Food Hydrocolloids 112 (2021) 106272

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Food Hydrocolloids
journal homepage: http://www.elsevier.com/locate/foodhyd

In vitro and in vivo digestibility from bionanocomposite edible films based

on native pumpkin flour/plum flour
Tomy J. Gutiérrez
Grupo de Materiales Compuestos Termoplásticos (CoMP), Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA), Facultad de Ingeniería,
Universidad Nacional de Mar del Plata (UNMdP) y Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Colón 10850, B7608FLC, Mar del Plata,
Argentina

A R T I C L E I N F O A B S T R A C T

Keywords: Pumpkin (Cucurbita maxima) flour was used as an unconventional food hydrocolloid source for the development
Food packaging of glycerol-plasticized edible films using the casting methodology. Four film systems were made from thermo­
Nutritional aspects plastic pumpkin flour (TPPF) to evaluate the effect of matrix concentration (2.5% and 5%) and the addition of a
Plasticizing effect
natural filler (bionanocomposite - ‘huesito’ plum (Spondias purpurea) flour), maintaining the same matrix:plas­
Resistant starch
Structure
ticizer:nanofiller ratio. A comprehensive study in terms of the structural, thermal, crystalline, physicochemical,
Thermoplastic flour microstructural and mechanical properties was carried out here. These properties were related to the results
obtained from the digestibility tests of the prepared edible films. This study showed that the increase in the
concentration of the constituents of edible films significantly affected their properties, despite maintaining the
same ratio of the constituents. A plasticizing effect was observed as a result of the increased concentration of the
pumpkin flour matrix. This effect allowed obtaining more hydrophobic and plastic materials, and less digestible.
It is worth noting that a new classification of resistant starch (RS) (type 6 RS) can be suggested based on the
results obtained or the redefinition of the concept of type 5 RS can also be recommended. As for the effect of the
addition of natural nanofiller, at a low concentration of the pumpkin flour matrix, natural nanofiller also caused
a plasticizing effect. An anti-plasticizing effect was, however, determined at a high concentration of the pumpkin
flour matrix. The addition of natural nanofiller in bionanocomposite films did not cause any significant effect on
their digestibility.

1. Introduction replacement of food packaging based on synthetic polymers, which

generates serious environmental problems (Gutiérrez & Alvarez, 2017).
The development of edible films and coatings made from food hy­ Our research group more recently has proposed modified (phosphated
drocolloids to be used as a polymeric matrix of food packaging has been and methylated) pumpkin (Cucurbita maxima) flour (phosphated and
widely studied during the last years in terms of their physicochemical, methylated) as a novel source of nonconventional food hydrocolloids for
mechanical and thermal properties (Álvarez, Famá, & Gutiérrez, 2017). the development of edible films and coatings (Gutiérrez, 2018b). In
Special attention has been given to the use of starch as a food hydro­ contrast, Jacek, Izabela, and Daria (2017), Przetaczek-Rożnowska and
colloid source for the development of these materials, due to their Fortuna (2017) and Przetaczek-Rożnowska (2017) only have reported
different advantages, which have been well established in the literature recently the comprehensive characterization of native and modified
(Gutiérrez, Herniou-Julien, Álvarez, & Alvarez, 2018). Nonetheless, pumpkin starches.
flour-based films have been shown to have better properties than According to Lovera, Pérez, and Laurentin (2017) nutritional studies
starch-based films manufactured from the same source of carbohydrate on starch are scarce and many involve limited studies of in vitro di­
polymers (Gutiérrez & Alvarez, 2017; Pelissari, Andrade-Mahecha, gestibility using a ‘pool’ of enzymes. In this sense, our research group
Sobral, & Menegalli, 2013). These results have been interesting and has reported some nutritional studies in terms of resistant starch (RS)
promising, since the production of flours is more economical than the content and in vitro digestibility in starch-based films (Gutiérrez, 2017b;
obtaining of starch, so the use of this resource as biopolymeric matrices Gutiérrez, Herniou-Julien, et al., 2018; Gutiérrez & Álvarez, 2016). To
of edible films and coatings, is a more feasible solution for the the author’s knowledge, this is the first work where nutritional aspects

E-mail addresses: tomy.gutierrez@fi.mdp.edu.ar, tomy.gutierrez@intema.gob.ar, tomy_gutierrez@yahoo.es.

https://doi.org/10.1016/j.foodhyd.2020.106272
Received 12 July 2020; Received in revised form 22 August 2020; Accepted 22 August 2020
Available online 27 August 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

