTG curves are presented as single
well-defined peaks, indicating a simple reaction mecha-
nism for the degradation of starch biopolymers (amylose
and amylopectin) [9, 13]. This degradation peak begins with
rapid dehydration and breakdown of hydroxyl groups on the
glucose rings to form water molecules. The main chain is
broken during this stage when the C–C–H, C–O and C–C
bonds are broken [9, 13]. The gaseous decomposition prod-
ucts consist mainly of ­H2O, CO, and ­CO2 [9, 14]. S1 and S2
showed ­Td like starch obtained with SBS [9, 15]. In the third
stage (T > 400 °C), inert carbon residues were formed, and
mass loss was stabilized [9, 13].
Starch from Ramon seed (Brosimum alicastrum) obtained by two extraction methods

Fig. 1  a Ramon (Brosimum

alicastrum) tree and b seeds

Fig. 2  SEM micrographs of
RSF, S1 and S2

13

Fig. 3  a TGA/DTGA curves of RSF, S1 and S2, b Gelatinization
curves of S1 and S2
Figure 3b shows the gelatinization phase transition for
S1 and S2 starches. The extracted starches showed similar
values of To, Tg, Tc and ­GELinterv values (Table 1). The tem-
peratures of endothermic transitions in the starches isolated
with the two methods are similar to those found for parota
[3]. The ΔHgel values were similar for both starches but
lower than those obtained for Perez-Pacheco et al. [1], indi-
cating that S1 and S2 require less energy to gelatinize. The
transition temperatures and gelatinization enthalpy of starch
are regulated by several factors such as degree and molecu-
lar architecture of crystallinity, and thus by the molecular
structure of amylopectin, starch composition, granule shape,
and heterogeneities within the granule population [16, 17].
Figure 4 shows the S1 and S2 spectra, and Table 2 pre-
sents the signal assignment. The FTIR spectra present a
band absorption at 3410–3389 ­cm−1 corresponding to the
Table 1  Thermal properties of Sample Td (°C) To (°C)
isolated starches
S1 325.09 ± 1.25 70.77 ± 1.45
S2 325.08 ± 1.26 73.92 ± 0.98
13
S. C. Pech‑Cohuo et al.
stretching vibrations of the hydroxyl groups (–OH) and con-
tributing to the inter and intra-molecular interactions of OH–;
which is a particular characteristic of the starch structure. An
intense band at 2930 ­cm−1 is attributable to stretching vibra-
tions of the C–H bonds. The water absorbed by the starch is
observed between 1650 and 1640 ­cm−1. The bands at 1421
and 1300 ­cm−1 correspond to C–H bonds. The region known
as “fingerprint” characteristic peaks of starch are observed at
1155, 1087, and 1019 ­cm−1, attributed to the vibrations of
glucose C–O–C bonds, and bands at 928, 862, 764, 709, 605,
and 573 ­cm−1, corresponding to the pyranose ring [3, 9].
In starch, the quantity of amorphous or crystalline region is
essential in predicting the response of this biopolymer for the
processing and development of materials [3]. Other authors
have reported that the wavelength band of 1047  ­cm−1 is
related to the ordered region and the band at 1022 ­cm−1 to the
amorphous component. The ratio (bands) of 1022:1047 ­cm−1
represents the order in the starches. In this report, the values
obtained for this ratio were 1.66 for S1 and S2, a value higher
compared with the Ramon starch obtained with SBS that was
1.04.
In plants, starch is stored as a discrete semicrystalline
granule and consists of two main components: linear amyl-
ose and the highly branched amylopectin. According to
their X-ray diffraction (XRD) patterns, there are A-, B-, and
C-type polymorphs of starch. The A-type starches are found
in cereal crops, B-type starches in plant tubers and some high
amylose cereal seeds, and C-type starches in legume seeds
and some rhizomes. A-type starch has two strong diffraction
peaks about 2θ ~ 15° and 23° and a doublet at around 2θ ~ 17°
and 18°, and B-type starch has one strongest diffraction peak
around 2θ ~ 17°, a few small peaks at around 2θ ~ 15°, 22°,
and 24°, and a characteristic peak at about 2θ ~ 5.6° 2 [22].
C-type starch has both A- and B-type crystallinity. The X-ray
diffraction patterns of the S1 and S2 are given in Fig. 5. In
S1 and S2 starch, the highest intensity XRD peaks were
2θ ~ 15°, 17°, 18°, 20°, 23°, and 26°, similar to observed
Moo-Huchim et al.[9]. Therefore, both starches exhibited
C-type crystals.
The percentage of crystallinity obtained was 31.89 ± 1.07%
and 29.61 ± 4.40% for S1 and S2, respectively. Similar values
were obtained with Ramon9 and Huaya [2] starch extracted
with SBS. The above could be attributed to the similarities in
crystallite size, amount of crystalline regions, the orientation
of the double helices within the crystalline domains, or the
extent of interaction between double helices [2, 9]
Tg (°C) Tc (°C) Gelinterv (°C) ΔHgel (J/g)
76.24 ± 1.12 81.35 ± 1.74 10.58 ± 1.60 12.63 ± 0.49
79.01 ± 0.71 83.59 ± 1.41 9.67 ± 1.21 12.70 ± 0.66
Starch from Ramon seed (Brosimum alicastrum) obtained by two extraction methods
Fig. 4  FTIR curves of S1 and S2
Table 2  FTIR bands assignment
of Ramon starch
Fig. 5  X-ray diffraction patterns of S1 and S2
Conclusion
Starch was obtained from Ramon seeds (Brosimum alicas-
trum) through two methodologies with different extraction
medium. Statistical analysis showed no significant differences
in the yield values. The morphology and physicochemical
properties were similar for both processes. However, further
analyses are required to determine the effect of the extraction
medium on other properties such as solubility and rheology.
Wave number ­(cm−1) Assignments for the starch molecule based on its molecular structure
3410–3389 Free, intermolecular, or intramolecular OH groups stretching vibration [3, 9, 18, 19]
2930 CH asymmetrical stretch vibrations in ­CH2 [3, 9, 20]
1650–1640 Water adsorbed; OH bending vibration [3, 9, 19, 21]
1421 CH2 bending of pyranose ring [3, 9, 21]
1300 In plane bending of CH groups [3, 9, 20]
1155 O–C stretch within the pyranose ring [19, 20]
1087 O–C stretch within the pyranose ring [20, 21]
1019 C–O stretch in the ring [20]
928 Pyranose ring, CO bending [20]
862 Pyranose ring, CH bending [20]
764 Pyranose ring, rocking of ­CH2 groups [20]
709 Pyranose ring, out of base bending of hydrogen bonded OH groups [20]
605 Pyranose ring, vibration structural OH groups [20]
573 Pyranose ring stretching vibration [18]
13

