membranes

Article
Characterization and Applications of the Pectin Extracted from
the Peel of Passiflora tripartita var. mollissima
Minerva Rentería-Ortega 1 , María de Lourdes Colín-Alvarez 1 , Víctor Alfonso Gaona-Sánchez 1 ,
Mayra C. Chalapud 2 , Alitzel Belém García-Hernández 3 , Erika Berenice León-Espinosa 1 ,
Mariana Valdespino-León 4 , Fatima Sarahi Serrano-Villa 5 and Georgina Calderón-Domínguez 5, *

1 Tecnológico Nacional de México/TES de San Felipe del Progreso, San Felipe del Progreso 50640, Mexico;
minerva.ro@sfelipeprogreso.tecnm.mx (M.R.-O.); marial.ca@sfelipeprogreso.tecnm.mx (M.d.L.C.-A.);
erikab.le@sfelipeprogreso.tecnm.mx (E.B.L.-E.)
2 Planta Piloto de Ingeniería Química–PLAPIQUI (UNS-CONICET), Bahía Blanca 8000, Argentina;
mchalapud@plapiqui.edu.ar
3 Departamento de Ciencias de la Alimentación, División de Ciencias Biológicas y de la Salud,
Universidad Autónoma Metropolitana Unidad Lerma, Lerma de Villada 52005, Mexico;
ali_ialee@outlook.com
4 Tecnológico Nacional de México/IT Superior de Cintalapa, Carretera Panamericana Km 995,
Cintalapa 30400, Mexico; valdespino@cintalapa.tecnm.mx
5 Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería
Bioquímica, Ciudad de México 07738, Mexico; fserranov2101tmp@alumnoguinda.mx
* Correspondence: gcalderon@ipn.mx

Abstract: The inadequate management of organic waste and excessive use of plastic containers cause
damage to the environment; therefore, different studies have been carried out to obtain new biomate-
rials from agricultural subproducts. The objective of this work was to evaluate the feasibility of using
the pectin extracted from the peel of Passiflora tripartita var. mollissima (PT), characterizing its type
Citation: Rentería-Ortega, M.; and viability for the production of edible biodegradable films. In addition, films of two thicknesses
Colín-Alvarez, M.d.L.; (23.45 ± 3.02 µm and 53.34 ± 2.28 µm) were prepared. The results indicated that PT is an excellent
Gaona-Sánchez, V.A.; Chalapud,
raw material for the extraction of pectin, with high yields (23.02 ± 0.02%), high galacturonic acid
M.C.; García-Hernández, A.B.;
content (65.43 ± 2.241%), neutral sugars (ribose, xylose, glucose) and a high degree of esterification
León-Espinosa, E.B.;
(76.93 ± 1.65%), classifying it as a high-methoxy pectin. Regarding the films, they were malleable
Valdespino-León, M.; Serrano-Villa,
and flexible, with a water vapor permeability from 2.57 × 10−10 ± 0.046 to 0.13 × 10−10 ± 0.029 g/s
F.S.; Calderón-Domínguez, G.
Characterization and Applications of mPa according to thickness, being similar to other Passiflora varieties of edible films. The pectin
the Pectin Extracted from the Peel of extraction yield from PT makes this fruit a promising material for pectin production and its chemical
Passiflora tripartita var. mollissima. composition a valuable additive for the food and pharmaceutical industries.
Membranes 2023, 13, 797. https://
doi.org/10.3390/membranes13090797 Keywords: extraction; characterization; pectin; films; Passiflora tripartita var. mollissima

Academic Editor: Isabel Coelhoso

Received: 26 July 2023

Revised: 9 September 2023 1. Introduction
Accepted: 11 September 2023
Currently, environmental concerns regarding the production of non-biodegradable
Published: 16 September 2023
packaging have increased interest in its replacement with biomaterials. These materials
are natural polymers, such as proteins and polysaccharides, which have been used in
developing bio-based packaging films and edible coatings [1]. Among these biopolymers,
Copyright: © 2023 by the authors.
pectin is widely used due to its high solubility in water, forming viscous solutions and,
Licensee MDPI, Basel, Switzerland. under appropriate conditions, its gelling capacity; this behavior varies depending on the
This article is an open access article number of carboxyl groups esterified with methanol [2].
distributed under the terms and Structurally, pectin is an acidic heteropolysaccharide, commonly named homogalac-
conditions of the Creative Commons turonan, composed mainly of galacturonic acid (GalA), forming a linear backbone of
Attribution (CC BY) license (https:// (1 → 4)-linked α-d-GalA residues [3,4]. The carboxyl residues of the GalA unit can be
creativecommons.org/licenses/by/ esterified with methanol, which alters the electrical characteristics of the molecule. The
4.0/). degree and pattern of methoxylation of homogalacturonan, as well as its molecular weight,

Membranes 2023, 13, 797. https://doi.org/10.3390/membranes13090797 https://www.mdpi.com/journal/membranes