are evaluated in terms of RS content and in vitro and in vivo digestibility edible films prepared by the casting methodology from using a 2.6:1
in starch-based films. A novel methodology using insects (weevils) has (matrix:glycerol) ratio and 500 mL of distilled water. Two groups of
been used in this study to evaluate in vivo digestibility (Lovera et al., films were made with matrix concentrations of 2.5% w/v and 5% w/v of
2017). The use of weevils to carry out in vivo digestibility bioassays lies flour, maintaining the same matrix:glycerol ratio with and without the
in the fact that they are easy to handle, reproducible, inexpensive and addition of 4% w/w plum flour with respect to the weight of matrix. The
not subject to ethical and legal regulations compared to in vivo studies film-forming solutions (FFS) were heated for 30 min at 90 ◦ C in a water
using animals (Law & Wells, 1989). Pérez-Navarrete, Betancur-Ancona, bath with constant stirring to ensure gelatinization (Gutiérrez & Álvarez,
Casotto, Carmona, and Tovar (2007) evaluated the in vitro digestibility 2016). Plum flour was added to some of the film systems 2 min before
of corn and bean flour using adult rice weevils (Sitophilus oryzae L., the end of the FFS process in order to preserve the anthocyanins (pig­
Coleoptera: Curculionidae). Lovera et al. (2017) also reported in vitro ments) and ensure a homogeneous distribution of the natural nanofiller.
digestibility in unconventional starches from different sources using the After gelatinization the FFS were degassed for 30 min by applying a
same kind of weevil. Nevertheless, the rice weevil was replaced by the vacuum. They were then poured into 40 × 30 cm stainless steel trays
furniture weevil (Tricorynus sp, Coleoptera: Anobiidae) in this study. maintaining a constant level and uniform thickness, and left in a tray
This modification was made since the furniture weevil can feed on all the dehydrator (Mitchell, USA, Model 645,159) for 24 h at 45 ◦ C. Once
components of the flour matrix (starch, fiber, protein and fat) unlike the dried, the four thermoplastic pumpkin flour (TPPF) film systems: at
rice weevil, which is only capable of digesting starch. In addition, the 2.5% (TPPF 2.5%), at 2.5% plus plum flour (TPPF 2.5%/P), at 5% (TPPF
Food and Agriculture Organization of the United Nations (FAO, 1985) 5%) and at 5% plus plum flour (TPPF 5%/P) were peeled off the trays.
has recognized that furniture weevils are one of the main pests in stored The resultant materials were conditioned with an atmosphere of satu­
starch-based foods. rated NaBr solution (aw ~0.575 at 25 ◦ C) for seven days before each test.
The novelty of this work lies in the exhaustive structural, physico­ During this period, the containers were protected from light in a dark
chemical, thermal, surface, morphological and mechanical character­ room in order to avoid photodegradation of the pigments.
ization of films made from an unconventional hydrocolloid source such
as native pumpkin flour. It is also worth noting that this study provides
some nutritional aspects in terms of RS content, and in vitro and in vivo 2.3. Film characterizations
digestibility, which has been poorly studied in starch-based food prod­
ucts. In addition, a novel methodology was used to determine the in vivo 2.3.1. Attenuated total reflectance Fourier transform infrared (ATR/FTIR)
digestibility of edible starch-based films. It is also important to note that spectroscopy
Gutiérrez (2018b) previously used only in vivo digestibility tests on The FTIR spectra were acquired with a Nicolet 8700 FTIR spec­
modified pumpkin flour-based films to study their ecotoxicity from an trometer (Thermo Scientific Instrument Co., Madison, Wisconsin, USA)
ecological approach. In contrast, this study delved into other di­ using the single reflection horizontal ATR accessory Smart Orbit, and a
gestibility tests to address this research towards the nutritional aspects diamond crystal at an incident angle of 45◦ , varying the absorption
of edible films made from native pumpkin flour. range from 650 to 4000 cm− 1, with 32 co-added scans at a 4 cm− 1
The goal of this work was to evaluate comprehensively the effect of resolution and room temperature (25 ◦ C).
the concentration of the pumpkin flour matrix, as well as the effect of the
addition of a natural nanofiller (bionanocomposite): ‘huesito’ plum 2.3.2. Thermogravimetric analysis (TGA) & differential scanning
(Spondias purpurea) flour obtained from peel of fruits. The hypothesis of calorimetry (DSC)
this work is based on the fact that the addition of the natural nanofiller The tests were carried out in a thermal analyzer (Shimadzu DTG-60,
used can act as a reinforcement of the matrix, while increasing the Japan). Each film systems, weighing between 3.98 and 8.61 mg, was
concentration of the pumpkin flour matrix could improve all the prop­ subjected to heating from room temperature up to 500 ◦ C at a constant
erties evaluated, especially those related to the nutritional aspects of the rate of 10 ◦ C/min under a nitrogen flow of 30 mL/min. Three replicates
films. This could increase the boom in edible flour-based films in the per sample were analyzed to ensure repeatability. The residual mass of
food industry. the materials was recalculated on dry basis and the different degradation
phases noted. The standard deviation (SD) was lower than 1% for all the
2. Experimental systems tested, and the representative curves of each one, were reported.
The DSC thermograms were acquired by a Mettler Toledo DSC 823,
2.1. Materials (Schwerzenbach, Switzerland) and used to determine the glass transi­
tion temperature (Tg) and the heat capacity change (ΔCp) associated
Native (unmodified) pumpkin (Cucurbita maxima) flour from the with the amorphous phase of all films in study. The Tg was measured as
edible part of the pumpkin, and the ‘huesito’ plum (Spondias purpurea) the temperature in the middle of the relaxation range and the ΔCp as the
flour from the fruit peel were obtained by the method described by difference in heat flow. Samples between 3.6 and 4.1 mg were weighed,
Pacheco (2001). The pumpkins and plums were acquired at a local placed individually in aluminum pans and hermetically sealed; an empty
market in Caracas, Venezuela. The flours were stored in dark containers pan was used as a reference. Initially, samples were heated at a rate of
for 1 week at room temperature (25 ◦ C) before preparing the films, in 20 ◦ C/min from 30 ◦ C to 110 ◦ C under nitrogen atmosphere to remove
order to avoid oxidative damage to the materials used. The native previous thermal history and moisture (Gutiérrez, Herniou-Julien, et al.,
pumpkin flour obtained was characterized in terms of its moisture 2018). They were then cooled down to − 70 ◦ C using an intra-cooler and
content (12 ± 4%), total proteins (5.37 ± 0.04%), crude fat (0.6 ± re-heated to 110 ◦ C at a heating rate of 10 ◦ C/min. Two samples of each
0.2%), ash (6.88 ± 0.06%), crude fiber (5.5 ± 0.2%), total carbohy­ film system were tested to ensure repeatability. All the thermograms
drates (75.2 ± 0.1%) and amylose content (28 ± 6%). The proximal shown refer to the second heating.
composition and the nanometer scale (~200 nm) of the plum flour was
previously reported by us elsewhere (Gutiérrez, 2018b). Food grade 2.3.3. Moisture content (MC), water activity (aw) & water solubility (WS)
glycerol (Aldrich, product code - G7893) was used as a plasticizer, and The MC was determined thermogravimetrically by cutting samples of
the plum flour as a natural nanofiller (bionanocomposite). each system into 2 × 2 cm pieces (approx. 0.5 g), being placed them in
an oven at 105 ◦ C for 24 h, and then weighing each one with an
2.2. Film formation analytical balance (Denver Instrument APX-200). The procedure was
performed in triplicate for each film system and the MC was calculated
Native pumpkin flour was used as matrix for the development of using Eq. (1):

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Fig. 1. (A) FTIR spectra of the different films studied in all the absorption range and (B) Normalized FTIR spectra in the absorption range corresponding to C–O group
(OH stretching) of the different films studied. Thermoplastic pumpkin flour (TPPF) film systems at: (a) 2.5% (TPPF 2.5%), (b) 2.5% plus plum flour (TPPF 2.5%/P),
(c) 5% (TPPF 5%) and (d) 5% plus plum flour (TPPF 5%/P).

MC (%) =
wi − wf
× 100 (1) The aw of the films was determined using a psychrometric aw meter
wi Aqualab Cx-2 (Decagon Devices, Pullman, USA) previously calibrated
with water at 25 ◦ C. The average value of three measurements was re­
where, wi and wf, are the initial and final weights of each sample. The ported ± SD.
results were expressed as mean values ± SD. The WS percentage of the films was determined gravimetrically.

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Square-shaped sample pieces (2 × 2 cm) of each film systems weighing

~0.5 g was dried in an oven at 105 ◦ C for 24 h. After that, its initial mass
was recorded, and the sample was placed in 50 mL of distilled water at
room temperature (22 ◦ C) for 24 h. The solution was then removed, and
the remaining films were again placed in an oven at 105 ◦ C for 24 h.
Finally, its final weight was recorded and the WS percentage was
determined using Eq. (2):
wi − wf
WS (%) = × 100 (2)
w

where, wi and wf, are the initial and final weights, respectively, of each
sample. The experiments were carried out in triplicate and the results
expressed as mean values ± SD.

