International Journal of Biological Macromolecules 263 (2024) 130230

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

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

Enhancing pectin extraction from orange peel through citric acid-assisted

optimization based on a dual response
Maricarmen Iñiguez-Moreno a, b, José Juan Pablo Pizaña-Aranda a, b, Diana Ramírez-Gamboa a, b,
Claudia Angélica Ramírez-Herrera b, Rafael G. Araújo a, b, Elda A. Flores-Contreras a, b,
Hafiz M.N. Iqbal a, b, Roberto Parra-Saldívar a, b, Elda M. Melchor-Martínez a, b, *
a
Tecnologico de Monterrey, School and Engineering and Science, 64849, Mexico
b
Tecnologico de Monterrey, Institute of Advanced Materials for Sustainable Manufacturing, 64849, Mexico

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

Keywords: Pectin is widely used in several products in the industry. Conventionally, strong and harmful acids are used for its
Pectin extraction extraction. This study optimized the extraction of orange peel’s pectin using citric acid, considering yield and
Valorization orange peels degree of esterification (DE) as response variables. Proximal analyses were performed, and the samples were
Box Behnken design
subjected to a Box-Behnken design on three central points, considering as variables the temperature, time, and
pH. The results of proximate analyses of the orange peels revealed 11.76 % moisture content, 87.26 % volatiles,
0.09 % ash, 50.45 % soluble carbohydrates, 70.60 % total carbohydrates, 0.89 % fixed carbon, 5.35 % lipids, and
36.75 mg GAE/g of phenolic compounds. The resulting second-order polynomial model described the relation of
the input and output variables related to each other. The best performance to obtain a higher yield (18.18 %) of
high methoxyl pectin (DE 50 %) was set at 100 ◦ C/30 min/pH 2.48. Pectin showed antioxidant properties by
ABTS and DPPH assays and similar thermal properties to the commercial polymer. Its equivalent weight was
1219.51 mol/g, and the methoxyl and anhydrouronic acid were 2.23 and 67.10 %, respectively. Hence, pectin
extraction with citric acid results in a high-quality polymer and could be used as a gelling agent, stabilizer, or
texturizer in food products.

1. Introduction
Citrus fruits are one of the world’s most common crops. Fortunella,
Clymendia, Microcitrus, Eremocitrus, Poncirus, and Citrus are the six
genera belonging to this group. Orange is the main traded citrus fruit,
accounting for more than half of global citrus production. Other citrus
with commercial importance include tangerines, lemons, grapefruits,
mandarins, and pomelos [1]. Citrus sinensis commonly known as orange
sweet is processed for the production of juice and jams [2]. In 2021 > 75
million tons of oranges were produced worldwide [3], and approxi­
mately 40 % of the initial weight of the orange is wasted during the juice
extraction process, resulting in millions of tons of organic residues
generated each year globally [1,2]. Waste from citrus processing in­
cludes peels, seeds, juice, and pomace [1]. Orange peels are an impor­
tant source of essential oil, phenolic compounds, limonoids, dietary
fiber, proteins, and polymers like hemicellulose, cellulose, lignin, and
pectin [2,4]. Valorization of orange peels for pectin extraction is gaining
popularity, as this process not only helps reduce waste generation but
also provides a natural and sustainable alternative to synthetic com­
pounds [1]. Conventionally, pectin is extracted by inorganic acids such
as hydrochloric and sulfuric acids which can be harmful to the envi­
ronment and may have negative health impacts and affect the quality of
the polymer [5]. Green extractions using safer solvents such as citric
acid are an attractive option for consumers who prioritize natural
products [4,6]. In addition to this, the obtention of pectin from orange
peels adds value to these waste products, reducing the amount of waste
generated [7].
Pectin is a complex carbohydrate that represents around 30 % of the
dry weight of citrus peels, which are one of the main sources used for its
obtention [8]. The three components of this biopolymer are
rhamnogalacturonan-I, rhamnogalacturonan-II, and homogalacturonan
(HGA). The primary component of pectin is HGA, which is made up