Membranes 2023, 13, 797 2 of 16

are essential parameters that determine the functional attributes of different pectins [5],
giving rise to pectins’ classification into two groups: high and low methoxyl, dependent on
the esterified carboxylic groups percentage.
High-methoxyl pectins are those that have more than 50% of esterified carboxylic
groups [6], form gels in aqueous systems under pH conditions between 2.8 and 3.5 and
have a soluble solids content between 60% and 70%. Low-methoxyl pectin is characterized
by generating gel in the presence of polyvalent salts or in low-soluble solids systems, with
a wide pH range [7].
The primary source of commercial pectin is citrus fruit peel and apple pomace, since
they provide high yields (6–23%) [8]; however, the search to find other new sources is still
ongoing. In this sense, the extraction of pectin from banana peel [9], prickly pear fruit
and leaves [10–12], tejocote [13,14], cocoa [15], pineapple residues [16], guava [17] and
different varieties of passion fruit peel [18–21], among others, has been mentioned, with
all of them having low pectin yields (1.15–3.38%), with the exception of passion fruit with
higher values (21.25–23.86%).
The genus Passiflora includes more than 500 species, and the most known are as fol-
lows: Passiflora edulis Sims, Passiflora ligularis Juss, Passiflora alta Curtis, Passiflora mollisima,
and Passiflora edulis var. flavicarpa Degenerer [22]. From these species, P. mollissima (Kunth)
L. H. Bailey, commonly known as “curuba de Castilla” or “banana passion fruit” [23],
has been cited as a good source of vitamins A, B and C, with high antioxidant activity, as
measured using FRAP, ABTS and phenolic compound content, as well as a good pectin
source [23].
Regarding pectin extraction from passion fruit and its characterization, most reports
are based mainly on Passiflora edulis f. flavicarpa Degener [19] and just a few on Passiflora
mollissima [24], including, in most of the studies, the physicochemical properties of fruits
at different stages of maturation, without characterizing the pectin. Furthermore, there is
no information that allows one to establish whether the pectin extracted from Passiflora
tripartita passion fruit var. mollissima is of the high- or low-methoxyl type. Likewise, there
are no reports on applications, such as the preparation of biodegradable or edible films,
based on this pectin. The objective of this work was to evaluate the feasibility of using
the pectin extracted from the shell of “Passiflora tripartita var. mollissima to develop an
edible-biodegradable film” for food applications, evaluating the pectin yield, characterizing
its type and the feasibility of film production.

2. Materials and Methods

2.1. Materials
The fruits of Passiflora tripartita var. mollissima were obtained from the community of
Guarda la Lagunita (Las Canoas), belonging to the municipality of San José del Rincón,
State of Mexico. Citric pectin (P-1935, Sigma-Aldrich, México) was also used as a control
sample. Glycerol (G5516, Sigma-Aldrich, Toluca, México), tween 20 (P1379, Sigma-Aldrich,
Mex), hydrochloric acid (320331, Sigma-Aldrich, Toluca, México.), 96◦ ethanol and distilled
water were also used.

2.2. Methods
2.2.1. Pectin Extraction
The extraction of pectin from the peel of “Passiflora tripartita var. mollissima” was
carried out following the methodology reported by Chumbes (2010) [25], with some modi-
fications. The extraction was carried out via acid hydrolysis, maintaining a 1:1 shell/water
ratio, at 80 ◦ C for 30 min, adjusting the pH to 3.5 with HCl. The solids were separated
via centrifugation (2900× g; Metrix Lab Dynamica, Roterdam, The Netherlands), and the
supernatant was precipitated with ethanol 96% in a 1:1 ratio while stirring gently. Subse-
quently, the precipitated solids were washed with ethanol 96% in a 1:1 ratio, dehydrated at
Membranes 2023, 13, 797 3 of 16

30 ◦ C for 12 h and milled (KRUPS GX4100) until a fine powder was obtained. Finally, the
pectin yield was calculated according to Equation (1).

Pectinpowder(g)
Efficiency = × 100 (1)
Initial shell sample ( g)

2.2.2. Characterization of the Pectin of Passiflora tripartita var. mollissima

Methoxyl Group Content
The methoxyl group content was determined according to the methodology reported
by Valdespino-León et al. (2020) [4]. Powder pectin (250 mg) was mixed in 50 mL of CO2 -
free water until completely dissolved, then titrated with 0.5 N NaOH using phenolphthalein
until the formation of a faint pink color (initial titration), and then 10 mL of NaOH 0.5N
was added and shaken vigorously and allowed to settle for 15 min. Subsequently, 10 mL of
0.5 N HCl was added until the pink coloration disappeared. Finally, a second titration with
0.5 N NaOH was carried out until the faint pink color was maintained (final titration).
The methoxyl group percentage was calculated (Equation (2)) considering that each
mL of 0.5 N NaOH of the final titration is equivalent to 15.52 mg of methoxyl (OCH3 ).
mg OCH3
consumed NaOH f inal volume (ml ) × 15.52 ml NaOH × 100
%R − OCH 3 = (2)
pectin weight (mg)

Degree of Esterification
Two solutions, one of Passiflora tripartita var. mollissima (PPT) and the other of citric
pectin (PC) in CO2 -free water at a concentration of 0.1% (w/v), as reported by Valdespino-
León et al. (2020) [4], were prepared. An aliquot of 10 mL was taken and titrated with
0.1 N NaOH using phenol phthalein as indicator (initial titration), and then 20 mL of 0.5 N
NaOH was added to neutralize the solution. Finally, the final titration was carried out by
placing 0.1 N NaOH until reaching a weak pink coloration. The calculation of the degree of
esterification was carried out according to Equation (3).

B
DE = × 100 (3)
A+B
where DE is the degree of esterification, A is the volume spent on titration A and B is the
volume spent on titration B.

Acidity
Regarding the free acidity or acidity percentage, 20 mL of solution was prepared at a
concentration of 0.5% (w/v) of powdered pectin (PPT or PC) in distilled water. The solution
was heated to 70 ◦ C in a boiling water bath. It was titrated with 0.1 N NaOH using 1%
phenolphthalein as the indicator [26]. The results were calculated according to Equation
4 and expressed in terms of meq of free carboxyl using the meq of citric acid (mEAC)
(A.O.A.C. 925.34) as a reference.