2.3.4. X-ray diffraction (XRD)

X-ray diffractograms of the different film systems were acquired
using an X-ray diffractometer (Siemens D 5000) (monochromatic Cu Kα
Fig. 2. TGA curves of the different films studied based on thermoplastic
radiation source - λ = 1.5406 Å) operating at a voltage of 40 kV and
pumpkin flour (TPPF) at: (a) 2.5% (TPPF 2.5%), (b) 2.5% plus plum flour (TPPF
2.5%/P), (c) 5% (TPPF 5%) and (d) 5% plus plum flour (TPPF 5%/P), in
current 30 mA. Scattered radiation was detected in a 2θ angular range
dry basis. from 3 to 33◦ at a scanning rate of 1◦ /min. The distances between the
planes of the crystals d (Å) were calculated from the diffraction angles
(o) measured from the X-ray diffractograms according to Bragg’s law:

Fig. 3. Heating thermograms of the different films studied based on thermoplastic pumpkin flour (TPPF) at: (a) 2.5% (TPPF 2.5%), (b) 2.5% plus plum flour (TPPF
2.5%/P), (c) 5% (TPPF 5%) and (d) 5% plus plum flour (TPPF 5%/P).

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Table 1 kHz was used to obtain topographic images of the films. The tips were 2
Moisture content (MC), water activity (aw), water solubility (WS), water contact mm long and V-shaped with a spring constant of 0.2 N/m, and were
angle (WCA), thickness (e), opacity (Op) and color parameters of the different positioned over the sample under ambient conditions. The AFM images
films evaluated. were taken at the center and periphery of the surface of the films. The
Parameters TPPF 2.5% TPPF 2.5%/ TPPF 5% TPPF 5%/P surface analyzed was the side of the surface exposed to the drying air
P during film preparation. The average roughness (Ra) of the exposed
MC (%) 52 ± 2b 30 ± 5a 25 ± 3a 29 ± 5a dried surfaces was also determined. The AFM images were processed
aw 0.602 ± 0.603 ± 0.588 ± 0.588 ± with PicoView image software.
0.001b 0.001b 0.001a 0.002a An USB Digital Microscope (model DIGMIC200X, China - contact
WS (%) 76.3 ± 0.4c 76 ± 2b,c,d 71.0 ± 0.5a 74.1 ± 0.7b
WCA (o) 30 ± 5a 43 ± 3c 34 ± 3a,b 27.6 ± 0.6a
angle goniometer) equipped with an Image Analysis Software 220X 2.0
e (mm) 0.18 ± 0.01a 0.18 ± 0.02a 0.20 ± 0.06a 0.27 ± MP video, with 0.01◦ precision and operating at 25 ◦ C and under
0.02a,b ambient conditions was used to determine the WCAs (θ). A drop of
Op 0.98 ± 0.01a 1.1 ± 0.1b 1.23 ± 0.02c 1.55 ± distilled water (2 μL) was placed on the surface exposed to the drying air
0.02d
during film preparation using a syringe (KDL Corp., Shanghai, China).
L* 20.4 ± 0.3a 22.63 ± 22.98 ± 20.10 ±
0.05b 0.04c 0.03a WCAs were then calculated from the intersection of the liquid-solid
a* - 0.9 ± 0.1a 2.11 ± 0.02b 2.23 ± 0.08c 4.43 ± interface (drop of water-surface of the film) and the liquid-vapor inter­
0.04d face (tangent to the boundary of the drop) (Gutiérrez & González, 2017).
b* 17.3 ± 0.6a 31.6 ± 0.2c 32.2 ± 0.1d 28.2 ± 0.3b The WCA were acquired taking note of the recommendations made by
Color differences 74.8 ± 0.5a 77.06 ± 76.99 ± 78.29 ±
Gutiérrez and González (2016) in order to avoid false results caused by
(ΔE) 0.09b 0.06b 0.07c
Whiteness index 18.6 ± 0.5c 16.40 ± 16.48 ± 15.15 ± phenomena such as dehydration, dissolution and swelling. The WCAs
(WI) 0.09b 0.06b 0.06a were expressed as the average value of 12 measurements per film ± SD.
Yellow index (YI) 74 ± 3a 112.3 ± 0.4b 113.0 ± 0.2c 117.3 ± A representative image of each film system was also taken.
0.7d

Equal letters in the same row indicate no statistically significant differences (p ≤ 2.3.6. Thickness (e), opacity (Op) & color parameters
0.05). Film systems based on thermoplastic pumpkin flour (TPPF) at: 2.5% The e of each flour film was determined by measuring samples at
(TPPF 2.5%), 2.5% plus plum flour (TPPF 2.5%/P), 5% (TPPF 5%) and 5% plus eighteen different locations per sample using a micrometer (Liuling,
plum flour (TPPF 5%/P). Shanghai, China) with an accuracy of ±0.001 mm. Results were re­
ported as mean values ± SD.
The Op was determined by the procedure described by Gutiérrez
(2017a). The ultraviolet (UV) and visible light barrier properties of dried
films were measured using a UV–Vis spectrophotometer (u-2001, Japan)
at selected wavelengths between 400 and 800 nm. Film opacity was then
measured at 600 nm and calculated using the following equation given
by Sukhija, Singh, and Riar (2016):
Opacity = A600/ (4)
e

where: A600 = the absorbance at 600 nm and “e” = film thickness (mm).
The following color parameters of the films: L* where L* = 0 in­
dicates black and L* = 100 white), a* (position between red and green)
where negative values indicate green while positive values indicate
magenta, and b* (position between yellow and blue) where negative
values indicate blue and positive values indicate yellow, were measured
using a Macbeth® colorimeter (Color-Eye 2445 model, illuminant D65
and 10◦ observer) standardized with a white reference plate (L* = 93.54,
Fig. 4. X-ray diffraction patterns of the different films studied based on ther­ a* = − 0.81 and b* = 1.58). Color differences (ΔE) were measured by the
moplastic pumpkin flour (TPPF) at: 2.5% (TPPF 2.5%), 2.5% plus plum flour
magnitude of the vector resulting from the three components: brightness
(TPPF 2.5%/P), 5% (TPPF 5%) and 5% plus plum flour (TPPF 5%/P).
difference (ΔL), red-green chromaticity difference (Δa) and yellow-blue
chromaticity difference (Δb) (Gutiérrez, Ponce, & Alvarez, 2017), were
d(Å) =
nλ
(3) calculated by the following equation:
2sin (θ) √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE = Δa2 + Δb2 + ΔL2 (5)
where λ is the Cu Kα radiation wavelength, and n the order of reflection.
For the calculations n was taken as 1. The thicknesses of the samples on where Δa = a*reference plate - a*sample, b*reference plate - b*sample and ΔL =
the slides were ~0.21 mm. L*reference plate - L*sample.
The yellowness index (YI) which, as its name suggests, determines
2.3.5. Optical microscopy, atomic force microscopy (AFM) & water contact the degree of yellowness of a substance, was calculated according to
angle (WCA) ASTM D1925-70 (1988), using the CIELAB scale:
Small pieces (2 cm × 1 cm) of each film system were mounted onto
100 (1277X − 1067Z)
glass slides and observed with an optical microscope (Olympus BX60M, YI = (6)
Y
Japan) at 50 × of magnification. A video camera imaging system
(Olympus IMAGE RS) was used to examine and photograph the film where X, Y and Z are the tristimulus values in the CIELAB scale.
surfaces exposed to the drying air. At least three microphotographs of
each system were taken. 2.3.7. Field emission scanning electron microscopy (FESEM) & uniaxial
An Agilent 5500 AFM in the Acoustic AC Mode (AAC Mode) with tensile tests
silicon nitride (Si3N4) tips and a cantilever oscillation frequency of 155 The morphology of the cryo-fractured surface of each film system

