International Journal of Biological Macromolecules 146 (2020) 431–443

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Effects of pressurized hot water extraction on the yield and chemical

characterization of pectins from Campomanesia xanthocarpa Berg fruits
Isabela Pereira Dias a,1, Shayla Fernanda Barbieri a,1, Damian Estuardo López Fetzer c,
Marcos Lúcio Corazza c, Joana Léa Meira Silveira a,b,⁎
a
Postgraduate Program in Biochemistry Sciences, Sector of Biological Sciences, Federal University of Paraná, Curitiba, PR 81.531-980, Brazil
b
Department of Biochemistry and Molecular Biology, Federal University of Paraná, CEP 81.531-980 Curitiba, PR, Brazil
c
Department of Chemical Engineering, Federal University of Paraná, CEP 81.531-980 Curitiba, PR, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Pressurized hot water extraction (PHWE), known as a “green” extraction technique, was used to obtain polysac-
Received 8 November 2019 charide from the pulp of gabiroba (Campomanesia xanthocarpa Berg) fruits. The effects of pressure, temperature,
Received in revised form 18 December 2019 and flow rate on pectin yields were analyzed through a full factorial design experiment 23. The optimal extraction
Accepted 30 December 2019
conditions to achieve maximum pectin yield (5.70 wt%) were pressure of 150 bar, temperature of 120 °C, and
Available online 3 January 2020
flow rate of 1.5 mL min−1. The high pressure (100 bar) promoted an increase in galacturonic acid content
Keywords:
(36.0%) compared to conventional hot water extraction (CEGP) with 25.7%. Differences in the proportion of
Gabiroba homogalacturonan (HG) and rhamnogalacturonan (RG-I) domains ranging from 16.3 to 35.4% and 61.7 to
Pressurized hot water extraction (PHWE) 80.1%, respectively, were observed for each pectin sample according to the extraction conditions. The mono-
Full factorial design experimental dimensional (13C-NMR) and bi-dimensional (1H/13C HSQC-NMR) analyses confirmed the presence of HG and
Pectin RG-I regions and indicated the presence of arabinogalactans type I (AG-I) and arabinogalactans type II (AG-II)
NMR analysis in the PHWE pectin samples, which was not found for pectins from gabiroba pulp obtained by CEGP. The results
HPSEC-MALLS-RI showed that PHWE proved to be a promising method for extracting pectins from gabiroba fruits.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction Pectin is structurally and functionally the most complex polysaccha-

Pectin is an important component of the diet, and it can be naturally
present in fruits and vegetables or intentionally added (INS 440) during
the processing and preparation of foods [1]. This polysaccharide is a
health-promoting functional ingredient widely used in the food indus-
try as an emulsifier, texturizer, thickener, and gelling agent [1,2]. In ad-
dition, it can be applied in biomedical [3], pharmaceutical [4], and
cosmetics fields [5,6].
Application of pectins is related to the diversity in their structure and
physicochemical properties such as monosaccharide composition, de-
gree of methyl-esterification, molecular weight, and gelation properties,
which are dependent on the pectin source and extraction method ap-
plied in its isolation from the plant material [1,5,7].
⁎ Corresponding author at: Postgraduate Program in Biochemistry Sciences, Sector of
Biological Sciences, Federal University of Paraná, Curitiba, PR 81531-990, Brazil.
E-mail address: jlms12@ufpr.br (J.L.M. Silveira).
1
These authors equally contributed to this work.
ride in plant cell walls, having important functions in plant growth,
morphology, development, and defense [8]. It is generally composed
of the following: homogalacturonan (HG), a linear polymer formed by
D-galacturonic acid residues α(1→4) linked, which may be partially
methyl-esterified at C-6 and acetyl-esterified at O-2 and/or O-3 position
[7,9]; rhamnogalacturonan I (RG-I), a repeating disaccharide [→2)-α-L-
Rhap-(1→4)-α-D-GalAp-(1→]n with neutral side chains mainly com-
posed of arabinans, galactans, and arabinogalactans (AG) [9,10]; and
rhamnogalacturonan II (RG-II), which comprises a homogalacturonic
backbone branched by complex side chains containing the rare mono-
saccharides apiose, O-methyl-xylose, and O-methylfucose [5,8].
The industrial production of pectins is an important ally to agribusi-
ness and can favor the ecosystem as an alternative for adding value to
agrowaste and solid residues extracted from citrus peels (85.5%),
apple pomace (14.0%), and beet pulp (0.5%) [7,11]. On a commercial
scale, pectin production requires large amounts of raw material includ-
ing acid solvents such as sulfuric, nitric, and hydrochloric associated
with high-temperature (60–100 °C) processing. However, these

https://doi.org/10.1016/j.ijbiomac.2019.12.261
0141-8130/© 2020 Elsevier B.V. All rights reserved.
432 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443