% Acidity
Volume o f NaOH consumed x Normal NaOH × 0.006404 mEAC
mL NaOH × 100 (4)
= pectin weight (mg)

High-Performance Liquid Chromatography (HPLC)

The evaluation of the pectic substances was performed using high-performance liquid
chromatography (HPLC) according to the methodology reported by Valdespino-León et al.
(2020) [4]. Briefly, 500 mg of the sample (PPT or PC) was enzymatically hydrolyzed by
dissolving in 0.1 M citrate buffer (pH 4) in a 1:30 (w/v) ratio and adding 400 U (450 µL) of As-
pergillus niger pectinase (P4716-100KU, Sigma-Aldrich, St. Louis, MO, USA). Subsequently,
the samples were incubated in an orbital shaker (Barnstead International, MaxQ 4000,
Membranes 2023, 13, 797 4 of 16

Dubuque, IA, USA) at 30 ◦ C for 2.5 h and at 200 rpm; then, the hydrolyzed samples were
stored and refrigerated at 4 ◦ C for 24 h to allow for the precipitation of solids and recovering
the supernatants. Consecutively, 2 mL aliquots of the hydrolyzed samples were taken,
filtered through 0.2 µm syringe unit (Millex® , 13 mmØ CAT. SLGN013NL, Dublin, Ireland)
and placed in glass vials (2 mL). Regarding the calibration standards, solutions of 10 mg
of D (+) galacturonic acid monohydrate (47267 Sigma-Aldrich, St. Louis, MO, USA) and
simple sugars (including mannose (92683), rhamnose (83650), glucuronic acid (G5269),
glucose (G8270), galactose (PHR1206), xylose (PHR2102), arabinose (A3256) and fructose
(93183)) (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 0.01 M H2 SO4 , to prepare
a 0.01% solution, which was filtered in the same way as the pectin hydrolysates [27].
The samples were analyzed in an Agilent 1260 Infinity HPLC equipment (Agilent,
Santa Clara, CA, USA) with an IR detector and an Agilent Hi-Plex H column (7.7 × 300 mm,
8 µm, PL1170-6830, Agilent, Santa Clara, CA, USA) following the methodology reported
by Ball et al. (2011) [27] with some modifications. The mobile phase was 0.01 M H2 SO4
with a flow of 0.4 mL/min; the sample injection volume was 20 µL; and the analysis
temperature was 55 ◦ C, both in the column and in the detector. All samples and standards
were injected in triplicate, and the duration of each run was established as 25 min. All
chromatograms were analyzed using OpenLab CDS v. 4.0 Agilent (Agilent, Santa Clara,
CA, USA), reporting the presence of galacturonic acid and other sugars in the hydrolyzed
samples, as well as their retention times and concentrations.

2.2.3. Film Preparation

The pectin solution (surface tension: 34.7 ± 1.09 N/m; density: 0.97 ± 0.15 g/mL;
viscosity: 0.55 ± 0.04 Pa/s) was prepared following the methodology proposed by Gaona-
Sanchez et al. (2016) [28] with some modifications. Pectin samples (2 g of pectin) were
dissolved in 50 mL of distilled water by stirring at 850 rpm (Thermo Scientific SP131325
Vernon Hills, IL, USA) for 45 min at room temperature; then, glycerol (22% w/w) was added,
maintaining the agitation for 30 min. Finally, Tween® 20 was added in a ratio of 1:10 (w/w)
(Tween 20/pectin) at the same speed, temperature and time as described above.
Regarding the preparation of the films, the casting technique was used, in which the
pectin solution (7 and 14 mL) was poured into circular Teflon molds (8.0 cm in diameter)
and then dried at 30 ◦ C for 12 h in an oven (AFOS, Hull, East Yorkshire, England) and
stored at room temperature in a desiccator with Drierite™ anhydride desiccant (Drierite,
Xenia, OH, USA) until further analysis.

2.2.4. Characterization of Pectin Films

Color
The readings of the reflection spectrum of the films were carried out according to
the methodology reported by Gaona-Sánchez et al. (2015) [29] and Valdespino-León et al.
(2020) [4] using a colorimeter (Konica Minolta CR-400, NJ, USES), which was previously
calibrated with a reference plate (Y = 93.7, x = 0.3159, y = 0.3324). The result of the
measurements was the average of the reading made at five different points. The coordinates
of the CIELab color space were obtained, where L* represents lightness (values between
0 and 100), ±a* was the chromatic component from green (−) to red (+) and ±b* was the
chromatic component from blue (−) to yellow (+) [30]. In addition, the transparency of
the films (%T) was determined considering that L*=%T, assuming that a translucent film
will generate the same luminosity values (L*) as the white calibration plate (L* 0 = 100)
and that any difference will be the result of a more opaque material (L* < 100) [31]. For
the measurements, a standard white plate was used as the background, and statistical
analysis was performed with SigmaPlot 12.5 software using one-way ANOVA with p < 0.05.
Reported values are the average of each independent triplicate.
Membranes 2023, 13, 797 5 of 16

Texture
The determination of tensile strength (TS) was performed following the methodology
described by Ali et al. (2023) [32], with some modifications. A texture meter (Texture
Analyzer CT3, Brookfield™, Chandler, AZ, USA) with a 4500 g load cell programmed with
the TexturePro CV V1.6 software was used.
The films were cut into 70 × 33.9 mm rectangles and placed on specific double-grip
pieces (TA-DGA accessory) for the tensile test using the following conditions: activation
load of 450 g, speed of 0.3 mm/ s and return speed of 4.5 mm/s. The tensile strength
(MPa) was calculated by dividing the maximum force (N) at the breaking point by the cross-
sectional area (mm2 ) of the film block (Equation (5)), as described by Xie et al. (2023) [33],
while the elongation-at-break values were obtained by recording the elongation at break
divided by the initial length of the sample and multiplied by 100.

F
TS = (5)
Lxx
where TS is the tensile strength, L is the width (mm) and x is the thickness (mm) of the film.
The statistical analysis was performed with SigmaPlot 12.5 software using the one-way
ANOVA with p ≤ 0.05. The reported values are the average of each independent triplicate.
At least five independent samples were employed to assure reproducibility.