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Fig. 5. Optical micrographs of the films based on thermoplastic pumpkin flour (TPPF) at: (a) 2.5% (TPPF 2.5%), (b) 2.5% plus plum flour (TPPF 2.5%/P), (c) 5%
(TPPF 5%) and (d) 5% plus plum flour (TPPF 5%/P). At 50 × of magnification.

was observed using a FESEM Supra 55, Zeiss (Oberkochen, Germany) solutions at 1 and 13. At the end of the trial changes in appearance of
microscope operating at an acceleration voltage of 3 kV. The films were samples were recorded with the same Sony camera indicated above. At
previously cryo-fractured by immersion in liquid nitrogen, mounted on least six samples of each film system were tested in order to ensure good
bronze stubs and sputter coated (Sputter coater SPI Module, Santa Clara, reproducibility.
CA, USA) with a thin layer of gold for 35 s. All samples were observed at
a 2.0 k × magnification. 2.3.9. Determination of resistant starch (RS)
The uniaxial tensile tests were carried out in an Instron dynamom­ RS content was determined following the official AOAC (2003)
eter (Instron Ltd., High Wycombe, UK) (5 Lbs) working at a crosshead method. RS yield was determined as:
speed of 0.02 in/sec was used to obtain the load (N)-distance (mm)
Residue mass (g)
curves of the film systems evaluated. At least ten specimens of each RS (%) = × 100 (dry weight basis) (7)
Sample mass (g)
system were previously cut in a bone-shape with an effective area of
~10 mm long × ~0.5 mm width and cross-sectional area ~1.14 mm2 Analyses were performed in triplicate to ensure repeatability. Results
(the exact values differed slightly between samples) were tested at room were reported as average values ± SD.
temperature (25 ◦ C) following the ISO 527-2 (2012) norm. The
load-distance curves were then transformed into stress (MPa)-strain (%) 2.3.10. In vitro digestibility tests - starch hydrolysis index
curves to determine the following mechanical parameters: Young’s The in vitro rate of starch hydrolysis was evaluated using the meth­
modulus (E), maximum stress (σ m), strain at break (εb) and toughness odology proposed by Gutiérrez and Álvarez (2016). Data were plotted as
(T). The elastic modulus or E was measured from the slope of the linear degree of hydrolysis versus time curves. The glycemic index (GI) was
regression of the stress-strain curves where the σ m value is the maximum estimated using the model proposed by Goñi, Garcia-Alonso, and
point of each curve, and the εb corresponds to the maximum elongation Saura-Calixto (1997):
of the samples before rupture. T was determined as the area under the
GI = 39.21 + 0.803 × H90 (8)
stress-strain curves.
where H90 = percentage of starch hydrolysis at 90 min.
2.3.8. Response to pH changes & stability in acidic or alkaline solutions
To evaluate the response to pH changes of the films square shape
2.3.11. In vivo digestibility tests
samples (2.5 × 2.5 cm) of each film system were placed in Petri plates
The in vivo digestibility tests of films were carried out using the
containing 20 mL of solutions at pHs of 1, 7 and 13, prepared from HCl
furniture weevil (Tricorynus sp, Coleoptera: Anobiidae). Two biomarkers
(0.1 M) and NaOH (0.1 M). Film response was then evaluated from
were evaluated: survival rate and weight variations.
images taken with an 8.1 mega pixel Cyber-shot Sony camera; model
DSC-H3 (Tokyo, Japan).
2.3.11.1. Furniture weevil culture & diet preparation. The weevils were
The stability of the films in acidic and alkaline solutions was evalu­
maintained using the methodology described by Lovera et al. (2017).
ated by maintaining the film systems previously tested for the deter­
The films were dried in an oven at 105 ◦ C for 72 h before milling and
mination of the response to pH changes during 24 days at 25 ◦ C in pH

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Fig. 6. AFM images of the surface of the films based on thermoplastic pumpkin flour (TPPF) at: (a) 2.5% (TPPF 2.5%), (b) 2.5% plus plum flour (TPPF 2.5%/P), (c)
5% (TPPF 5%) and (d) 5% plus plum flour (TPPF 5%/P).

passing them through a 60-mesh sieve. This was done to avoid distor­ were shown as mean values ± SD. Differences between the mean values
tions in the results due to variations in the moisture content of the films, of the measured properties were compared using a multiple-range
as well as to guarantee a particle size that did not affect the values of the Tukey’s test. A significance level of 0.05 was used.
evaluated biomarkers.
3. Results and discussion
2.3.11.2. Survival rate & weight variations of the weevils. To carry out the
bioassays from weevils, they were deposited in four recipients (30 3.1. Attenuated total reflectance Fourier transform infrared (ATR/FTIR)
weevils per recipient) and fed with 3 g of the previously prepared diets. spectroscopy
The recipients were covered with a mesh to prevent the insects from
escaping. Bioassays were performed at room temperature (30 ◦ C) over a All evaluated film systems exhibited similar FTIR spectra (Fig. 1A).
period of 8 days. Every two days the weevils were weighed and any dead Similar results were similar reported by Gutiérrez (2017a) for cassava
ones discarded. The tests were carried out in triplicate. The biomarkers (Manihot esculenta C.) and taro (Colocasia esculenta L. Schott)
were then calculated using Eqs (9) and (10): starch-based films. Differences in the band intensities associated with
the stretching of the O-H bonds (3305 cm-1) were, however, observed
Survival rate (%) = Nlw/ × 100 (9)
Inw (Fig. 1B). A higher intensity of this band has been associated with a
greater degree of freedom to vibrate the O-H bond, as a consequence of
where Nlw = number of live weevils on day “n” and Inw = initial the sparse intermolecular hydrogen (H)-bonding interactions between
number of weevils. the starch chains (main component of flour) and the plasticizer (glyc­
Weight variation (%) = wf − wi
/
× 100 (10) erol) or the restricted intermolecular H-bonding interactions between
wi
the starch matrix and the added natural nanofiller (bionanocomposite -
where wi is initial average weight and wf the average weight on day “n”. plum flour), which has led to a greater hydrophilic nature of the
starch-based film systems (Gutiérrez, 2018c). With this in mind, the
TPPF 2.5% film can be predicted to be more hydrophilic than the TPPF
2.4. Statistical analysis 5% film. This suggests that the increase in the concentration of the
pumpkin flour matrix from 2.5% to 5% had a positive effect in reducing
Results were previously analyzed by t-student test, with a value of p water sensitivity, even using the same matrix:glycerol ratio, i.e. a higher
< 0.05 and confidence interval 95% to delete anomalous data. Analyses concentration of the matrix would increase the amount of compensatory
of variance (ANOVA) using OriginPro 8 (Version 8.5, Northampton, H-bonding interactions between the pumpkin flour matrix and the
USA) software were them employed to analyze the data, and the results