extraction conditions can lead to degradation of the polymer structure 2. Materials and methods
such as demethoxylation [12], as well as corrosion in equipment and
piping and environmental problems like water pollution by the acidic 2.1. Plant material
effluents produced [13,14].
In view of this, environmentally friendly technologies have been Campomanesia xanthocarpa fruits were collected in Irati-Paraná,
emerging as alternatives to the traditional acid extraction method. Brazil, located at coordinates 25° 25′ south latitude, 50° 36′ west longi-
Cleaner extraction techniques such as enzyme-assisted [14], tude, and 25° 17′ south latitude, 50° 30′ longitude west, in November
microwave-assisted [15,16], ultrasound-assisted [17,18], and pressur- 2018 during the ripening period. The fruits were selected and washed;
ized hot water extraction [19,20] have been employed in the isolation the peels and seeds were removed in a Macanuda removing device
of macromolecules from plants. (Model SPI-DMJI/2013), and the pulp sample was then stored in a
In this context, pressurized hot water extraction (PHWE), also called freezer at −20 °C. A moisture analyzer (Model-OHAUS MB25) was
subcritical water extraction (SWE), superheated water extraction, and used to evaluate the total solids content of the pulp.
pressurized liquid extraction (PLE) allow the fast and efficient extrac-
tion of macromolecules from plants using water as a solvent [21,22]. 2.2. Experimental design
In PHWE, water is maintained at a temperature between normal boiling
point (100 °C) and critical point (374 °C) under pressure that is high Response surface methodology (RSM) was used in the design of the
enough to keep the water in the liquid state [23,24]. In these conditions, experiment and to determine an adequate model for pectin extraction
the water exhibits physical advantages such as high diffusion, low vis- using the PHWE technique. Three-factor, two-level (−1, 1) full factorial
cosity, low surface tension, and increased vapor pressure. Such proper- design was chosen to investigate the effect of process variables and op-
ties provide effective mass transfer and higher solubility of more timize the pectin yield. In developing the regression equation, the tested
hydrophobic compounds [13,21,22,25]. factors were coded according to the equation:
PHWE has been mainly employed for extraction of phenolic
compounds [26], essential oils [26], lipids [27], and antioxidant Xi−X0
compounds [28]. Moreover, PHWE has been identified as a sustain- xi ¼ ð1Þ
ΔX
able approach to polysaccharide extraction, e.g., glucans from
mushrooms (Pleurotus ostreatus) [29] and hemicelluloses from
The variables considered were pressure (X1) ranging between 50
wood [30].
and 150 bar, temperature (X2) between 80 and 120 °C, and flow rate
Some studies have shown pectin extraction through PHWE in labo-
(X3) between 1.5 and 4.5 mL min−1 (Table 1). A total of 11 test runs
ratory scale under different conditions—temperature 90 to 175 °C, pres-
under these conditions, including three replicates for the central point,
sure 3 to 100 bar, and processing time 5 to 100 min—using different raw
were performed for the statistical modeling. The experimental data
materials: apple (Malus domestica) pomace, tangerine (Citrus reticulata)
were fitted to a first order polynomial equation to establish the relation-
peel [19], mango (Mangifera indica) peel [31], pomelo (Citrus grandis
ship between independent variables and responses. The generalized
Osbeck) peel [32,33], sugar beet (Beta vulgaris) pulp, [34] and cacao
form of the equation is:
(Theobroma cacao L.) pod husk [20]. In those studies, the authors mainly
evaluated the yield of pectins obtained and some chemical parameters
such as degree of methyl esterification, monosaccharide composition, Y ¼ β0 þ β1 X 1 þ β2 X 2 þ β3 X 3 þ β12 X 1 X 2 þ β13 X 1 X 3 þ β23 X 2 X 3 ð2Þ
and molar mass.
The use of a suitable extraction method alongside a good under- Table 1
standing of the individual and interactive effects of the process param- Full factorial design of experiments with independent variables and their levels, experi-
eters temperature, pressure, and flow rate (in a semi-batch or mental and predicted data (yield) of PEGP samples.
continuous extraction processes) are essential to maximizing pectin ex- Variables Unit Actual levels
traction yield. However, not only the pectin yields should be considered,
−1 0 +1
but also their structure and chemical composition because these are
known to determine their applications [7,13,25]. (X1) Pressure bar 50 100 150
(X2) Temperature °C 80 100 120
In our research group, Campomanesia xanthocarpa Berg (gabiroba), a
(X3) Flow rate mL min−1 1.5 3.0 4.5
Brazilian native species belongs to Myrtaceae family [35], was studied as
a source of polysaccharides [36,37] with food [37,38] and bioactive Runa X1 X2 X3 Yield (wt%) YPb (wt%)
properties [39]. Between the polysaccharides, a crude pectin from PEGP1 50 80 1.5 5.46 5.38
gabiroba pulp was obtained by hot water extraction using a batch pro- PEGP2 150 80 1.5 3.52 3.44
cess [37]. It was composed of 31.0% of HG with a degree of methyl- PEGP3 50 120 1.5 5.18 5.42
esterification of 60% and 65.3% RG-I. This structure presented different PEGP4 150 120 1.5 5.70 5.88
PEGP5 50 80 4.5 4.33 4.56
rheological properties, according to its concentration in solution, favor-
PEGP6 150 80 4.5 2.39 2.62
able to the application of gabiroba pectin in different systems [37]. Fur- PEGP7 50 120 4.5 4.72 4.60
thermore, the crude and purified gabiroba pectins presented antitumor PEGP8 150 120 4.5 5.12 5.06
potential, as demonstrated by their cytotoxic effect in human glioblas- PEGP9 100 100 3.0 4.87 4.62
toma cell lines [39]. PEGP10 100 100 3.0 4.63 4.62
PEGP11 100 100 3.0 4.92 4.62
Therefore, considering the biotechnological and therapeutic poten- CEGP 1.01 100 – 5.05 –
tial already presented by the gabiroba pectins, the aim of this study
was to extract these polysaccharides using a PHWE semi-batch tech- Validation
nique, evaluate the influence of extraction parameters (pressure, tem- Triplicates X1 X2 X3 Yield (wt%) Mean (wt%) SDc (wt%)
perature, and flow rate) by full factorial design using the pectin
PEGP4a 150 120 1.5 5.70 5.66 0.06
extraction yield as the response variable, in addition to determine and PEGP4b 5.71
compare the chemical composition of gabiroba pectins obtained by PEGP4c 5.59
PHWE at different conditions. Furthermore, to the best of our knowl- a
PHWE pectin samples.
edge, there are no reported data presented concerning the application b
Yield predicted by the statistical model.
of PHWE extraction for polysaccharide recovery from Myrtaceae fruits. c
SD: standard deviation; CEGP: conventional extraction gabiroba pectin.
 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443
where Y represents yield as response variable and X1, X2, and X3 are the
uncoded values of the independent variables of extraction: pressure
(bar), temperature (°C), and flow rate (mL min−1), respectively. β0,
β1, β2, β3, β12, β13, and β23 are constant coefficients where β0 is a con-
stant; β1, β2, and β3 are coefficients for linear terms, and β12, β13, and
β23 are the coefficients for interaction terms. The effects of process var-
iables were analyzed statistically using an analysis of variance (ANOVA).
The quality of the models was evaluated using the following statistical
tests: p-value (p=probability), regression coefficient (R2), and lack of
fit. The p-value, in this case, accounts for the probability of obtaining
model values either greater than or equal to the experimental result
[40].
2.3. Equipment and experimental procedure (PHWE method)
A semi-batch extraction was used to extract pectin from gabiroba
pulp with high pressure and hot water as solvent. A schematic diagram
of the experimental setup used in this work is presented in Fig. 1.
Around 25 g of thawed gabiroba pulp was placed inside the PHWE
extractor vessel (20.44 cm3 inner volume, length L = 17.2 cm, diameter
Φ = 1.24 cm). Both ends of the extractor were equipped with stainless
steel filters with pore sizes of 0.5 mm. The temperature was controlled
by a heating jacket equipped with electrical resistance and thermocou-
ple sensors. Water was pumped into the extraction vessel using a high-
pressure liquid pump (Eldex, model 2SM, USA) and the constant volu-
metric flow mode. The pressure was adjusted and controlled using a
back-pressure regulator (V3) (model KPB1SOA Swagelok, UK).
First, the vessel was loaded with the raw material, and the pretreat-
ment was applied: 550 mL of ethanol 99.9% was pumped at 80 °C and
50 bar using a constant flow rate around 4 mL min−1, resulting in the in-
soluble alcohol residues (AIR). Subsequently, ethanol was replaced by
18 MΩ.cm degassed ultrapure water, pH 6.9 (Master system-MS2000,
Gehaka, Brazil). After the adjusted extraction conditions (pressure, tem-
perature, and flow rate) were performed according to the pre-
established experimental design (Table 1), the system was kept in static
equilibration for 15 min. Afterward, in the static period, dynamic extrac-
tion was performed until there was 500 mL of aqueous extract.
The total volume of aqueous extract was collected at 20 °C from the
PHWE system and then centrifuged (5000 ×g, 4 °C for 15 min). The su-
pernatant was removed using a rotary evaporator vacuum (802,
Fisatom) at 60 °C and dialyzed using 6–8 kDa membrane (Spectrum
labs) against ultrapure water to conductivity similar to that of water
(0.05 μS cm−1). After dialysis, the retained membrane fraction was pre-
cipitated with ethanol PA (3:1 v/v) (Dipalcool), followed by centrifuga-
tion (5000 ×g, 4 °C for 25 min). Finally, the precipitate was washed
three times with ethanol PA (Sigma), dried in a vacuum oven
(EDGCON5P with FastVac DV-200N-250 vacuum pump) at room tem-
perature (25 °C), and named as PEGP (Fig. 2).
Fig. 1. Schematic diagram of the dynamic pressure hot water extraction system (PHWE). V1 and V2: cylinder valves; V3: back pressure regulator valve.
433
Pectin extraction yield was calculated as the ratio between the dry
weight of pectins (PEGP) after ethanolic precipitation and the dry
weight of gabiroba pulp as follows in Eq. (3):
weight of dried pectin ðgÞ
Pectin Yield ðwt%Þ ¼  100% ð3Þ
weight of dried pulp ðgÞ
2.4. Conventional extraction of gabiroba pectin (CEGP)
Conventional aqueous extraction was carried out to extract pectins
from gabiroba pulp in order to compare the yield and chemical structure
of these polysaccharides with the samples obtained by PHWE
extraction.
The CEGP was performed according to Barbieri [37] with some mod-
ifications. Gabiroba pulp (25 g) was submitted to treatment with 99%
ethanol under reflux for 30 min at 80 °C, providing the alcohol insoluble
residues (AIR). Afterwards, the pectins were extracted with ultrapure
water at 100 °C, 1:20 solid:liquid, for 2.77 h. These parameters corre-
spond to similar conditions at the central point present in the factorial
design (run PEGP9, PEGP10, and PEGP11 in Table 1), but under atmo-
spheric pressure (1.01 bar). The aqueous extracts were obtained by cen-
trifugation (12,000 ×g; 25 min at 4 °C), dialyzed (6–8 kDa), and the
volume was reduced under vacuum followed by treatment with 99%
ethanol (3:1, v/v) to precipitate the pectins. The precipitate was dried
in a vacuum oven (EDGCON5P with FastVac DV-200N-250 vacuum
pump) at room temperature (25 °C) and named as CEGP.
2.5. Determination of galacturonic acid and neutral monosaccharides
Uronic acid content was measured using the colorimetric m-
hydroxybiphenyl method with galacturonic acid solution
(0–250 g mL−1) as a standard according to Blumenkrantzand and
Asboe-Hansen [41]. The sulfuric acid/tetraborate was added to tubes
containing the sample, and the tubes were cooled in an ice-water
bath. Then, the tubes were shaken in a vortex mixer, heated in a boiling
water bath (6 min), and cooled in an ice-water bath. After adding re-
agent and shaking for 5 min, the absorbance of the samples was deter-
mined at 520 nm.
The identity of the uronic acid was determined by anion exchange
chromatography with pulse amperometric detection (HPAEC-PAD). Pec-
tins were hydrolyzed with 2 mol L−1 TFA (8 h, 100 °C), then dried and
washed three times with methanol until the acid was totally removed
[37]. Samples (1 mg mL−1) were filtered through a membrane of
0.22 μm and injected into a Thermo Scientific Dionex ICS-5000/DC/SP/
ASDV (USA) with CarboPac PA20 column (3 × 150 mm) according to
Nagel, Sirisakulwat, Carle, and Neidhart [42]. Data were collected and
434 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443
Fig. 2. Scheme of extraction of pectins from the pulp of gabiroba fruits by pressurized hot water extraction (PHWE).
analyzed using the ChromeleonTM 7.0 Chromatography Data System
software.
The neutral monosaccharides were determined by total acid hydro-
lysis with 2 mol L−1 TFA for 8 h at 100 °C, followed by conversion to
alditol acetates by NaBH4 reduction (100 °C for 10 min) [43] and acety-
lation with acetic anhydride (Ac2O)-pyridine (1:1 v/v, 1 mL) at 100 °C
for 30 min [44]. The resulting alditol acetates were extracted with
CHCl3, and the samples were analyzed in a Thermo Scientific Trace GC
Ultra gas chromatograph with a mixture of He and N2 with compressed
air as a carrier gas at 1 mL min−1 and using a DB-225-MS column
(0.32 mm internal diameter × 30 m × film thickness 0.25 μm) pro-
grammed from 100 °C to 230 °C at a heating rate of 60 °C min−1. The
alditol acetates were identified by their profiles, and retention times
were compared with standards.
The proportions of homogalacturonans (HG) and ramnogalacturonans
type I (RG-I) were calculated according to M'Sakni et al. [45] where:
HG ¼ GalA–Rha
RGI ¼ ½GalA ð%Þ−HG ð%Þ þ Rha þ Ara þ Gal
2.6. High performance size exclusion chromatography
The elution profile of the soluble polysaccharides was analyzed by
high performance size exclusion chromatography coupled with multi-
angle laser light scattering (DSP\\F, Wyatt Technology, USA) and refrac-
tive index detectors (Waters 2410, USA) (HPSEC-MALLS-RI). The chro-
matography was carried out on a Waters system containing four gel-
permeation columns packed with Ultrahydrogel® 2000, 500, 250, and
120 connected in series, with exclusion limits of 7 × 106, 4 × 105,
8 × 104, and 5 × 103 g mol−1, respectively. The flow rate was 0.6 mL min−1
with 0.1 mol L−1 NaNO2 in the mobile phase and 0.2 g L−1 NaN3 as a pre-
servative at a temperature of 25 °C. The data were collected and proc-
essed using Wyatt Technology ASTRA software, version 4.70.07. The
elution profile of pectin from citrus peels (PCP, Sigma Aldrich P9135)
was used as a standard for comparing the data obtained.
2.7. Nuclear magnetic resonance (NMR) spectroscopy
Mono-dimensional (13C- and 1H-) and bi-dimensional (1H/13C HSQC)
NMR spectra were acquired at 70 °C on a Bruker AVANCE III 400 NMR
(Bruker, Germany) spectrometer, operating at 9.5 T, observing 1H at
400.13 MHz and 13C at 100.61 MHz, and equipped with a 5 mm multinu-
clear inverse detection probe with z-gradient. The samples were solubi-
lized in D2O, and the chemical shifts were expressed as δ (ppm) using
the resonances of –CH3 groups of acetones (1H at δ 2.22; 13C at δ 30.20)
as internal references. All pulse programs were supplied by Bruker. Top-
Spin software, version 3.1, was used for the evaluation of data.
The values of degree of methyl-esterification (DM) were determined
by 1H‐NMR spectroscopy integrating the hydrogen areas corresponding
to H-1 and H-5 of unesterified α-D-GalAp units and H-1 and H-5 of ester-
ified α-D-GalAp units [46,47]. Briefly, the samples were deuterium-
exchanged three times by freeze drying with a D2O solution and finally
dissolved in D2O and transferred to a 5 mm NMR tube. The 1H‐NMR spec-
ð4Þ tra were acquired at 70 °C on a Bruker AVANCE III 400 NMR spectrometer,
observing 1H at 400.13 MHz. Chemical shifts were expressed as δ (ppm).
ð5Þ
2.8. Statistical analysis
Results were statistically evaluated by analysis of variance (ANOVA)
at 95% level of confidence using the Statistica 10 software (Statsoft Inc.,
USA) in order to identify significant differences between the responses
analyzed in the full factorial design (23). The same software was used
to construct the response surfaces.
3. Results and discussion
3.1. Effect of the variables on pectin yield using pressurized hot water ex-
traction (PHWE)
In this study, the full factorial design experimental 23 was applied to
investigate the effect of independent variables (pressure, temperature,
and flow rate) on extraction yield of PEGP samples (Table 1), which
 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443 435