Thickness
Films thickness was measured following the methodology reported by Arriaga (2019) [34]
using a digital micrometer (Fowler 54860-001 Electronic IP54. Shanghai, China), taking the
value that indicates the contact between the film and the probes. The measurements were
made at a minimum of three points (central and extreme) and in at least three independent
samples, reporting the average.

Water Vapor Permeability

This parameter was evaluated according to the methodology reported by Valdespino-
León et al. (2020) [4], which is based on the ASTM E-96 method. A permeability cell and a
cup with a lid were used, which were filled with distilled water; then, the film was placed
in the mouth of the cup, which, in turn, was placed inside a container with Drierite™
anhydride desiccant (Drierite, Xenia, OH, USA) at 30 ◦ C. The weight was measured for 4 h
on an analytical balance with an accuracy of 0.0001 g (OHAUS® Pioneer ™, Parsippany, NJ,
USA). The data were plotted, and the slope of the curve was calculated (weight vs time)
(R2 > 0.99), obtaining the water vapor transmission rate (WVTR, gs−1 m−2 ) and dividing
the value by the tested film area. WVP was calculated according to the combined laws of
Fick and Henry for the diffusion of gases through films according to Equation 6, where
x is the thickness of the film (m), and ∆P corresponds to the difference in vapor pressure
within the system, whose value is 4246.9 Pa, corresponding to the saturation pressure of
water at the saturation temperature at which the system is located (30 ◦ C) (Table A4 water
saturated: temperature table) [35].

X
WVP = WVTR. (6)
∆P

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry (DSC) was carried out on DSC 2000 equipment
equipment (TA Instruments, New Castle, DE, USA). Samples (5–6 mg) were placed in
a standard aluminum container with a perforated lid and heated from 5 to 350 ◦ C at
a heating rate of 10 ◦ C min−1 in a nitrogen atmosphere with a set flow rate of 20 mL
min−1 . An empty aluminum tray (<10 mg) was used as a reference probe. The experiments
were performed in independent triplicates. In this analysis, the denaturation temperature
Membranes 2023, 13, 797 6 of 16

(TD), the decomposition temperature (TDS), the melting temperature (Tm) and the glass
transition temperature (Tg) are reported [36].

Statistical Analysis
All the analyses were made in independent triplicates, and the results were presented
as mean values. Statistical differences were detected using one-way analysis of variance
(ANOVA, Tukey’s test), and a value of p < 0.05 indicated statistical significance using the
software SigmaPlot 12.5.

3. Results and Discussion

3.1. Pectin Extraction Yield
The pectin extraction yield from Passiflora tripartita var. mollissima reached 23.2 ± 0.05%
on a dry basis. This yield value is higher than that reported by Charchalac et al. (2008) [37]
for passion fruit peel pectin (Passiflora edulis var. flavicarpa, 12.7–21.3%), Passiflora edulis f.
flavicarpa passion fruit (7.52 ± 0.05%, [38] and Passiflora edulis f. flavicarpa Degener fruits
(18.45%) [39]. It is also higher than the results of passion fruit peels reported by Kulkarni
and Vijayanand (2010) [40], who cited values between 5.78% and 9.02%. Comparing with
other pectin sources, the obtained yield can be considered very high, as most reports
cited values below 10 percent, for example, for shells from curuba (9.7%), guava (1%)
and badea (1.8%) [2], with the exception of apple pomace [41], which presented much
higher values (7–23%). According to the above, Muñoz and Cuesta (2012) [18] mentioned
that the yields can vary according to the fruits, maturity, extraction method, time and
extraction temperature.

3.2. Pectin Chemical Characterization

In the literature, pectin is considered to be a high-methoxyl pectin when the percentage
of galacturonic acid, the methoxyl degree and the degree of esterification are higher than
65%, 6.7% and 50%, respectively [4]. In this sense, Table 1 shows the chemical character-
ization Passiflora tripartita var. mollissima pectin (PPT), compared with citrus pectin (PC)
as a control, with both of them being significantly different (p < 0.05) and classified as
high-methoxyl pectins.

Table 1. Chemical characterization of Passiflora tripartita var. mollissima and citric pectins.

Parameter PPT PC
a
% R-OCH3 7.8 ± 0.28 11.6 ± 0.29 b
% AGal 65.4 ± 2.24 a 72.4 ± 1.83 b
DE 76.9 ± 1.66 a 95.9 ± 3.05 b
% Acidity (citric acid meq) 0.64 ± 0.06 a 1.1 ± 0.07 b
PPT: Passiflora tripartita var. mollissima pectin; PC: citric pectin. The results represent the average of three repetitions.
Results followed by different letters in each row indicate significant difference (p < 0.05) according to Tukey’s test.
PPT Passiflora tripartita var. mollissima pectin, PC citric pectin, R-OCH3 methoxylation percentage (%), DE degree
of esterification (%).

According to the above, the percentage of galacturonic acid in PPT (65.4 ± 2.2) was
lower than that of PC (72.4 ± 1.8), with similar values to those reported by Lin et al.
(2020) [38] and Freitas de Oliveira et al. (2016) [42] on pectin from Passiflora edulis f. flavicarpa
and Passiflora edulis Sims f. flavicarpa Degener (68.53 ± 1.40 and 66.27 ± 0.98), mentioning
that the values depend on the extraction treatment, the degree of maturation, as well as the
region where the fruits were obtained. In the same way, the degree of methoxylation (ME)
of PPT was lower than that of PC. However, both were larger than 7%, being classified as
high-methoxyl pectins, which agrees with the results reported by other authors for pectin
obtained from Passiflora edulis f. flavicarpa. However, the PPT values are lower than those
reported for other passion fruit peels, associated with a de-esterification with HCl during
the extraction process, which affects its percentage and decreases methoxylation.
Membranes 2023, 13, 797 7 of 16