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Fig. 7. FESEM micrographs of the cryogenic fracture surface of the different films studied based on thermoplastic pumpkin flour (TPPF) at: (a) 2.5% (TPPF 2.5%), (b)
2.5% plus plum flour (TPPF 2.5%/P), (c) 5% (TPPF 5%) and (d) 5% plus plum flour (TPPF 5%/P). At 2.0 k × of magnification.

plasticizer (glycerol). The TPPF 2.5%/P film had a lower band intensity
than the TPPF 2.5% film (Fig. 1B), i.e. that a reduction in water sensi­
tivity for the first film system compared to the second can be expected.
This means that the addition of the natural nanofiller had a positive
effect in reducing the water sensitivity in film systems at low concen­
trations (2.5%). However, an inverse trend was observed with
increasing matrix concentration (compared film systems: TPPF 5% and
TPPF 5%/P), even maintaining the same matrix:plasticizer:nanofiller
ratio used for the TPPF 2.5% and TPPF 2.5%/P film systems.

3.2. Thermogravimetric analysis (TGA) & differential scanning

calorimetry (DSC)

The TGA curves of the analyzed film systems showed three stages of
thermal degradation (Fig. 2), which have been well described in the
literature (Sun, Liu, Ji, Hou, & Dong, 2018): 1) evaporation of the free
water and adsorbed moisture, 2) thermal degradation of the starch-rich
Fig. 8. Stress (σ) – strain (ε) curves of the different films studied based on phase, which also contains glycerol and 3) the elimination of the starch
thermoplastic pumpkin flour (TPPF) at: (a) 2.5% (TPPF 2.5%), (b) 2.5% plus polyhydroxyl groups, as well as depolymerization and oxidation of the
plum flour (TPPF 2.5%/P), (c) 5% (TPPF 5%) and (d) 5% plus plum flour (TPPF partially degraded starch matrix, thus yielding carbon and ash.
5%/P). Films containing a higher concentration of the pumpkin flour matrix
show an increase in thermal resistance in the range associated with the
glycerol-rich phase. This was observed independent of the addition or
Table 2 not of the added natural nanofiller (see box at the top right of Fig. 2). It is
Parameters of the uniaxial tensile tests: strain at break (εb), maximum stress (σm), worth noting that this fact was observed using the same matrix:glycerol:
Young’s modulus (E) and toughness (T).
nanofiller ratio. Thus, an increase in the concentration of the pumpkin
Materials εb (%) σm (MPa) E (MPa) T ( × 105) (J/m3) flour matrix improved the intermolecular H-bonding interactions be­
TPPF 2.5% 11.6 ± 0.9a
0.13 ± 0.01a
2.3 ± 0.4a
0.01 ± 0.10a tween the pumpkin flour matrix and glycerol. This would be in line with
TPPF 2.5%/P 19 ± 2b 0.15 ± 0.01a 2.0 ± 0.6a 0.01 ± 0.10a the results predicted from the FTIR spectra (see section 3.1).
TPPF 5% 19 ± 2b 0.28 ± 0.02c 2.1 ± 0.3a 0.03 ± 0.10a The addition of the natural nanofiller improved the thermal resis­
TPPF 5%/P 19 ± 2b 0.21 ± 0.04b 2.3 0.2a 0.02 ± 0.10a
tance in the glycerol-rich phase of the TPPF 2.5%/P film compared to
±

Equal letters in the same column indicate no statistically significant difference the TPPF 2.5% film (at low concentration of pumpkin flour matrix). An
(p ≤ 0.05). Film systems based on thermoplastic pumpkin flour (TPPF) at: 2.5% opposite trend was, however, observed by increasing the concentration
(TPPF 2.5%), 2.5% plus plum flour (TPPF 2.5%/P), 5% (TPPF 5%) and 5% plus of the pumpkin flour matrix from 2.5% to 5%, even maintaining the
plum flour (TPPF 5%/P). same matrix:plasticizer:nanofiller ratio used for the TPPF 2.5% and

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Fig. 9. Response at different pH conditions of the films studied based on thermoplastic pumpkin flour (TPPF) at: (a) 2.5% (TPPF 2.5%), (b) 2.5% plus plum flour
(TPPF 2.5%/P), (c) 5% (TPPF 5%) and (d) 5% plus plum flour (TPPF 5%/P).