Supplementary Information  The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 557/s​ 43580-0​ 21-0​ 0134-w.
Acknowledgements  Postdoctoral Program for Indigenous Mexican
Women in Science, Technology, Engineering, and Mathematics, of
Mexican Council of Science and Technology—Center for Research and
Higher Studies in Social Anthropology—International Development
Research Center-Canada. The technical support from Santiago Duarte-
Aranda (SEM, FTIR), Wilberth Herrera Kao (DSC) and Daniel Aguilar
(XRD) is highly appreciated. The XRD analysis was performed at the
National Laboratory of Nano and Biomaterials (financed by Fomix-
Yucatan and Conacyt-Mexico), CINVESTAV-IPN Merida Unit. The
authors thank Dr. Patricia Quintana for the access to LANNBIO.
10. A. Palacios-Fonseca, J. Castro-Rosas, C. Gómez-Aldapa, T.
Data availability  All data generated or analyzed during this study are
included in this published article and its supplementary information
files.
12. N. Lindeboom, P.R. Chang, R.T. Tyler, Starch-Stärke. 56(3–4),
Declarations  89 (2004)
13. S. Kouser, A. Kareem, R. Kshirsagar, Int. Res. J. Pharm. 8(4), 113
Conflict of interest  The authors declare no conflict of interest.
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