was calculated on the basis of the dry pulp weight (16.45% total solids). rate) is shown below in Eq. (6):
Eleven runs for eight factorial points and three replicates at the central
point (100 bar, 100 °C, and 3.0 mL min−1) were carried out. The design Pectin Yield ¼ 4:62−0:37ðX 1 Þ þ 0:62ðX 2 Þ−0:42ðX 3 Þ þ 0:60ðX 1 X 2 Þ ð6Þ
of the experiments, the pectin extraction yield, and predicted responses
(YP) are given in Table 1. where X1 (pressure), X2 (temperature), and X3 (flow rate) are the coded
The yield of the PEGP samples ranged from 2.39 wt% to 5.70 wt% ac- variables.
cording to the PHWE conditions applied (Table 1). The optimal extrac- Eq. (6) and the data from the variance analysis (ANOVA) presented
tion conditions for achieving maximum yield were pressure of in Table 2 demonstrate that the yield of the PEGP samples was affected
150 bar, temperature of 120 °C, and flow rate of 1.5 mL min−1, with by the three variables (pressure, temperature, and flow rate) through a
which it was possible to obtain a pectin yield of up to 5.70 wt% in the linear effect.
PEGP4 sample. Under these conditions, the analyses were performed The individual and interactive effects that the process variables
in triplicate (PEGP4 a; b; c) showing a mean value of 5.66 ± 0.06 wt% (pressure, temperature, and flow rate) had on the PEGP yield are also
(Table 1). The experimental yield was close to the predicted yield shown in Fig. 3 by three-dimensional (3D) surface plots: when the
(5.88 wt%), which demonstrates the validation of the optimized slope of the response surface is relatively steep, it means that the re-
condition. sponse value has greater effect with the change of extraction conditions.
In addition, to confirm the reproducibility of the experimental de- In this work, the 3D response surfaces were obtained by keeping one of
sign, the central point (PEGP 9–11) was analyzed in triplicate. The re- the variables constant at zero level while varying the other two vari-
sults obtained in these analyses showed a similar yield for each ables (Fig. 3).
sample, with mean value 4.80 wt% ± 0.15 wt% (Table 1). In Fig. 3a the response surface shows the correlation between pres-
In order to compare the efficiency of the PHWE extraction, a semi- sure and temperature variables. The pectin yield increased with the in-
batch process with hot aqueous extraction commonly used at labora- crease in temperature and pressure to 120 °C and 150 bar, respectively.
tory scale (batch process) for pectin extraction from gabiroba pulp According to the literature, a high temperature associated with pressure
(CEGP) was performed under conditions of 1.01 bar, 100 °C, and (that maintains water in the liquid state) affects the mass transfer rate
2.77 h, similar to conditions used for the PEGP9, PEGP10, and PEGP11 to favor extraction, enhancing the solubility of the solute and the diffu-
samples at central point (100 bar, 100 °C, and 3.0 mL min−1, related sion coefficient [13,22,29]. These conditions may cause cell deformation
to 2.77 h) in the experimental design. The yield obtained by conven- and cell membrane damage that facilitates the solvent's permeability in
tional extraction was 5.05 wt%, close to that obtained at the central the plant tissues, promoting the extraction of pectins that are more
point (4.80 wt% average). However, it can be observed that under tightly bound to the cell wall [48]. Some authors such as Chen, Fu, and
higher pressure and temperature conditions (150 bar and 120 °C), Luo [34] also describe the influence of pressure on pectin yield, showing
there was an increase of up to 5.70 wt% in the pectin yield. that a pressure around 100 bar combined with a temperature of 120 °C
Table 2 presents the analysis of variance (ANOVA) used to evaluate for 30 min constituted an ideal condition for obtaining a high yield of
the significance of each variable on extraction yield of PEGP samples sugar beet pulp pectin (24.63%) by PHWE.
present in the full factorial design experimental 23. Flow rate was also a significant variable to be considered in pectin
As can be seen, the p-values of linear coefficients (X1: pressure; X2: extraction by PHWE. Fig. 3b and c show that the highest yield was
temperature; X3: flow rate, Table 2) and interaction term coefficients achieved when a lower flow rate (1.5 mL min−1) was used. The flow
X1X2 were lower than 0.05 (p b 0.05), indicating the significant effects rate is directly related to the total extraction time, which in this work
of these parameters on PHWE pectin yield. The lack of fit tests were ranged from 1.85 h (4.5 mL min−1) to 5.55 h (1.5 mL min−1), to obtain
not significant (p = 0.252), indicating that the models had adequate ac- a total 500 mL of aqueous extract for each run. Different authors have
curacy for predicting pectin yield using any combination of independent described time as an important variable in pectin extraction [49–51] be-
factors within the range of this study. The large values of the coefficients cause extended contact times between extracting solvent and plant ma-
of determination (R2) at 0.96 and adjusted R2 at 0.93 showed that the terial provide greater mass transfer of solid particles in the solution [52].
model can be used with a strong confidence level to predict the extrac- However, some studies show that excessive time exposure under high
tion process (Table 2). temperatures leads to degradation of the pectin chain molecules
The linear equation was obtained by removing non-significant terms [32,53].
(p N 0.05) such as the interaction between pressure (X1) and flow rate As seen in Fig. 3c, the highest yield was achieved at the highest tem-
(X3), with p = 0.810, and interaction between temperature (X2) and perature of 120 °C and at the lower flow rate of 1.5 mL min−1. This may
flow rate (X3), with p = 0.109. The relationship between the response have been due to changes in the physical properties of water at different
(yield) and independent variables (pressure, temperature, and flow temperatures. According to the NIST database [54], higher temperatures
reduce the viscosity of liquid solvents; the water's viscosity decreased
from 3.5 × 104 Pa s to 2.3 × 104 Pa s when the temperature was in-
creased from 80 °C to 120 °C, allowing a better penetration of matrix
Table 2
ANOVA analysis for the regression model of the PEGP sample yields. particles and improving the efficiency of the extraction process [55]. In
addition, increases in vapor pressure and rapid thermal desorption of
SS DF MS F-value p-value
target compounds from matrices could enhance extraction efficiency
X1 1.095 1 1.095 45.6 0.021 [25].
X2 3.150 1 3.150 131.1 0.008 Other studies using PHWE to obtain pectins also observed tempera-
X3 1.361 1 1.361 56.6 0.017
ture as a significant variable [19,32,34,48,56]. Liew et al. [32] showed
X1 X2 2.880 1 2.880 119.8 0.008
X1 X3 0.002 1 0.002 0.1 0.810 that pectin yields obtained from pomelo (Citrus grandis Osbeck) peels
X2 X3 0.186 1 0.186 7.7 0.109 at 120 °C were at least three times (4.3 to 20.4%) higher than the yields
Lack of fit 0.143 2 0.071 3.0 0.252 at 90 °C.
Pure error 0.048 2 0.024
The results obtained through the experimental design (Table 1),
Total SS 8.865 10
R2 0.96 Eq. (6), and response surface (Fig. 3) showed that the extraction condi-
Adjusted R2 0.93 tions pressure, temperature, and flow rate affected the pectin yield ob-
X1: pressure; X2: temperature; X3: flow rate; SS: sum of squares; DF: degrees of freedom;
tained from gabiroba pulp by PHWE. According to these data, it was
MS: mean square; R2: regression coefficient; F-value: Fisher value; p-value: probability observed that high pressure (150 bar) and high temperature (120 °C)
value. combined with a lower flow rate (1.5 mL min−1/5.55 h) that provides
436 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443