TPPF 2.5%/P film systems. This fact suggests that the added natural 3.1). An opposite effect (anti-plasticizing effect) was observed by
nanofiller improved the compensatory H-bonding interactions at low increasing the concentration of the pumpkin flour matrix (compared
concentrations of pumpkin flour matrix (2.5%), but a contrary effect was film systems: TPPF 5% (Fig. 3c) and TPPF 5%/P (Fig. 3d)). This is also in
evident by increasing the concentration of pumpkin flour matrix to 5%. line with the observations obtained from the FTIR spectra (see section
This agrees with the results observed from the FTIR spectra, i.e. at low 3.1). Similar results were also reported by Chang, Abd Karim, and Seow
concentration of pumpkin flour matrix (2.5%). Thus, the natural nano­ (2006) for tapioca starch films plasticized with different concentrations
filler improved the intermolecular H-bonding interactions between the of glycerol: 0, 2.5, 5, 10, 15 and 20%.
glycerol, matrix and natural nanofiller, thus reducing the degrees of It is important to note again that all films were made using the same
freedom of the OH groups of glycerol and starch, since they would be matrix:plasticizer:nanofiller ratio. Thus, the interactions given between
involved in the formation of compensatory H-bonding interactions be­ the constituents of the films depend on their concentration and not on
tween them. This would eventually lead to a lower water susceptibility their ratio.
for the TPPF 2.5%/P film compared to TPPF 2.5% film. An inverse trend
could thus be expected due to the increase in the concentration of 3.3. Moisture content (MC), water activity (aw) & water solubility (WS)
pumpkin flour matrix (5%).
On the other hand, the TPPF 5% film had a lower glass transition The TPPF 5% film showed significantly lower MC, aw and WS values
temperature (Tg) (28 ◦ C) than the TPPF 2.5% film (33 ◦ C) (Fig. 3a and c). (p ≤ 0.05) than the TPPF 2.5% film (Table 1). Thus, the increase in the
As is well known, a reduction in Tg values is associated with an increase concentration of the pumpkin flour matrix reduced the water sensitivity
in the mobility of amorphous polymer chains (Gutiérrez, Suniaga, of these films. These results were expected based on the analyzes per­
Monsalve, & García, 2016). This behavior is typical of a plasticizing formed from the FTIR spectra (see section 3.1).
effect (Medina Jaramillo, Gutiérrez, Goyanes, Bernal, & Famá, 2016). Keeping in mind the previous discussions, the TPPF 2.5%/P film
Thus, an increase in the concentration of the pumpkin flour matrix led to should be a more hydrophobic material than the TPPF 2.5% film. In this
a plasticizing effect, despite the fact that all the films were manufactured sense, the TPPF 2.5%/P film showed MC values significantly lower (p ≤
using the same matrix:plasticizer:nanofiller ratio. This fact could be 0.05) than the TPPF 2.5% film. No statistically significant differences (p
explained due to the better compensatory H-bonding interactions given ≥ 0.05) were, however, observed in terms of aw and WS values for the
in the TPPF 5% film compared to TPPF 2.5% film, as a result the plas­ TPPF 2.5% and TPPF 2.5%/P films (Table 1).
ticizing effect was observed. These results fit well with the analyzes With respect to the effect of natural nanofiller at high concentrations
previously done here. It is also worth noting that the TPPF 5% film was of the pumpkin flour matrix (compared film systems: TPPF 5% and TPPF
more stable than the TPPF 2.5% film. This was observed by the increase 5%/P), the TPPF 5% film was expected to be a less hydrophilic material
in the heat capacity change (ΔCp) values for the TPPF 5% film with than the TPPF 5%/P film. Lower WS values (p ≤ 0.05) for the TPPF 5%
respect to the TPPF 2.5% film. It could hitherto be suggested that an film compared to the TPPF 5%/P well support the expected results
increase in the concentration of the pumpkin flour matrix improves the (Table 1). The TPPF 5%/P film was slightly more water soluble than the
compensatory H-bonding interactions, resulting in more stable films, as TPPF 5% film (p ≥ 0.05) (Table 1). This is in line with the expected
a consequence of the observed plasticizing effect. results. No statistically significant differences (p ≥ 0.05) were, however,
As regards the effect of the addition of natural nanofiller, at low determined in the MC and aw values for the TPPF 5% and TPPF 5%/P
concentrations of the pumpkin flour matrix (compared film systems: films (Table 1).
TPPF 2.5% (Fig. 3a) and TPPF 2.5%/P (Fig. 3b)), the natural nanofiller
caused a plasticizing effect, resulting in more stable films, due to the
formation of better compensatory H-bonding interactions (see section

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Fig. 10. Digital photographs of the different films evaluated after 9 and 24 days immersed in alkaline medium. Film systems based on thermoplastic pumpkin flour
(TPPF) at: 2.5% (TPPF 2.5%), 2.5% plus plum flour (TPPF 2.5%/P), 5% (TPPF 5%) and 5% plus plum flour (TPPF 5%/P).

3.4. X-ray diffraction (XRD) García-Tejeda et al. (2013) indicated that the peak located around
d-spacings ≅ 16.9 Å for edible films based on native and oxidized ba­
All the film systems analyzed showed X-ray diffraction patterns nana starch may be associated with interactions between the short
typical of semi-crystalline biopolymers (Fig. 4). All films were almost external amylopectin chains and the glycerol. The peaks with d-spacings
totally amorphous (crystallinity percentage < 1%). These results ≅ 4.4 Å, 4.7 Å, 6.5 Å and 6.9 Å are indicative of a V-type structure, and
confirm that the starch contained in the pumpkin flour matrix was are associated with glycerol-starch H-bonding interactions (Gutiérrez &
completely gelatinized (Merino, Mansilla, Gutiérrez, Casalongué, & Alvarez, 2018). These results suggest that the properties of the films
Alvarez, 2018). made are determined by the compensatory H-bonding interactions.
The peaks displayed at d-spacings ≅ 4.1 Å, 4.9 Å and 5.1 Å were
attributed to the presence of a type A structure, while the peaks
exhibited at d-spacings ≅ 3.7 Å, 4.1 Å and 16.1 Å were related to the
type B structure (Gutiérrez & Álvarez, 2016). In particular,

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

Fig. 11. (A) Resistant starch content and (B) in vitro α-amylolysis curves of the different films studied. (C) Survival rate and (D) weight variations of weevils fed from
a diet based on the different films studied. Film systems based on thermoplastic pumpkin flour (TPPF) at: 2.5% (TPPF 2.5%), 2.5% plus plum flour (TPPF 2.5%/P),
5% (TPPF 5%) and 5% plus plum flour (TPPF 5%/P).