On the other hand, the results of the degree of esterification (DE) in both pectin
samples were higher than 50% and similar to those reported by Freitas de Oliveira et al.
in 2016 [42] (68.8% ± 0.57 to 77.4% ± 0.52), who mentioned that depending on the source
and on the experimental conditions applied during the extraction process, the pectin will
have different characteristics; in addition to the above, Mendoza-Vargas et al. (2017) [43]
reported that during ripening, the tissues of the fruits present a variation in the soluble
pectin content, and when the fruits are ripe, the pectin is fully esterified; adding to the
above, Cerón-Salazar and Cardona-Alzate, (2011) [44] mentioned that in the immature
state, the pectin is fully esterified, which gives the tissue greater rigidity. They also cited
that at early stages of maturation, higher pectin yields are obtained with a high methoxyl
percent, similar to that reported in this study [45].
Regarding the percentage of acidity, PPT presented a lower value than PC. The re-
sults are probably related to the galacturonic acid content of the pectin and possibly to
a homogalacturonan skeleton, as reported by Valdespino-León et al. (2020) [4]. In this
same sense, Cabarcas et al. (2012) [9] mentioned that pectins are neutral in their natural
state; in solution, they have an acid character, which depends on the medium and the
degree of esterification. However, the results are higher than those reported in 2016 by
Campo-Vera et al. [46] (0.32 ± 0.13 to 0.43 ± 0.05). The differences are likely due to the
parameters used in terms of the temperature and time of hydrolysis, affecting both the
degree of esterification and the acidity, as reported by Durán et al. (2012) [47]. In addition
to the above, Rodríguez-Mora et al. (2022) [15] reported a direct relationship between free
acidity and extraction pH, which varies between 2.8 and 3.4 as a function of the degree of
esterification. Therefore, the highest levels of acidity occur when the extraction medium
shows extreme acidity conditions; in this sense, Cabarcas et al. (2012) [9] mentioned that the
free acidity increases as the extraction pH is more acidic, causing a change in the chemical
nature of the carboxyl groups, decreasing their state of form (salts or esters) and increasing
their presence as acid groups.

High-Performance Liquid Chromatography (HPLC)

The enzymatic hydrolysis of both pectins (PC and PPT) generated significant fractions
of glucuronic and galacturonic acids that correspond to the elution peaks at 13.62 ± 0.01 min
and 14.03 ± 0.01 min, respectively, confirming what was reported by Valdespino-León et al.
(2020) [4], who expressed that the presence of glucuronic acid together with galacturonic
acid can constitute the pectic fraction of fruits and vegetables. In the case of the pectin
obtained from the shell of Passiflora tripartita var. mollissima, it can be seen that the intensity
of the glucuronic acid peak is higher than that of galacturonic acid (Figure 1), which implies
that the purification process carried out is insufficient to eliminate this component, which
is considered a contaminant in pectin extraction [48].
When evaluating the concentration of the constituent sugars of the pectins, it was ob-
served that citrus pectin (PC) is mainly composed of galacturonic acid (27.047 ± 0.149 mg/mL)
and glucuronic acid (709.47± 0.88 mg/mL), with small proportions of other reducing sug-
ars, such as xylose (2.688 ± 0.015 mg/mL), rhamnose (0.275 ± 0.003 mg/mL) and arabinose
(0.119 ± 0.001 mg/mL), reflecting a predominantly linear homogalacturonan structure [49],
with some substitutions of rhamnogalacturonan I [50], which, when hydrolyzed with
pectinase, promotes the release of galacturonic acid units and other sugars.
Regarding the concentration of galacturonic acid present in PPT (13.933 ± 0.412 mg/mL),
it was lower than that of PC; however, the concentration of glucuronic acid considerably
exceeded this, indicating that pectin has a lower purity compared to PC [51], which means
that a purifying step is needed.
Membranes 2023,
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Figure 1. HPLC
HPLC chromatogram
chromatogramofofhydrolyzed
hydrolyzedcitric
citricpectin
pectin(PC)
(PC)and
andPassiflora tripartita
Passiflora mollissima
var.var.
tripartita mollis-
sima pectin
pectin (PPT)(PPT) samples.
samples. a PC; abPC;
PPT.b Retention
PPT. Retention
times: times: 13.61= ±
GlcA =GlcA 13.61
0.02±min,
0.02 GaIA
min, GaIA
= 14.03 ± 0.01± min;
= 14.03 0.01
min; Glucose
Glucose = 14.54
= 14.54 ± 0.03
± 0.03 min;min; Xylose
Xylose = 15.36
= 15.36 ± 0.01
± 0.01 min;min; Rhamnose
Rhamnose = 16.128
= 16.128 ± 0.05
± 0.05 min,
min, arabinose
arabinose =
= 16.63 ± 0.01 min.
16.63 ± 0.01 min.