a longer contact time between solvent and raw material were shown to
be the most favorable conditions for obtaining the highest yield of
gabiroba pectin (5.70 wt%) observed in the PEGP4 sample (Table 1).
However, in addition to its high yield, the chemical composition of the
polysaccharide of interest must be taken into account as it directly re-
flects its application.
Thus, the PEGP4 sample obtained as an optimal condition by exper-
imental design, the PEGP2 sample (150 bar, 80 °C, and 1.5 mL min−1/
5.55 h) that presented the lowest content of galacturonic acid (GalA,
Table 3), and the PEGP10 sample (100 bar, 100 °C, and 3.0 mL min−1/
2.77 h; at central point) that presented a higher GalA content
(Table 3) in relation to the tested conditions, were selected to study
their chemical composition. The PWHE samples was also compared
with the CEGP—obtained by conventional extraction with hot water
(1.01 bar, 100 °C, and 2.77 h)—to evaluate the influence of extraction
processes on the chemical composition of these pectins.

3.2. Chemical characterization of gabiroba pectins

The influence of extraction conditions on the chemical structure of

pectins extracted from gabiroba pulp through PHWE was evaluated by
colorimetric, spectrometric, and spectroscopic techniques.
The monosaccharide analyses (Table 3) revealed that all samples of
pectic polysaccharides obtained from the pulp of gabiroba fruits pre-
sented arabinose (Ara: 44.3 to 59.7%), followed by galacturonic acid
(GalA: 17.2 to 36.1%) and galactose (Gal: 8.9 to 18.7%) as the major
monosaccharides, varying their proportions according to the extraction
conditions. In addition, minor amounts of rhamnose (Rha: 0.6 to 1.5%),
xylose (Xyl: 0.3 to 1.3%), mannose (Man: 0.5 to 2.8%), glucose (Glc: 0.5
to 1.6%), and fucose (Fuc: 0.1 to 0.3%) were also present in these pectic
structures. The small amounts of Xyl, Man, and Glc possibly came from
hemicelluloses detected in the gabiroba pulp [36].
These results are in agreement with the monosaccharide composi-
tion of gabiroba pectins recently described by Barbieri et al. [37].
These authors used hot water aqueous extraction in a batch processes
for 28 h, giving a crude fraction pectin mainly composed by 54.5% Ara,
33.5% GalA, and 7.6% Gal. According to the literature, similar monosac-
charide composition was observed in water extraction pectins from
other fruits belonging to the Myrtaceae family such as araçá (Psidium
catteleianum), which presented Ara (50.3%), GalA (30%), and Gal
(10.4%) [57], and in a mixture of two species of guavira
(Campomanesia pubescens and C. adamantium) composed of Ara
(46.7%), GalA (44.6%), and Gal (5.5%) [58].
Some studies have shown that the content of GalA may vary accord-
ing to raw material and the extraction conditions [19,33,51]. In this
work, the GalA content of pectins obtained by PHWE was affected by
pressure and temperature varying between 17.2% and 36.1% (Table 3).
It was observed that a lower temperature (80 °C) promoted the ex-
traction of pectins with lower GalA content (17.2%) in the PEGP2 sample
compared to the other tested conditions. Already, when the gabiroba
pectin was extracted at a higher temperature (100 °C) and 100 bar, an
increase in GalA content to 36.0% in the PEGP10 sample was observed
(Table 3). However, when the temperature was increased to 120 °C in
the PEGP4 sample, a slight decrease in the GalA content to 34.7% was ob-
served (Table 3). This reduction is probably associated with elevated ex-
traction temperature that may also lead to degradation or
depolymerization of the pectin. Wang et al. [19] also showed a decrease
in GalA content from 44.4% to 40.1% in apple pomace pectin when the
temperature of subcritical water extraction (PHWE) increased from
130 °C to 150 °C, respectively.

Fig. 3. Response surface plot showing the effect of the interaction between
(a) temperature and pressure, (b) pressure and flow rate, and (c) flow rate and
temperature on the yield of the PEGP samples.
 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443 437

Under atmospheric pressure (1.01 bar) for 2.77 h, the CEGP sample
obtained by conventional aqueous extraction at 100 °C presented a
minor GalA content of 25.7% (Table 3) as compared to the PEGP10 sam-
ple extracted by PHWE in the same conditions (100 °C, 2.77 h), but
under the higher pressure of 100 bar, which showed a 10.3% increase
in GalA content (36.0%).
The differences in the proportions of homogalacturonan (HG) and
rhamnogalacturonan (RG-I) domains in the pectin samples were evalu-
ated by applying Eqs. (4) and (5) [45] and using the monosaccharide
composition data (Table 3).
All the gabiroba pectin samples obtained from PHWE (PEGP 1-11) and
CEGP presented a main chain consisting mainly of the RG-I domain,
which ranged from 61.7% to 80.1% depending on the extraction conditions
(Table 3). According to the literature [59], a higher proportion of RG-I-rich
pectin can be obtained by using water as a solvent when compared to ex-
traction using mineral acids at pH 1–3 and 80–90 °C (the process com-
monly used to obtain commercial HG-rich pectins) because the hot acid
results in the hydrolysis of the neutral side chains of the RG-I region.
The PEGP2 sample presented a high RG-I (80.1%) and low HG
(16.3%) content compared to the other pectin samples obtained by
PHWE. However, when the pectin was extracted at a high temperature
(PEGP4 sample), an 18.1% decrease in the RG-I composing its structure,
compared to the PEGP2 sample, was observed.
Differences of pressure applied at conventional extraction (CEGP)
and PHWE (PEGP10) at 1.01 bar and 100 bar, respectively, suggest
that under atmospheric pressure the extraction of pectins formed
mainly by the RG-I domain is favored, as observed in the CEGP sample
with RG-I (73.6%) and HG (24.2%) proportions, while the use of higher
pressure leads to extraction of pectins composed of a lower proportion
of RG-I (62.8%) and a higher proportion of HG (35.4%) compared to
CEGP. According to the literature [8–10], the RG-I proportion reflects
the rhamnose content present in the main pectin chain along with arab-
inose and galactose that compose the neutral side chains. The differ-
ences in Ara and Gal content could indicate changes in the side chains.
Therefore, as evidenced by monosaccharide composition (Table 3), the
PHWE samples presented a decrease in Ara and Gal content that sug-
gests a break in the side chains of gabiroba pectins.
The pectin samples (Table 1) from gabiroba pulp were also analyzed
by high performance size exclusion chromatography (HPSEC-MALLS-
RI) (Fig. 4) to verify the macromolecular distribution.
All the PHWE pectin samples resulted in heterogeneous elution pro-
files (Supplementary Fig. S1). The chromatograms for CEGP (Fig. 4a),
PEGP2 (Fig. 4b), PEGP4 (Fig. 4c), PEGP10 (Fig. 4d), and PCP (Fig. 4e)
are presented to show qualitative differences between elution profile
peaks.
The chromatograms show four peaks detected by RI at around 40 (I),
45 (II), 50 (III), and 54 min (VI), indicating the presence of high and low
mass populations that make up the structure of these pectins. The light-
scattering detector at 90° (LS) shows a major peak around 40 min for all
samples analyzed, corresponding to the largest molar mass and the first
peak (I) detected by refractive index (RI), which confirms the ability to
obtain high molar mass pectin populations through PHWE and CEGP.
According to our previous study, which isolated a
homogalacturonan from gabiroba fruits after a fractionation process
[37], it is possible that this pectin population eluted in peak (I), corre-
sponding to the HG region. This hypothesis can be confirmed by com-
paring the gabiroba pectin samples with the elution profile of a pectin
sample obtained from citrus peel (called PCP) used as a commercial
standard (Sigma Aldrich). A homogeneous elution profile of the PCP—
mainly formed by GalA (81.9%), followed by Gal (11.5%), Ara (3.7%),
and Rha (1.6%)—corresponding to 80.3% of HG can be observed by RI
and LS detectors in Fig. 4e, with a major peak detected at 42 min (I), cor-
roborating the data obtained in this study. The major peaks present in
the elution profile, ranging between 45 and 54 min as detected by re-
fractive index (RI) for CEGP, PEGP2, PEGP4, and PEGP10 samples, prob-
ably correspond to the RG-I region being in agreement with the
monosaccharide composition (Table 3).
The chemical structures of CEGP, PEGP2, PEGP4, and PEGP10 were
investigated by 13C-NMR (Fig. 5) and 1H/13C HSQC-NMR (Fig. 6,
Table 4), analyzing the influence of extraction conditions on the chem-
ical structure of pectins extracted from gabiroba pulp through PHWE.
The main peaks and correlations observed in each spectrum were com-
pared with the NMR data available in the literature [37,39,60–62].
For the CEGP (Fig. 5a), PEGP4 (Fig. 5c), and PEGP10 (Fig. 5d), the sig-
nals of the C-6 of unesterified α-D-GalAp units were assigned at around
δ 174.0 ppm. Signals of methyl-esterified α-D-GalAp units could not be
observed in the 13C‐NMR spectrum for all samples; however, signals of
methyl and acetyl groups linked to the α-D-GalAp units appear close to
δ 52.9 and δ 20.0 ppm, respectively. For PEGP2 (Fig. 5b), the signals at-
tributed to the presence of α-D-GalAp units are not observed, probably
due to a low amount of GalA (17.2%, Table 3).
The 13C-NMR also indicated the presence of arabinogalactans (AG)
in the CEGP and PEGP samples. Signals around δ 109.0, δ 107.0, and
106.0 ppm were attributed to α-L-Araf (C-1), while signals for anomeric
carbon (C-1) of (1→4)-linked-β-D-Galp units were found at δ
104.3 ppm. The signal around δ 16.0 ppm was assigned to α-L-Rhap
(1→2)-linked units (Fig. 5), showing the presence of the RG-I region
in agreement with the monosaccharide composition (Table 3).
The 1H/13C HSQC-NMR spectrum shows the correlations between
carbon and hydrogen with signals at different intensities for each