3.5. Optical microscopy, atomic force microscopy (AFM) & water contact film could also be attributed to a higher surface roughness in the former
angle (WCA) compared to the latter. Therefore, the surface roughness caused physical
impediments to the collapse of the drop of water. This fits well with the
All the manufactured films were almost totally amorphous (Fig. 5). postulates provided by Vogler (1998). On the other hand, the TPPF
This fits well with the low crystallinity values (<1%) obtained from XRD 2.5%/P film showed WCA values significantly higher (p ≤ 0.05) than the
patterns of these same film systems (see section 3.4). TPPF 2.5% film (Table 1). This can be well explained by: 1) the better
The maximum roughness values for the tested films showed the compensatory H-bonding interactions for the TPPF 2.5%/P film
following ascending order: TPPF 2.5% (70 nm) < TPPF 2.5%/P (0.325 compared to the TPPF 2.5% film and 2) the higher roughness from the
μm) < TPPF 5% (0.4 μm) < TPPF 5%/P (0.8 μm) (Fig. 6). Surface TPPF 2.5%/P film with respect to the TPPF 2.5% film. It should be noted
roughness values on the micrometer scale were also reported by Fazeli, that at high concentrations of the pumpkin flour matrix (TPPF 5% and
Keley, and Biazar (2018) for cellulose nanofiber-reinforced starch-based TPPF 5%/P films), although the matrix:plasticizer:nanofiller ratio was
composite films. The bionanocomposite films (TPPF 2.5%/P and TPPF the same, this trend was opposite compared to films made at low con­
5%/P) were rougher than their analogous non-bionanocomposite films centrations of the pumpkin flour matrix (TPPF 2.5% and TPPF 2.5%/P
(TPPF 2.5% and TPPF 5%). A similar trend was observed by Balak­ films). The lower WCA values (p ≤ 0.05) for the TPPF 5%/P film
rishnan, Gopi, M S, and Thomas (2018) for cellulose-reinforced pota­ compared to the TPPF 5% film (Table 1) could thus be related to the
to-based bionanocomposite films. anti-plasticizing effect caused by the natural nanofiller at high concen­
According to Gutiérrez, Ollier, and Alvarez (2018), the surface trations of the pumpkin flour matrix (see section 3.2), thus creating a
roughness, the chemical interactions given below the surface of the more hydrophilic surface, as a consequence of the pumpkin flour mol­
materials and the surface energy influence the WCA values. It is also well ecules more sensitive to water. It is important to note that the surface
known that rougher and more hydrophobic biomaterial surfaces lead to roughness values for TPPF 5% and TPPF 5%/P films were unable to
higher WCA values (Vogler, 1998). Keeping this in view, the TPPF 5% explain well the WCA results for these film systems.
film showed a slight non-significant increase (p ≥ 0.05) in WCA values
compared to the TPPF 2.5% film (Table 1). This slight increase in WCA
3.6. Thickness (e), opacity (op) & color parameters
values could be explained due to the better compensatory H-bonding
interactions given within the material. Therefore, the polar groups of the
The concentration of the pumpkin flour matrix and the addition of
pumpkin flour matrix would not be available for interactions with the
the natural nanofiller did not show statistically significant effects (p ≥
water molecules, thus increasing the WCA values. This fits well with the
0.05) on the e values for the films studied (Table 1). Therefore, the Op
results already discussed from the FTIR spectra (see section 3.1). The
values and the mechanical properties are independent of the e values.
higher WCA values for the TPPF 5% film compared to the TPPF 2.5%
The Op values ranged from 0.98 to 1.55 for the film systems studied

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

(Table 1). Similar Op values were recently reported by Abral et al. 5% (0.28 ± 0.02 MPa) film with respect to the TPPF 5%/P (0.21 ± 0.04
(2019) for sago starch-based films. A statistically significant increase (p MPa) film.
≤ 0.05) in the Op values was observed as a result of the increase in the All the films studied showed Young’s modulus (E) and toughness (T)
concentration of the pumpkin flour matrix and the addition of natural values around 2.2 MPa and 0.02 × 105 J/m3, respectively (Table 2). No
nanofiller (Table 1). Interestingly, a direct relationship between the statistically significant differences (p ≥ 0.05) were, however, observed
surface roughness and Op values was observed: rougher materials were for the film systems tested.
more opaque (see section 3.5). These results suggest that light can be
scattered by surface irregularities before passing through the material. 3.8. Response to pH changes & stability in acidic or alkaline solutions
Similar results were obtained by Gutiérrez and González (2016) for
edible films made from cassava and taro starch. Unfortunately, the bionanocomposite films containing the natural
On the other hand, the color parameters indicated that the TPPF 5% nanofiller (plum flour - TPPF 2.5%/P and TPPF 5%/P films) did not
film tended to be a darker, redder and yellower material than the TPPF show noticeable changes of coloration with the naked eye as a response
2.5% film (Table 1). A similar trend was observed by adding natural to the different pHs of the medium (Fig. 9). Therefore, the anthocyanins
nanofiller at a low concentration of the pumpkin flour matrix (compared contained in the natural nanofiller did not exhibit the expected bato­
film systems: TPPF 2.5% and TPPF 2.5%/P) (Table 1), while at a high chromic effect (Gutiérrez, 2018a), i.e. no pH-sensitive bio­
concentration of the pumpkin flour matrix, the effect of adding natural nanocomposite films (smart films) were obtained. However, other
nanofiller tended to make the materials whiter, redder and bluer research groups have achieved to develop pH-sensitive films from starch
(compared film systems: TPPF 5% and TPPF 5%/P) (Table 1). and anthocyanin extracts (Jiang et al., 2020; Zhang, Huang, Yan, Hu, &
Ren, 2020; Zhang, Sun, Cao, & Wang, 2020). For this reason, the absence
3.7. Field emission scanning electron microscopy (FESEM) & uniaxial of the expected batochromic effect can be explained by: 1) the antho­
tensile tests cyanins also had strong H-bonding interactions with the pumpkin flour
matrix, thus leading to the chromophoric functional groups from the
All film samples showed a completely amorphous microstructures, anthocyanins not being available and 2) the concentration of anthocy­
and no evidence of ungelatinized starch granules or retrograded starch anins contained in the natural nanofiller was too low to give appreciable
particles was observed (Fig. 7). This fits well with the results obtained changes in coloration.
from the XRD patterns (see section 3.4). In addition, the bio­ On the other hand, all the film systems were stable (did not show
nanocomposite films showed a homogeneous distribution of natural swelling) in alkaline medium for at least 8 days, at the end of the test (24
fillers within the pumpkin flour matrix (Fig. 7b and d). Therefore, the days), all the materials showed loss of their structural integrity (Fig. 10).
methodology for the preparation of the film samples was properly In contrast, all films were stable for at least 24 days in acidic medium.
selected. Therefore, the prepared films can preferably be recommended to be in
The stress (σ)-strain (ε) curves from the films studied showed a very contact with slightly acidic foods such as meat and citrus fruits. The
small elastic region followed by a region almost totally dominated by the highest stability of all films in acidic medium compared to alkaline
plastic behavior of these materials (Fig. 8). A similar mechanical medium can be attributed to the fact that hydroxyl ions can break H-
behavior was observed by do Evangelho et al. (2019) for orange bonding interactions more easily than protons (Gutiérrez, Morales,
essential oil-containing corn starch-based films. Pérez, Tapia, & Famá, 2015). This fits well with all previous discussions.
The TPPF 5% film (strain at break, εb = 19 ± 2%) was significantly
more plastic (p ≤ 0.05) than the TPPF 2.5% film (εb = 11.6 ± 0.9%) 3.9. Determination of resistant starch (RS) & in vitro and in vivo
(Table 2). This agrees well with the plasticizing effect observed, due to digestibility tests
increased compensatory H-bonding interactions, as a result of increased
concentration of pumpkin flour matrix (see section 3.2). Therefore, the The TPPF 5% films showed significantly higher RS values (p ≤ 0.05)
starch chains contained in the pumpkin flour matrix are more mobile for than the TPPF 2.5% films (Fig. 11A). This could be related to the in­
the TPPF 5% film compared to the TPPF 2.5% film during uniaxial crease in the concentration of the pumpkin flour matrix. Taking into
tensile tests, thus observing a more material plastic. account the classification given by Goñi, Garcia-Diz, Mañas, and
As for the effect of natural nanofiller at low concentrations of the Saura-Calixto (1996), TPPF 5% films could have a high probability of
pumpkin flour matrix, the TPPF 2.5%/P film (εb = 19 ± 2%) was sta­ providing health benefits, while TPPF 2.5% films would have an inter­
tistically more plastic (p ≤ 0.05) than the TPPF 2.5% film (εb = 11.6 ± mediate probability.
0.9%) (Table 2). This is in line with the plasticizing effect observed at Interestingly, since the structural properties in the studied film sys­
low concentrations of the pumpkin flour matrix by adding natural tems were exhaustively analyzed. The type of RS determined would not
nanofiller (see section 3.2). No statistically significant differences (p ≥ fit well under the current classification of the different RS types: phys­
0.05) were, however, observed in terms of the εb values for the TPPF 5% ically inaccessible starch (type 1 RS, RS1), resistant granules (type 2 RS,
and TPPF 5%/P films. Therefore, the expected anti-plasticizing effect RS2), retrograded starch (type 3 RS, RS3), physically or chemically
(see section 3.2), as a result of the addition of natural nanofiller at high modified starch (type 4 RS, RS4 - starch with non-starch bonds) and
concentrations of the pumpkin flour matrix, did not cause significant starches containing amylose-lipid complexes (type 5 RS, RS5) (Gutiér­
variations in the εb values. rez, 2018d). Another type of RS could thus be suggested. This new type
The maximum stress (σm) values showed that an increase in the of RS could be called RS6 or the type 5 RS could also be redefined, since
concentration of the pumpkin flour matrix led to the development of this new type of RS would be related to better compensatory H-bonding
significantly more resistant materials (p ≤ 0.05) (compared film sys­ interactions with any other molecule capable of establishing this type of
tems: TPPF 2.5% (0.13 ± 0.01 MPa) and TPPF 5% (0.28 ± 0.02 MPa)) interactions, i.e. not only the H-bonds given in the amylose-lipid com­
(Table 2). plexes. In particular, the RS determined in this study was associated with
The addition of the natural nanofiller did not meet the desired per­ the best compensatory H-bonding interactions between the pumpkin
formance (reinforcement material), since at low concentrations of the flour matrix and the plasticizer (glycerol). Hernández, Emaldi, and
pumpkin flour matrix no statistically significant differences (p ≥ 0.05) Tovar (2008) also observed that those starch-based films establishing
were observed in terms of the σm values for the TPPF 2.5% (0.13 ± 0.01 better H-bonding interactions between starch and glycerol, led to higher
MPa) and TPPF 2.5%/P (0.15 ± 0.01) films, while at high concentrations values of RS content.
of the pumpkin flour matrix, the addition of natural nanofiller caused a As expected, the TPPF 5% film exhibited significantly lower values
statistically significant reduction (p ≤ 0.05) in the σm values for the TPPF (p ≤ 0.05) of in vitro digestibility and estimated glycemic index (GI =