3.3. Characterization
Regarding the ofconcentration
Pectin Films of galacturonic acid present in PPT (13.933 ± 0.412
3.3.1.
mg/mL),Thickness
it was lower than that of PC; however, the concentration of glucuronic acid con-
siderably exceeded
Regarding the this, indicating
thickness of thethat pectin
films, this has a lower as
parameter, purity compared
expected, to PC [51],
was dependent
which
on the means
solution that a purifying
volume step isThe
employed. needed.
films made with 7 mL of the solution presented
thickness values of 23.45 ± 3.02 µ (PPT1), while those made with 14 mL of solution had
3.3. Characterization
53.34 ± 2.28 µ (PPT2), of Pectin Films
the latter being almost twice that of the former, and with the small
differences possibly
3.3.1. Thickness due to the drying rate at which the film was formed. The thicker film
also showed
Regarding larger
the flexibility,
thickness of less
theluminosity,
films, this more permeability
parameter, to water
as expected, wasvapor (Tableon
dependent 2)
and it was easier to remove from the plate (Figure 2). These values for PPT2
the solution volume employed. The films made with 7 mL of the solution presented thick- are similar
to those reported by Younis et al. (2019) [52] in chitosan-based films (54.37 µ), mentioning
ness values of 23.45 ± 3.02 µ (PPT1), while those made with 14 mL of solution had 53.34 ±
that as the thickness of the film increases, the diameter of the pores also increases. In
2.28 µ (PPT2), the latter being almost twice that of the former, and with the small differ-
addition to the above, Nascimento et al. (2012) [53] reported higher values (133 and 185 µ)
ences possibly due to the drying rate at which the film was formed. The thicker film also
in the thickness of starch films or mesocarp flour; this result is associated with a more
showed larger flexibility, less luminosity, more permeability to water vapor (Table 2) and
significant amount of solution poured onto the plates and more solids. These results prove
it was easier to remove from the plate (Figure 2). These values for PPT2 are similar to those
Membranes 2023, 13, x FOR PEER REVIEW
the feasibility of developing PPT films with similar physical properties to those reported 9 of 16
reported by Younis et al. (2019) [52] in chitosan-based films (54.37 µ), mentioning that as
for other pectin films prepared using the casting technique.
the thickness of the film increases, the diameter of the pores also increases. In addition to
the above, Nascimento et al. (2012) [53] reported higher values (133 and 185 µ) in the
thickness of starch films or mesocarp flour; this result is associated with a more significant
amount of solution poured onto the plates and more solids. These results prove the feasi-
bility of developing PPT films with similar physical properties to those reported for other
pectin films prepared using the casting technique.

Figure2.2. Simple
Figure Simple films
films based
based on
on pectin
pectin extracted
extracted from
from the
the peel
peel of
of “Passiflora
“Passiflora tripartita
tripartita var.
var. mollissima”.
mollissima”.
(A): PPT1: film with a thickness of 23.45 ± 3.02 µ; (B): PPT2 film with a thickness of 53.342.28
A: PPT1: film with a thickness of 23.45 ± 3.02 µ; B: PPT2 film with a thickness of 53.34 ± µ. µ.
± 2.28

Table 2. Properties of the films of Passiflora tripartita var. mollissima.

Properties/Samples PPT1 PPT2

Color
L* 92.12 ± 2.21 a 85.24 ± 1.33 b
Membranes 2023, 13, 797 9 of 16

Table 2. Properties of the films of Passiflora tripartita var. mollissima.

Properties/Samples PPT1 PPT2

Color
L* 92.12 ± 2.21 a 85.24 ± 1.33 b
a* 11.72± 0.54 a 9.27± 0.30 b
b* 45.62 ± 3.17 a 65.61 ± 2.21 b
Mechanical properties
Deformation modulus (MPa) 6.68 ± 0.13 a 3.7 ± 0.17 b
Ts (MPa) 4.07 ± 0.45 a 4.80 ± 0.33 b
Toughness (J/m3 ) 3.7 ± 0.36 a 1.87 ± 0.21 b
Elongation at break 22.7 ± 1.6 a 19.62 ± 2.05 b
Barrier properties
WVP (g/s·m·Pa) 0.128 × 10−10 ± 0.029 a 3.187 × 10−10 ± 0.080 b
PPT1: film with a thickness of 23.45 ± 3.02 µ, PPT2 film with a thickness of 53.34 ± 2.28 µ. Values followed by the
same letter (a,b) within the same column are not significantly different (p > 0.05) according to Tukey’s multiple
range test.

3.3.2. Color and Texture

Regarding the color parameters (L*, a*, b*) of the films (Table 2), samples show a
significant difference (p < 0.05) in luminosity (L*) (PPT1: 92.12; PP2: 85.24), red-green
coordinates (a*) (PPT1: 11.72± 0.54; PP2: 9.27 ± 0.30) and the yellow-blue color (b*) (PPT1:
45.62 ± 3.17; PPT2: 65.61 ± 2.21).
The results agree with the visual appearance of the films since PPT1 is more transparent
than PPT2, being almost colorless (transparent), which coincides with the high values
obtained close to 100 reported for the parameter L*. At the same time, the significant
difference found in b* is possibly associated with the increase in the solution concentration
since the films became more yellowish. In contrast, for the values of a*, both films go from
green to red. This finding indicated that, since the green color of the films became lighter
and yellow became the dominant color, the color changes likely to come from the brownish
character of the peel peptides of Passiflora tripartita var. mollissima denote a change in
coloration, as reported by [34].
Brion-Espinoza et al. (2021) reported similar results [54] in edible films of pectin added
with peptides from jackfruit leaves, obtaining values for L* from 91.31±0.01 to 94.01 ± 0.02
and b* from 5.27 ± 0.93 to 13.25 ± 0.059, attributed to the peptide compounds of jackfruit.
In another work, Saurabh et al. (2015) [55] mentioned that the reduction in the values of
L* and a* indicates an increase in the darkness and greenness of the films based on guar
gum, while an increase in b* values means an increase in yellowing. According to the color
values obtained, the films could be helpful for the protection of photosensitive compounds
when applied as a coating, reducing the intensity of light that passes through them or even
incorporating dyes that attract the consumer.
The mechanical properties of the films (deformation modulus, tensile strength (TS)
and toughness) can be associated with their chemical structures [56]. The PPT1 and PPT2
films presented significant differences (p < 0.05), as shown in Table 2. The deformation
modulus for PPT1 was higher (6.68 ± 0.13 Mpa) than for PPT2 (3.7 ± 0.17 Mpa), which
could be related to the thickness of the film, since Márquez et al. (2008) [57] reported that
films tend to be more brittle and deform more quickly when they are thinner; likewise,
Trujillo (2014) [58] mentioned that the films made from more concentrated solutions of
glycerol generate an increase in thickness, tension and deformation at the break. At the
same time, Sood et al. (2022) [59] mentioned that the thickness of the film depends on the
preparation method, the drying conditions, the composition of the film and the interaction
between the components, in addition to being influenced by the structure of the films
developed during drying, directly influencing the mechanical properties of said film.
Regarding tensile strength, PPT1 films presented lower values (4.07 ± 0.45 MPa)
than PPT2 films (4.80 ± 0.33 MPa) and a higher toughness (3.7 ± 0.36). These results
were attributed to the fact that the PPT2 pectin film contains more polar groups, which
Membranes 2023, 13, 797 10 of 16