Table 3
Monosaccharide composition of pectins obtained from the gabiroba pulp.

Monosaccharide composition (%)a

Run X1b X2 b X3b GalAc Rha Fuc Ara Xyl Man Gal Glc HGd RG-Id

CEGP 1.01 100 – 25.7 1.5 0.3 54.4 0.3 1.0 15.8 1.0 24.2 73.2
PEGP1 50 80 1.5 34.0 1.2 0.1 44.4 0.9 2.8 14.9 1.6 32.8 61.7
PEGP2 150 80 1.5 17.2 0.9 0.2 59.7 0.4 1.7 18.7 1.3 16.3 80.1
PEGP3 50 120 1.5 24.5 0.6 0.3 57.7 1.0 1.5 13.5 1.1 23.9 72.3
PEGP4 150 120 1.5 34.8 1.2 0.2 44.4 1.2 1.7 15.2 1.3 33.6 62.0
PEGP5 50 80 4.5 35.2 1.0 0.2 44.3 0.9 0.7 16.9 0.8 34.2 63.4
PEGP6 150 80 4.5 29.6 1.1 0.1 54.1 0.8 0.6 13.0 0.6 28.5 69.4
PEGP7 50 120 4.5 30.8 1.3 0.2 46.2 1.0 1.4 17.9 1.1 29.5 66.7
PEGP8 150 120 4.5 29.6 0.8 0.2 49.3 1.3 1.0 16.4 1.2 28.8 67.4
PEGP9 100 100 3.0 36.1 1.0 0.1 49.5 0.6 0.6 11.4 0.5 35.0 63.0
PEGP10 100 100 3.0 36.0 0.7 0.1 52.4 1.0 0.5 8.9 0.5 35.4 62.6
PEGP11 100 100 3.0 35.0 0.6 0.1 46.6 0.7 0.5 16.0 0.5 34.4 63.8

CEGP: conventional extraction gabiroba pectin. PEGP1-11: PHWE pectin samples.

a
% of peak area of monosaccharide composition relative to the total peak area, determined by GLC.
b
X1: pressure (bar); X2: temperature (°C); X3: flow rate (mL min−1).
c
Uronic acids, determined using the m-hydroxybiphenyl method [41]. GalA was identified by HPAEC-PAD.
d
HG/RG-I according to M'sakni et al. [45].
438 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443

monosaccharide unit that makes up the structure of gabiroba pectins
extracted by PHWE under different pressure, temperature, and flow
rate conditions. The spectrums of CEGP, PEGP2, PEGP4, and PEGP10
(Fig. 6a, b, c, and d, respectively; Table 4) revealed C-1/H-1 correlations
from HG assigned to →4)-α-D-6MeGalAp-(1→ (unit A) at δ 100.1/4.96
and of →4)-α-D-GalAp-(1→ (unit B) at δ 99.5/5.14 units. The remaining
methyl-esterified units of α-D-GalAp ring correlations were assigned at
δ 68.5/3.77 (C-2/H-2), δ 68.9/3.91 (C-3/H-3), δ 78.2/4.45 (C-4/H-4), and
δ 70.5/5.06 (C-5/H-5) units, while the signals at δ 68.4/3.75 (C-2/H-2), δ
68.4/3.98 (C-3/H-3), δ 78.2/4.45 (O-substituted C-4/H-4), and δ 71.6/
4.70 (C-5/H-5) were attributed to α-D-GalAp unesterified units. The sig-
nal at δ 52.8/3.82 corresponding to CH3-C6 of →4)-α-D-6MeGalAp-(1→
was observed for all samples [61,63].
The methyl esterification degree (DM) of pectin samples obtained
by PHWE extraction and CEGP were determined by 1H-NMR spectros-
copy. All samples presented a high degree of methyl-esterification
(DM N 50%), which showed 79.6%, 68.3%, 52.0%, 61.8% for the CEGP,
PEGP2, PEGP4, PEGP10, respectively.
In addition to the typical signals of homogalacturonan, signals attrib-
uted to RG-I backbone were assigned at δ 98.7/4.93 (C-1/H-1), δ 16.5/
1.25 (C-6/H-6) to unsubstituted →2)-α-L-Rhap-(1→ (unit C) and at
16.5/1.28 ppm to substituted →2,4)-α-L-Rhap-(1→ (unit D) (Fig. 6;
Table 4) [37,64].
The 1D and 2D NMR spectra also provide information about the
arabinose components of CEGP, PEGP2, PEGP4, and PEGP10 samples.
Unusual signals at δ 101.6/5.09 (C-1/H-1), δ 74.4/4.04 (C-3/H-3), δ

Fig. 4. HPSEC elution profile of gabiroba pectins obtained from the pulp of gabiroba fruits according to the experimental design. Refractive index (RI). Light scattering (LS 90°). (a) CEGP,
(b) PEGP2, (c) PEGP4, (d) PEGP10, and (e) PCP.
 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443 439

Fig. 5. 13C-NMR spectrum of (a) CEGP, (b) PEGP2, (c) PEGP4, and (d) PEGP10. Samples were dissolved in deuterium oxide (D2O) and data collected at probe temperature of 70 °C. Acetone
(δ30.2) was used as internal standard. Chemical shifts are expressed in δ, ppm.
440 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443

82.2/3.90 (C-4/H-4), and δ 63.2/3.79 (C-5/H-5) were assigned to termi-

nal β-L-Araf-(1→ (unit E). Despite few reports of the presence of the t-
β-L-Araf-(1→ units, this signal was also recently observed for pectins
obtained from guavira pomace [56], a fruit that belongs to the
Myrtaceae family as does the gabiroba fruit.
The terminal of Ara in the α configuration (unit F) was observed
at δ 107.0/5.15 (C-1/H-1). Furthermore, the other anomeric signals
—(C-1/H-1) at δ 107.6/5.08, δ 107.0/5.17, and δ 107.1/5.20 ppm—
were attributed to →5)-α-L-Araf-(1→ (G), →3)-α-L-Araf-(1→ (H)
and →3,5)-α-L-Araf-(1→ (I) units, respectively (Fig. 6, Table 4)
[58,60].
The 1H/13C HSQC-NMR spectrum (Fig. 6) also shows the correlation
at δ 104.3/4.63 ppm assigned to nonreducing terminal β-D-galactose
units (J), whereas the anomeric signals at δ 96.5/4.58 and δ 92.4/
5.30 ppm indicated the presence of α and β, reducing end galactose
units (units K and L). The intense correlations in the type-I
arabinogalactan (AG-I) backbone were observed at δ 104.3/4.61, δ
71.9/3.68, δ 73.4/3.75, δ 77.6/4.14, δ 74.6, 3.70, and δ 61.0/3.81 ppm,
which was attributed to C-1/H-1, C-2/H-2, C-3/H-3, C-4/H-4, C-5/H-5,
and C-6/H-6 from →4)-β-D-Galp-(1→ (unit M) [37,61,63]. AG-I has
been obtained by aqueous extraction from fruits such as starfruit
(Averrhoa carambola L.) [64], cubiu (Solanum sessiliflorum D.) [63], and
tamarillo (Solanum betaceum) [65].
For the PEGP2, PEGP4, and PEGP10 samples, other correlations
were observed in the HSQC spectrum (Fig. 6b, c, d; Table 4). The sig-
nals assigned at δ 103.0/4.48, δ 103.0/4.53, and δ 102.6/4.53 ppm,
corresponding to →3,6)-β-D-Galp-(1→ (N), →3)-β-D-Galp-(1→
(O), and →6)-β-D-Galp-(1→ (P) units, respectively, are character-
istic components of type-II arabinogalactans [61,62,66]. These
polysaccharides consist of a (1→3)-linked-β-D-Galp backbone con-
taining short side chains of α-L-Araf-(1→6)-[β-D-Galp-(1→6)] n .
The galactosyl residues of these side chains can be substituted
with α-L-Araf-(1→3) units [10].
The signals attributed to AG-II were not observed in the sample ex-
tracted with water by the conventional method without the use of
high pressure, as shown in the CEGP spectrum (Fig. 5a).
The investigation of the chemical structure of pectins obtained
through monosaccharide composition as well as HPSEC-MALLS-RI,
13
C-NMR, and 1H/13C HSQC analyses confirmed that it is possible to ex-
tract pectins from the gabiroba pulp using the PHWE technique. Al-
though all samples obtained by PHWE presented a main chain
composed mainly by RG-I regions with a minor proportion of HG, the
HG/RG-I ratio ranged in relation to the extraction condition.
In evaluating the differences in PHWE samples in low temperature
conditions (80 °C), it was possible to obtain a low yield (3.52 wt%)
and low GalA content (17.2%) as observed in the PEGP2 sample. In addi-
tion, the GalA signals in the HSQC spectrum showed low intensity for
the HG compared to the other samples. When the temperature in-
creased to 120 °C, an increase in the yield and GalA content of the sam-
ples was observed. An optimal condition, demonstrated by
experimental design, can be reached to obtain a higher pectin yield
(5.70 wt%) from the PEGP4 sample. However, according to results ob-
tained in yield that were in agreement with the chemical structure, it
is possible to suggest that the most favorable condition for pectin ex-
traction from gabiroba pulp is at 100 bar of pressure and 100 °C of tem-
perature as tested at PEGP10 (central point) (Table 1, Table 3). In these
milder conditions, a high GalA content (36.0%) and yield of 4.63% of
PEGP10 sample was obtained without changes of chemical composition,
with the presence of RG-I region with a minor proportion of HG, in ad-
dition to AG-I and AG-II, as shown by NMR analyses.