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T.J. Gutiérrez Food Hydrocolloids 112 (2021) 106272

52.1) than the TPPF 5% film (GI = 61.7) (Fig. 11B). These results are References
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bionanocomposite films. In addition to this, an anti-plasticizing effect films made from cassava and taro starch. Innovative Food Science & Emerging
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was observed by adding natural nanofiller at a high concentration of Gutiérrez, T. J. (2017b). Surface and nutraceutical properties of edible films made from
pumpkin flour matrix, thus obtaining a more hydrophilic material. Films starchy sources with and without added blackberry pulp. Carbohydrate Polymers,
made from thermoplastic pumpkin flour (TPPF) could be suggested as 165, 169–179. https://doi.org/10.1016/j.carbpol.2017.02.016.
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slightly acidic food packaging materials such as citrus fruits and meat. blackberry pulp. Journal of Polymers and the Environment, 26(6), 2374–2391. https://
By last, a new classification of resistant starch (RS) (type 6 RS) can be doi.org/10.1007/s10924-017-1134-y.
suggested based on the results obtained here or the redefinition of the Gutiérrez, T. J. (2018b). Are modified pumpkin flour/plum flour nanocomposite films
biodegradable and compostable? Food Hydrocolloids, 83, 397–410. https://doi.org/
concept of type 5 RS can also be recommended.
10.1016/j.foodhyd.2018.05.035.
Gutiérrez, T. J. (2018c). Biological macromolecule composite films made from sagu
CRediT authorship contribution statement starch and flour/poly(ε-caprolactone) blends processed by blending/thermo
molding. Journal of Polymers and the Environment, 26(9), 3902–3912. https://doi.
org/10.1007/s10924-018-1268-6.
Tomy J. Gutiérrez: fully carried out this study.. Gutiérrez, T. J. (2018d). Plantain flours as potential raw materials for the development of
gluten-free functional foods. Carbohydrate Polymers, 202, 265–279. https://doi.org/
Declaration of competing interest 10.1016/j.carbpol.2018.08.121.
Gutiérrez, T. J., & Álvarez, K. (2016). Physico-chemical properties and in vitro
digestibility of edible films made from plantain flour with added Aloe vera gel.
The author declares no conflict of interest. Journal of Functional Foods, 26, 750–762. https://doi.org/10.1016/j.jff.2016.08.054.
Gutiérrez, T. J., & Alvarez, V. A. (2017). Films made by blending poly(ε-caprolactone)
with starch and flour from sagu rhizome grown at the venezuelan amazons. Journal
Acknowledgements of Polymers and the Environment, 25(3), 701–716. https://doi.org/10.1007/s10924-
016-0861-9.
The author would like to thank the Consejo Nacional de Inves­ Gutiérrez, T. J., & Alvarez, V. A. (2018). Bionanocomposite films developed from corn
starch and natural and modified nano-clays with or without added blueberry extract.
tigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Food Hydrocolloids, 77, 407–420. https://doi.org/10.1016/j.foodhyd.2017.10.017.
Mar del Plata (UNMdP) and Agencia Nacional de Promoción Científica y Gutiérrez, T. J., & González, G. (2016). Effects of exposure to pulsed light on surface and
Tecnológica (ANPCyT) (grant PICT-2017-1362) for financial support. structural properties of edible films made from cassava and taro starch. Food and
Bioprocess Technology, 9(11), 1812–1824. https://doi.org/10.1007/s11947-016-
Also, Dr. Mirian Carmona-Rodríguez for her valuable contribution. I am
1765-3.
also very grateful to Dr. Gema González and M. Sc. Antonio Monsalve Gutiérrez, T. J., & González, G. (2017). Effect of cross-linking with Aloe vera gel on
from the Venezuelan Institute for Scientific Research, for allowing me to surface and physicochemical properties of edible films made from plantain flour.
carry out the acquisition of AFM images in their laboratory. Food Biophysics, 12(1), 11–22. https://doi.org/10.1007/s11483-016-9458-z.
Gutiérrez, T. J., Herniou-Julien, C., Álvarez, K., & Alvarez, V. A. (2018). Structural
properties and in vitro digestibility of edible and pH-sensitive films made from

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