results in a more significant number of hydrogen bonds and a tighter internal structure.
In this sense, Fu et al. (2022) [60] mentioned that the polymer chains were intertwined to
form stronger intermolecular hydrogen bonding network structures, resulting in better
mechanical properties of the films. Segura-Ceniceros et al. (2006) [61] presented similar
results in films made with papain and pectin from passionflower edulis with a thickness
of 40 microns, mentioning that the lower the TS of the films, the more fragile and difficult
to manipulate. Similar information was reported by Valdespino-León et al. (2021) [4] for
citrus pectin films (4.80 ± 0.33 Mpa) and by Sood et al. (2022) [59] for films composed of red
grapefruit peel pectin, casein and egg albumin (1.34–9.65 MPa). These authors expressed
that the tensile strength is related to the interaction between the polymers within the matrix
and the constituents of the film, as well as the method of preparation. In the same way,
López and Checa (2019) [62] related the mechanical properties to the effect of the plasticizer
since it modifies the structure of the network formed by the biopolymer, achieving films
with high elasticity but reducing the resistance of the materials.

3.3.3. Water Vapor Permeability

The application of biopolymer-based edible films seeks to reduce the exchange of
moisture between food and the surrounding atmosphere or between two components of a
food product [63]. Table 2 shows the water vapor permeability for PPT1 and PPT2 samples,
where the values ranged from 0.128 × 10−10 ± 0.029 to 3.187 × 10−10 ± 0.080 g/s·m·Pa,
respectively. In this sense, the lowest WVP values and the thinnest films are observed in the
PPT1 sample (Table 2), representing a film that could protect most food products. However,
the films did not follow a direct relationship with thickness, which was possibly a result of
the drying process and film composition, mainly the higher quantity of exposed hydrophilic
groups in the thicker samples. In this regard, Morillon et al. (2002) [64] mentioned that the
increase in film permeability with thickness could be related to hydrophilic compounds,
and Nguyen et al. (2014) [65] reported that an increase in the concentration of polar groups
causes an increase in the availability of free hydroxyl groups in the film matrix to react
with water, thus increasing the moisture sensitivity of the films. Therefore, the thickness
of the film is a crucial parameter in the calculation of water vapor permeability values; in
addition, the influence of the thickness varies with the composition of the film, as reported
by Thakur et al. (2017) [66], but it remains the main affecting factor [67,68].
On the other hand, Salazar et al. 2015 [69] reported a water vapor permeability value
of 0.758 × 10−10 g/m *s* Pa for a film of nopal mucilage, gelatin and beeswax, a lower
value than that obtained for PPT2 films of pectin from Passiflora tripartita var. mollissima but
higher than PPT1.

3.3.4. Differential Scanning Calorimetry (DSC)

Figure 3 shows the differential scanning calorimetric curves of pectin films of Passiflora
tripartita var. mollissima at different thicknesses (A: 23.45 ± 3.02 µ; B: 53.34 ± 2.28 µ). Both
thermograms had similar behavior, which was expected since the components are the same.
Although the same peaks are observed in both thermograms, in B, there is a displacement,
which is probably due to the thickness effect, indicating that the structure formed in the
thinner film is less compact; therefore, it would require less energy to release the moisture,
having lower transition temperatures. This could also be explained by the largest resistance
to heat transfer of the thicker film, as expressed in heat conduction Fourier’s law. On the
other hand, both samples presented three inflection points; the first was observed around
105.36 and 108.79 ◦ C, respectively, the second point around 220.19–221.68 ◦ C and, finally,
the third was around 330.08 and 331.99 ◦ C.
 displacement, which is probably due to the thickness effect, indicating that the stru
formed in the thinner film is less compact; therefore, it would require less energy to re
the moisture, having lower transition temperatures. This could also be explained b
largest resistance to heat transfer of the thicker film, as expressed in heat conduction
rier’s law. On the other hand, both samples presented three inflection points; the firs
Membranes 2023, 13, 797 11 of 16
observed around 105.36 and 108.79 °C, respectively, the second point around 22
221.68 °C and, finally, the third was around 330.08 and 331.99 °C.

Figure 3. DSC Figure 3. DSC of

thermograms thermograms
the film of of the film of
“Passiflora “Passiflora
tripartita var. tripartita var. mollissima”.
mollissima”. A: film
(A): film with a with a
ness of 23.45 ± 3.02 µ; B: film with a thickness of 53.34
thickness of 23.45 ± 3.02 µ; (B): film with a thickness of 53.34 ± 2.28 µ. ± 2.28 µ.