Fig. 6. 1H/13C HSQC-NMR correlation map of (a) CEGP, (b) PEGP2, (c) PEGP4, and
(d) PEGP10. Samples were dissolved in deuterium oxide (D2O) and data collected at
probe temperature of 70 °C. Acetone (δ30.2/2.22) was used as internal standard.
Chemical shifts are expressed in δ, ppm.
Table 4
1
H and 13C NMR chemical shifts of gabiroba pectins.

Glycosil units Nucleus Chemical shifts, δ (ppm)

1 2 3 4 5 6 -OCH3

I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443
13
A →4)-α-D-6MeGalAp-(1→ C 100.1 68.5 68.9 78.2 70.5 nd 52.8
1
H 4.96 3.77 3.91 4.45 5.06 3.81
13
B →4)-α-D-GalAp-(1→ C 99.5 68.4 68.4 78.2 71.6 nd –
1
H 5.14 3.75 3.98 4.45 4.70
13
C →2)-α-L-Rhap-(1→ C 98.7 nd nd nd nd 16.5
1
H 4.93 1.25
13
D →2,4)-α-L-Rhap-(1→ C nd nd nd nd nd 16.5
1
H 1.28
13
E t-β-L-Araf-(1→ C 101.6 nd 74.4 82.2 63.2 –
1
H 5.09 4.04 3.90 3.79
13
F t-α-L-Araf-(1→ C 107.0 79.7 76.5 84.1 61.4 –
1
H 5.15 4.28 3.98 3.98 3.75
13
G →5)-α-L-Araf-(1→ C 107.6 81.1 76.7 82.2 66.4 –
1
H 5.08 4.13 4.01 4.19 3.81
13
H →3)-α-L-Araf-(1→ C 107.0 79.9 83.9 82.5 61.3 –
1
H 5.17 4.36 3.99 4.17 3.81
13
I →3,5)-α-L-Araf-(1→ C 107.1 80.9 nd 82.2 66.7 –
1
H 5.20 4.22 3.90 3.92
13
J t-β-D-Galp-(1→ C 104.3 71.8 73.5 69.0 74.8 60.9
1
H 4.63 3.53 3.78 3.90 3.66 3.75
13
K →4)-β-D-Galp C 96.5 72.4 71.9 77.4 74.5 60.9
1
H 4.58 3.58 3.76 4.12 3.82 3.71
13
L →4)-α-D-Galp C 92.4 nd nd nd nd nd
1
H 5.30
13
M →4)-β-D-Galp-(1→ C 104.3 71.9 73.4 77.6 74.6 61.0
1
H 4.61 3.68 3.75 4.14 3.70 3.81
13
N →3,6)-β-D-Galp-(1→ C 103.0 70.4 81.5 nd 74.5 69.0
1
H 4.48 4.23 3.85 3.81 3.91
13
O →3)-β-D-Galp-(1→ C 103.0 70.8 81.9 nd 73.4 62.7
1
H 4.53 4.30 3.79 3.75 3.82
13
p →6)-β-D-Galp-(1→ C 102.6 71.1 72.2 nd 73.6 69.0
1
H 4.53 4.47 3.68 3.88 3.91

nd: not determined.

441
442 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443
4. Conclusions
Pectin was extracted from gabiroba pulp by pressurized hot water
(PHWE), and the yield and chemical structure were affected by pres-
sure, temperature, and flow rate conditions. According to the experi-
mental design, the linear equation, and the surface response, the use
of 150 bar pressure and temperature of 120 °C led to a higher pectin
yield (5.70 wt%). Furthermore, this high pressure also promoted an in-
crease of 10.3% in galacturonic acid content compared to conventional
hot water extraction. Gabiroba pectin samples extracted through
PHWE presented a high degree of methyl-esterification with a main
chain composed mainly by RG-I regions with a minor proportion of
homogalacturonans. 13C-NMR and 1H/13C HSQC data also suggest the
presence of AG-I and AG-II in the gabiroba pulp extracted by PHWE.
AG-II structures were not observed in the CEGP sample extracted with
water without high pressure. Overall, considering the quantity and
quality of the extracted pectins, it can be suggested that PHWE is a
promising, environmentally friendly technique for the extraction of
these polysaccharides.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2019.12.261.
Acknowledgements
The authors gratefully acknowledge the following Brazilian agencies
for financial support: the National Council for Scientific and Technolog-
ical Development - CNPq, the Coordination for the Improvement of
Higher Education Personnel - CAPES, the Araucaria Foundation, the
Nanoglicobiotec and Ministry of Science and Technology/CNPq, and
the Federal University of Parana - Brazil. J.L.M.S. is a research member
of the CNPq Foundation (n° 476950/2013-9; 308296/2015-0; 309225/
2018-3); M.L.C is a research member of the CNPq Foundation (Grant
num. 305393/2016-2); I.P.D is the beneficiary of a post-graduation
scholarship (n° 1798682) provided by CAPES, and S.F.B. is the benefi-
ciary of a post-doctoral scholarship from Coordination of Superior Level
Staff Improvement - CAPES, n° 88887.335103/2019-00. The authors
would like to thank the NMR Center of UFPR for recording the NMR
spectra, the Brazilian Agricultural Research Corporation/Embrapa For-
estry, Rossana Catie Bueno de Godoy, Cristiane Helm, and Maria Cristina
Medeiros Mazza for providing the gabiroba pulp.
References
[1] S.Y. Chan, W.S. Choo, D.J. Young, X.J. Loh, Pectin as a rheology modifier: origin, struc-
ture, commercial production and rheology, Carbohydr. Polym. 161 (2017) 118–139,
https://doi.org/10.1016/j.carbpol.2016.12.033.
[2] Z.J. Kermani, A. Shpigelman, H.T.T. Pham, M. Ann, A.M.V. Loey, M.E. Hendrickx, Func-
tional properties of citric acid extracted mango peel pectin as related to its chemical
structure, Food Hydrocoll. 44 (2015) 424–434, https://doi.org/10.1016/j.foodhyd.
2014.10.018.
[3] F. Munarin, M.C. Tanzi, P. Petrini, Advances in biomedical applications of pectin gels,
Int. J. Biol. Macromol. 51 (4) (2012) 681–689, https://doi.org/10.1016/j.ijbiomac.
2012.07.002.
[4] G. Smistad, S. Bøyum, S.J. Alund, A.B.C. Samuelsen, M. Hiorth, The potential of pectin
as a stabilizer for liposomal drug delivery systems, Carbohydr. Polym. 90 (3) (2012)
1337–1344, https://doi.org/10.1016/j.carbpol.2012.07.002.
[5] F. Naqash, F.A. Masoodi, S.A. Rather, S.M. Wani, A. Gani, Emerging concepts in the
nutraceutical and functional properties of pectin—a review, Carbohydr. Polym. 168
(2017) 227–239, https://doi.org/10.1016/j.carbpol.2017.03.058.
[6] A. Noreen, Z.-I.-U. Nazlic, J. Akrama, I. Rasulb, N.Y. Manshaa, R. Iqbald, S. Tabasuma,
M. Zubera, K.M. Zia, Pectins functionalized biomaterials; a new viable approach for
biomedical applications: a review, Int. J. Biol. Macromol. 101 (2017) 254–272,
https://doi.org/10.1016/j.ijbiomac.2017.03.029.
[7] F. Dranca, M. Oroian, Extraction, purification and characterization of pectin from al-
ternative sources with potential technological applications, Food Res. Int. 113
(2018) 327–350, https://doi.org/10.1016/j.foodres.2018.06.065.
[8] D. Mohnen, Pectin structure and biosynthesis, Curr. Opin. Plant Biol. 11 (3) (2008)
266–277, https://doi.org/10.1016/j.pbi.2008.03.006.
[9] B.M. Yapo, Pectic substances: from simple pectic polysaccharides to complex pectins
- a new hypothetical model, Carbohydr. Polym. 86 (2) (2011) 373–385, https://doi.
org/10.1016/j.carbpol.2011.05.065.
[10] A.J. Voragen, G.J. Coenen, R. Verhoef, H. Schols, Pectin, a versatile polysaccharide
present in plant cell walls, Struct. Chem. 20 (2009) 263–275, https://doi.org/10.
1007/s11224-009-9442.
[11] R. Ciriminna, A. Fidalgo, R. Delisi, L.M. Ilharco, M. Pagliaro, Pectin production and
global market, Agro Food Ind. Hi Tec. 27 (5) (2016) 17–20.
[12] U. Einhorn-Stoll, H. Kunzek, Thermoanalytical characterisation of processing-
dependent structural changes and state transitions of citrus pectin, Food Hydrocoll.
23 (1) (2009) 40–52, https://doi.org/10.1016/j.foodhyd.2007.11.009.
[13] L.R. Adetunji, A. Adekunle, V. Orsat, V. Raghavan, Advances in the pectin production
process using novel extraction techniques: a review, Food Hydrocoll. 62 (2017)
239–250, https://doi.org/10.1016/j.foodhyd.2016.08.015.
[14] M. Marić, A.N. Grassino, Z. Zhuc, F.J. Barba, M. Brnčić, S.R. Brnčić, An overview of the
traditional and innovative approaches for pectin extraction from plant food wastes
and by-products: ultrasound-, microwaves-, and enzyme-assisted extraction,
Trends Food Sci. Tech. 76 (2018) 28–37, https://doi.org/10.1016/j.tifs.2018.03.022.
[15] S. Rahmati, A. Abdullah, O.L. Kang, Effects of different microwave intensity on the
extraction yield and physicochemical properties of pectin from dragon fruit
(Hylocereus polyrhizus) peels, Bioact. Carbohydr. Diet. Fibre 18 (2019) 1–8, https://
doi.org/10.1016/j.bcdf.2019.100186.
[16] P. Rodsamran, R. Sothornvit, Microwave heating extraction of pectin from lime peel:
characterization and properties compared with the conventional heating method,
Food Chem. 278 (2019) 364–372, https://doi.org/10.1016/j.foodchem.2018.11.067.
[17] B.B.V. Guandalini, N.P. Rodrigues, L.D.F. Marczak, Sequential extraction of phenolics
and pectin from mango peel assisted by ultrasound, Food Res. Int. 119 (2019)
455–461, https://doi.org/10.1016/j.foodres.2018.12.011.
[18] M. Kazemi, F. Khodaiyan, S.S. Hosseini, Eggplant peel as a high potential source of
high methylated pectin: ultrasonic extraction optimization and characterization,
LWT - Food Sci. Tech. 105 (2019) 182–189, https://doi.org/10.1016/j.lwt.2019.01.
060.
[19] X. Wang, Q. Chen, X. Lü, Pectin extracted from apple pomace and citrus peel by sub-
critical water, Food Hydrocoll. 38 (2014) 129–137, https://doi.org/10.1016/j.
foodhyd.2013.12.003.
[20] N. Muñoz-Almagro, L. Valadez-Carmona, J.A. Mendiola, E. Ibáñez, M. Villamiel,
Structural characterisation of pectin obtained from cacao pod husk. Comparison of
conventional and subcritical water extraction, Carbohydr. Polym. 217 (2019)
69–78, https://doi.org/10.1016/j.carbpol.2019.04.040.
[21] M. Plaza, C. Turner, Pressurized hot water extraction of bioactives, TrAC-Trend Anal.
Chem. 71 (2015) 39–54, https://doi.org/10.1016/j.trac.2015.02.022.
[22] M. Plaza, M.L. Marina, Pressurized hot water extraction of bioactives, TrAC-Trend
Anal. Chem. 116 (2019) 236–247, https://doi.org/10.1016/j.trac.2019.03.024.
[23] C.C. Teo, S.N. Tan, J.W.H. Yong, C.S. Hew, E.S. Ong, Pressurized hot water extraction
(PHWE), J. Chromatogr. A 1217 (16) (2010) 2484–2494, https://doi.org/10.1016/j.
chroma.2009.12.050.
[24] A. Mustafa, C. Turner, Pressurized liquid extraction as a green approach in food and
herbal plants extraction: a review, Anal. Chim. Acta 703 (1) (2011) 8–18, https://
doi.org/10.1016/j.aca.2011.07.018.
[25] S.M. Zakaria, S.M.M. Kamal, Subcritical water extraction of bioactive compounds
from plants and algae: applications in pharmaceutical and food ingredients, Food
Eng. Rev. 8 (1) (2016) 23–34, https://doi.org/10.1007/s12393-015-9119-x.
[26] M. Çam, E. Yuksel, H. Alasalvar, B. Basyigit, H. Sen, M. Yilmaztekin, A. Ahhmed, O.
Sagdıc, Simultaneous extraction of phenolics and essential oil from peppermint by
pressurized hot water extraction, J. Food Sci. Technol. 56 (1) (2019) 200–207,
https://doi.org/10.1007/s13197-018-3475-5.
[27] H.K. Reddy, T. Muppaneni, Y. Sun, Y. LI, S. Ponnusamy, P.D. Patil, P. Dailey, T. Schaub,
O.F. Holguin, B. Dungan, P. Cooke, P. Lammers, W. Voorhies, X. Lu, S. Deng, Subcrit-
ical water extraction of lipids from wet algae for biodiesel production, Fuel 13
(2014) 73–81, https://doi.org/10.1016/j.fuel.2014.04.081.
[28] M. Plaza, V. Abrahamsson, C. Turner, Extraction and neoformation of antioxidant
compounds by pressurized hot water extraction from apple byproducts, J. Agric.
Food Chem. 61 (2013) 5500–5510, https://doi.org/10.1016/j.fuel.2014.04.081.
[29] F.R. Smiderle, D. Morales, A. Gil-Ramírez, L.I. Jesus, B. Gilbert-López, M. Iacomini, C.
Soler-Rivas, et al., Evaluation of microwave-assisted and pressurized liquid extrac-
tions to obtain d-glucans from mushrooms, Carbohydr. Polym. 156 (2017)
165–174, https://doi.org/10.1016/j.carbpol.2016.09.029.
[30] G. Gallina, Á. Cabeza, H. Grénman, P. Biasi, J. García-Serna, T. Salmi, Hemicellulose