According to theAccording to the above,

above, exothermic exothermic
peaks peaks with abetween
with a temperature temperature
220.19between
and 220.1
221.68 ◦ C according
221.68 to°C Nisar
according
et al. to(2018)
Nisar[70] et al.
are(2018)
related [70]
to are
the related
thermaltodegradation
the thermalofdegradati
polymers andpolymers and pectin.
pectin. These valuesThese valuesto
are similar arethose
similar to thosebyreported
reported Linaresby Linares
(2015) [71](2015) [71
and Muñoz (2016)Muñoz [72](2016) [72] in commercial
in commercial pectin and pectin
hawthorn and pectin.
hawthorn pectin. work,
In another In another
Nisarwork, Ni
al.mentioned
et al. (2018) [70] (2018) [70]that at 230 ◦ C,
mentioned that at 230
there is an°C, there is antransition
exothermic exothermic peaktransition
in citrus peak in
pectin, responsible forresponsible
pectin, its degradation, a value
for its similar toathat
degradation, valuereported
similarby toMuñoz (2016) [72],
that reported by Muñoz (
with degradation ◦ C. In this regard, Pasini-
[72],temperatures
with degradation ranging between 220
temperatures and 240
ranging between 220 and 240 °C. In this re
Cabello et al. (2015) [73] reported
Pasini-Cabello et al.that the[73]
(2015) endothermic
reported that pre-peaks, before the degradation
the endothermic pre-peaks, before the
temperatures,radation
are related to a conformational change that could be the transformation
temperatures, are related to a conformational change that could be the tra
of the more stable
mation 4Cof
1 chair conformation
the more stable 4C1of the conformation
chair galacturonan of ring
thethrough a 1,4 B boat
galacturonan ring through
conformation to theconformation
boat reverse 1C4 chair to theconformation.
reverse 1C4 chair conformation.
In another study, Rezvanian
In another study, et al. (2017) [74]
Rezvanian reported
et al. (2017) that
[74] citrus
reported pectin
thatfilms
citruswith
pectin films
sodium alginate exhibited
sodium twoexhibited
alginate stages of two thermal degradation.
stages of thermal The first stageThe
degradation. wasfirst
up to
stage was
125 ◦ C, attributed to the loss of different types of water, including free
125 °C, attributed to the loss of different types of water, including free waterwater (released at (relea
40 to 60 ◦ C), water interacting with hydroxyl groups (lost up to 120 ◦ C) and water bound
40 to 60 °C), water interacting with hydroxyl groups (lost up to 120 °C) and water b
to carboxyl groups (up to 160 ◦ C), and the second stage between 185 and 370 ◦ C.
to carboxyl groups (up to 160 °C), and the second stage between 185 and 370 °C.
In this sense, Del Angel (2019) [75] indicated that from 290 ◦ C to 423 ◦ C, the decompo-
sition of polymeric residues occurs, and a high Tg is possibly attributed to the plasticizing
effect of these sugars and good chemical stability of the films [76], as well as intramolecular
and intermolecular interactions and steric effects [77]; therefore, the high value of Tg in the
films of Passiflora tripartita var. mollissima could be governed by its rigid structure or by the
presence of sugars [78], while low Tg values imply excellent flexibility of the films, even at
refrigeration temperatures [77].

4. Conclusions
It was confirmed that it is possible to obtain pectin from the shell of Passiflora tripartita
var. mollissima using the acid hydrolysis technique, with a good extraction yield, resulting
in high-methoxyl pectin, with glucuronic acid, galacturonic acid, glucose, xylose and
arabinose, characteristic pectin components. In addition to the above, it was possible to
make simple films using the plate casting technique; the films are malleable and flexible,
with a greenish-yellowish tendency, which highlights the feasibility of this technique for
Membranes 2023, 13, 797 12 of 16

the production of films of different thicknesses and allows for the uniformity of the films.
In addition, the color of the films could favor its use for the protection of photosensitive
compounds. On the other hand, the films presented good mechanical properties, being
resistant films. The thickness and characteristics of the film affected the thermal stability and
the diffusion rate of water vapor, presenting a low permeability to water vapor, suggesting
a good homogenization of the polymeric matrix and, therefore, a high barrier to water
vapor. Likewise, the films were continuous, with certain imperfections, and bright designs
were visualized that could indicate the presence of macromolecular aggregates. Finally,
pectin was successfully extracted from Passiflora tripartita var. mollissima, which is a little-
consumed and -studied fruit and contributes to a reduction in environmental impacts,
diversifying the materials for the extraction of feasible compounds in the elaboration of
films, with possible applications in the areas of medicine, pharmaceuticals and food.

Author Contributions: Conceptualization: M.R.-O. and G.C.-D.; methodology: E.B.L.-E., M.R.-O.,

M.V.-L., V.A.G.-S. and G.C.-D.; software: M.V.-L., M.R.-O. and M.C.C.; validation: M.R.-O. and
G.C.-D.; formal analysis: A.B.G.-H., M.C.C., M.d.L.C.-A. and F.S.S.-V.; investigation: A.B.G.-H.,
M.R.-O., V.A.G.-S. and G.C.-D.; resources: M.R.-O., V.A.G.-S. and G.C.-D.; Original draft preparation:
V.A.G.-S. and M.R.-O.; writing—reviewing and editing: M.R.-O., M.V.-L., F.S.S.-V. and G.C.-D.;
visualization: G.C.-D.; supervision: G.C.-D.; project administration: M.R.-O. and G.C.-D. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded through the proyects; 20221471; 20221510; 20230985; and
20230993, from the Instituto Politécnico Nacional (IPN, Mexico) a proyect CAT2022-0011 from catedras
COMECYT and FICDTEM-2023-97 from COMECYT-Estado de México.
Informed Consent Statement: Informed consent was obtained from all subjects involved in this study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Minerva Rentería-Ortega thanks Tecnológico de Estudios Superiores de San
Felipe del Progreso and Instituto Politécnico Nacional for economical support. Fátima Sarahí Serrano
Villa thanks Instituto Politécnico Nacional (BEIFI-IPN) and CONACYT, and Alitzel Belem García
Hernández thanks COMECyT (CAT2022-0011) for the scholarships provided.
Conflicts of Interest: The authors declare no conflict of interest.

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