extraction by hot pressurized water pretreatment at 160 °C for 10 different
woods: yield and molecular weight, The J. Supercrit. Fluid. 133 (2017) 716–725,
https://doi.org/10.1016/j.supflu.2017.10.001.
[31] H. Xia, A.S. Matharu, Unavoidable food supply chain waste: acid-free pectin extrac-
tion from mango peel via subcritical water, Faraday Discuss. 202 (2017) 31–42,
https://doi.org/10.1039/C7FD00035A.
[32] S.Q. Liew, W.H. Teoh, C.K. Tan, R.Y.G.C. Ngoh, Subcritical water extraction of low
methoxyl pectin from pomelo (Citrus grandis (L.) Osbeck) peels, Int. J. Biol.
Macromol. 116 (2018) 128–135, https://doi.org/10.1016/j.ijbiomac.2018.05.013.
[33] S.Q. Liew, W.H. Teoh, R. Yusoff, G.C. Ngoh, Comparisons of process intensifying
methods in the extraction of pectin from pomelo peel, Chem. Eng. Process. 143
(2019) https://doi.org/10.1016/j.cep.2019.107586.
[34] H.M. Chen, X. Fu, Z.G. Luo, Properties and extraction of pectin-enriched materials
from sugar beet pulp by ultrasonic-assisted treatment combined with subcritical
water, Food Chem. 168 (2015) 302–310, https://doi.org/10.1016/j.foodchem.2014.
07.078.
[35] G.M. Barroso, Sistemática das magnoliophytas, in: G.M. Barroso (Ed.), Sistemática
de Angiospermas Do Brasil-Parte II, Editora Universidade de São Paulo, São Paulo
1978, pp. 114–126.
[36] S.F. Barbieri, A.C. Ruthes, C.L.O. Petkowicz, R.C.B. Godoy, G.L. Sassaki, A. Santana-
Filho, Extraction, purification and structural characterization of a
 I.P. Dias et al. / International Journal of Biological Macromolecules 146 (2020) 431–443
galactoglucomannan from the gabiroba fruit (Campomanesia xanthocarpa Berg),
Myrtaceae family, Carbohydr. Polym. 174 (2017) 887–895, https://doi.org/10.
1016/j.carbpol.2017.07.015.
[37] S.F. Barbieri, S.C. Amaral, A.C. Ruthes, C.L.O. Petkowicz, N.C. Kerkhoven, E.R.A. Silva,
J.L.M. Silveira, Pectins from the pulp of gabiroba (Campomanesia xanthocarpa
Berg): structural characterization and rheological behavior, Carbohydr. Polym. 214
(2019) 250–258, https://doi.org/10.1016/j.carbpol.2019.03.045.
[38] S.F. Barbieri, C.L.O. Petkowicz, R.C.B. Godoy, H.C.M. Azeredo, C.R.C. Franco, J.L.M.
Silveira, Pulp and jam of gabiroba (Campomanesia xanthocarpa Berg): characteriza-
tion and rheological properties, Food Chem. 263 (2018) 292–299, https://doi.org/
10.1016/j.foodchem.2018.05.004.
[39] S.C. Amaral, S.F. Barbieri, A.C. Ruthes, J.M. Bark, S.M.B. Winnischofer, J.L.M. Silveira,
Cytotoxic effect of crude and purified pectins from Campomanesia xanthocarpa
Berg on human glioblastoma cells, Carbohydr. Polym. 224 (2019) https://doi.org/
10.1016/j.carbpol.2019.115140.
[40] G.E.P. Box, W.G. Hunter, J.S. Hunter, Statistics for Experimenters: An Introduction to
Design, Data Analysis and Model Building, John Wiley and Sons Inc, New York, 1978.
[41] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of
uronic acids, Anal. Biochem. 54 (2) (1973) 484–489, https://doi.org/10.1016/0003-
2697(73)90377-1.
[42] A. Nagel, S. Sirisakulwat, R. Carle, S. Neidhart, An acetate-hydroxide gradient for the
quantitation of the neutral sugar and uronic acid profile of pectins by HPAECPAD
without postcolumn pH adjustment, J. Agr. Food Chem. 62 (2014) 2037–2048,
https://doi.org/10.1021/jf404626d.
[43] M.L. Wolfrom, A. Thompson, Reduction with sodium borohydride, in: R.L. Whistler,
M.L. Wolfrom, J.N. BeMiller (Eds.), Methods in Carbohydrate Chemistry, Academic
Press Inc, New York and London 1963, pp. 65–68.
[44] M.L. Wolfrom, A. Thompson, Acetylation, in: R.L. Whistler, M.L. Wolfrom, J.N.
BeMiller (Eds.), Methods in Carbohydrate Chemistry, Academic Press Inc, New
York and London 1963, pp. 211–215.
[45] N.H. M’sakni, H. Majdoub, S. Roudesli, L. Picton, D.L. Cerf, C. Rihouey, C. Morvan,
Composition, structure and solution properties of polysaccharides extracted from
leaves of Mesembryanthenum crystallinum, Eur. Polym. J. 42 (4) (2006) 786–795,
https://doi.org/10.1016/j.eurpolymj.2005.09.014.
[46] H. Grasdalen, O.E. Bakøy, B. Larsen, Determination of the degree of esterification and
the distribution of methylated and free carboxyl groups in pectins by 1H NMR spec-
troscopy, Carbohydr. Res. 184 (1988) 183–191, https://doi.org/10.1016/0008-6215
(88)80016-8.
[47] C. Rosenbohm, I. Lundt, T.M.I.E. Christensen, N.W.G. Young, Chemically methylated
and reduced pectins: preparation, characterisation by 1H NMR spectroscopy, enzy-
matic degradation, and gelling properties, Carbohydr. Res. 338 (7) (2003) 637–649,
https://doi.org/10.1016/S0008-6215(02)00440-8.
[48] X. Guo, D. Han, H. Xi, L. Rao, X. Liao, X. Hu, J. Wu, Extraction of pectin from navel or-
ange peel assisted by ultra-high pressure, microwave or traditional heating: a com-
parison, Carbohydr. Polym. 88 (2) (2012) 441–448, https://doi.org/10.1016/j.
carbpol.2011.12.026.
[49] S.S. Hosseini, F. Khodaiyan, M.S. Yarmand, Optimization of microwave assisted ex-
traction of pectin from sour orange peel and its physicochemical properties,
Carbohydr. Polym. 140 (2016) 59–65, https://doi.org/10.1016/j.carbpol.2015.12.
051.
[50] E. Chaharbaghi, F. Khodaiyan, S.S. Hossein, Optimization of pectin extraction from
pistachio green hull as a new source, Carbohydr. Polym. 173 (2017) 107–113,
https://doi.org/10.1016/j.carbpol.2017.05.047.
[51] C. Colodel, L.C. Vriesmann, R.F. Teófilo, C.L.O. Petkowicz, Extraction of pectin from
ponkan (Citrus reticulata Blanco cv. Ponkan) peel: optimization and structural
443
characterization, Int. J. Biol. Macromol. 117 (2018) 385–391, https://doi.org/10.
1016/j.ijbiomac.2018.05.048.
[52] B. Pasandide, F. Khodaiyan, Z.E. Mousavi, S.S. Hosseini, Optimization of aqueous pec-
tin extraction from Citrus medica peel, Carbohydr. Polym. 178 (2017) 27–33, https://
doi.org/10.1016/j.carbpol.2017.08.098.
[53] J.P. Maran, Statistical optimization of aqueous extraction of pectin from waste du-
rian rinds, Int. J. Biol. Macromol. 73 (2015) 92–98, https://doi.org/10.1016/j.
ijbiomac.2014.10.050.
[54] NIST, National Institute of Standards and Technology, https://webbook.nist.gov/
chemistry/fluid/ 2017 (accessed 14 October 2019).
[55] N. Nastić, J. Švarc-Gajić, C. Delerue-Matos, S. Morais, M.F. Barroso, M.M. Moreira,
Subcritical water extraction of antioxidants from mountain germander (Teucrium
montanum L.), J. Supercrit. Fluids 138 (2018) 200–206, https://doi.org/10.1016/j.
supflu.2018.04.019.
[56] K. Klinchongkon, N. Chanthong, K. Ruchain, P. Khuwijitjaru, S. Adachi, Effect of eth-
anol addition on subcritical water extraction of pectic polysaccharides from passion
fruit peel, J. Food Process. Preserv. 41 (2017) 1–9, https://doi.org/10.1111/jfpp.
13138.
[57] L.C. Vriesmann, C.L.O. Petkowicz, P.I.B. Carneiro, M.E. Costa, E. Beleski-Carneiro,
Acidic polysaccharides from Psidium cattleianum (Araçá), Braz. Arch. Biol. Techn.
52 (2) (2009) 259–264, https://doi.org/10.1590/S1516-89132009000200001.
[58] V.S. Schneider, M. Iacomini, L.M.C. Cordeiro, β-L-Araf-containing arabinan and
glucuronoxylan from guavira fruit pomace, Carbohydr. Res. 481 (2019) 16–22,
https://doi.org/10.1016/j.carres.2019.06.005.
[59] Y. Mao, R. Lei, J. Ryan, F.A. Rodriguez, B. Rastall, A. Chatzifragkou, C. Winkworth-
Smith, S.E. Harding, R. Ibbett, E. Binner, Understanding the influence of processing
conditions on the extraction of rhamnogalacturonan-I “hairy” pectin from sugar
beet pulp, Food Chem: X 2 (2019) https://doi.org/10.1016/j.fochx.2019.100026.
[60] R.R. Klosterhoff, J.M. Bark, N.M. Glänzel, M. Iacomini, G.R. Martinez, S.M.B.
Winnischofer, L.M.C. Cordeiro, Structure and intracellular antioxidant activity of
pectic polysaccharide from acerola (Malpighia emarginata), Int. J. Biol. Macromol.
106 (2018) 473–480, https://doi.org/10.1016/j.ijbiomac.2017.08.032.
[61] G.E. Nascimento, M. Iacomini, L.M.C. Cordeiro, New findings on green sweet pepper
(Capsicum annum) pectins: Rhamnogalacturonan and type I and II arabinogalactans,
Carbohydr. Polym. 171 (2017) 292–299, https://doi.org/10.1016/j.carbpol.2017.05.
029.
[62] J.M. Rodrigues, M.E.R. Duarte, M.D. Noseda, Modified soybean meal polysaccharide
with high adhesion capacity to Salmonella, Int. J. Biol. Macromol. 139 (2019)
1074–1084, https://doi.org/10.1016/j.ijbiomac.2019.08.038.
[63] C. Colodel, R.M.G. Bagatin, T.M. Tavares, C.L.O. Petkowicz, Cell wall polysaccharides
from pulp and peel of cubiu: a pectin-richfruit, Carbohydr. Polym. 174 (2017)
226–234, https://doi.org/10.1016/j.carbpol.2017.06.052.
[64] C.L. Leivas, M. Iacomini, L.M.C. Cordeiro, Structural characterization of a
rhamnogalacturonan I-arabinan-type I arabinogalactan macromolecule from
starfruit (Averrhoa carambola L.), Carbohydr. Polym. 121 (2015) 224–230, https://
doi.org/10.1016/j.carbpol.2014.12.034.
[65] G.E. Nascimento, C.R. Corso, M.F.P. Werner, C.H. Baggio, M. Iacomini, L.M.C. Cordeiro,
Structure of an arabinogalactan from the edible tropical fruit tamarillo (Solanum
betaceum) and its antinociceptive activity, Carbohydr. Polym. 116 (2015)
300–306, https://doi.org/10.1016/j.carbpol.2014.03.032.
[66] C.S. Tamiello-Rosa, T.M. Cantu-Jungles, M. Iacomini, L.M.C. Cordeiro, Pectins from
cashew apple fruit (Anacardium occidentale): extraction and chemical characteriza-
tion, Carbohydr. Res. 483 (2019) https://doi.org/10.1016/j.carres.2019.107752.