* Corresponding author at: Tecnologico de Monterrey, School and Engineering and Science, 64849, Mexico; Tecnologico de Monterrey, Institute of Advanced
Materials for Sustainable Manufacturing, 64849, Mexico.
E-mail address: elda.melchor@tec.mx (E.M. Melchor-Martínez).

https://doi.org/10.1016/j.ijbiomac.2024.130230
Received 2 October 2023; Received in revised form 26 January 2024; Accepted 14 February 2024
Available online 17 February 2024
0141-8130/© 2024 Elsevier B.V. All rights reserved.
M. Iñiguez-Moreno et al.
primarily of (1 → 4)-α-D-galactopyranosyluronic acid units in linear
chains [9]. The primary criterion for categorizing pectin is the degree of
esterification (DE) of the carboxyl groups of the galacturonic acid units,
which are methyl-esterified and occasionally acetyl-esterified. Based on
DE pectin is classified into two types high methoxyl pectin (HMP, ≥50
%) and low methoxyl pectin (LMP, < 50 %) [10]. LMP requires the
presence of divalent cations such as calcium to form a gel. Whereas,
HMP can form a gel without divalent cations, typically in the presence of
sugar and acid [10,11]. HMP, on the other hand, forms a gel structure in
the presence of high sugar (> 55 % of co-solutes) and low pH (< 3.5)
without the need for additional calcium ions [12]. The application of
pectin in the industry is highly dependent on DE, which affects the
behavior of pectin, especially in the presence of sugars and divalent and
trivalent ions such as calcium ions [10,13].
Conventional extraction of pectin contemplates three key factors to
achieve high extraction yields of a high-quality polymer [14]. These
factors include temperature in the range of 75–100 ◦ C, the use of acid
(inorganic/organic) solutions with pH values between 1.5 and 4, and the
reaction time that can vary between 1 and 3 h. The variation in the values
of these parameters depends on the raw material from where the pectin is
to be extracted [15–18]. Usually, the use of inorganic acids such as sul­
furic, hydrochloric, or nitric acid, generates excellent extraction yields but
presents various environmental drawbacks. Besides, these acids also can
affect the properties of the extracted pectin, which has repercussions on
the final application. Alternatives for this process are focused on the use of
organic acids such as citric, acetic, malic, and tartaric, among others,
representing an eco-friendly for the obtention of food-grade polymers
from renewable sources [19]. Even organic acids present lower hydrolysis
capacities, due to their low dissociation constants, they result in high-
quality pectin with a low depolymerization [6]. Optimal extraction con­
ditions based on a specific response, such as pectin yield and quality are
relevant. Comparing low-temperature values or extraction times will
severely affect the extraction yield. On the contrary, high temperatures
and long extraction times can hinder the precipitation of pectin derived
from the degradation of the pectin chains, or in extreme cases it can be
completely degraded into oligomers or monomers [20].
Extraction of pectin from natural sources such as orange peels is a
complex and challenging process that requires optimization. Mathemat­
ical models can help estimate the yield and DE during the pectin extrac­
tion from natural sources, enabling process improvement and/or scale-up.
Response surface methodology (RSM) is one of the main reported math­
ematical approaches for the optimization of pectin extraction, owing to
this tool enabling the study of the relation between a collection of
quantitative experimental conditions and response variables [18]. Box-
Behnken Design (BBD) is quite an effective mathematical tool that min­
imizes the number of trial runs needed to get the best results, examining
the effects of three or more factors on a response variable at the same
time. The applications of RMS and BBD have been used to determine the
ideal values for the input variables for the pectin and bioactive com­
pounds extraction from several sources including orange peel, guava
pomace, and so on [21,22]. Besides, these statistical tools allow for
increasing the yield and quality of the extracted pectin [4,8]. This
knowledge can then be used to develop more efficient extraction methods
or to optimize other related processes which is a significant improvement
that can lead to increased profits for manufacturers. This study aimed to
optimize the pectin extraction using citric acid and a dual-response (yield
and DE) based on BBD and RSM and the characterization of the physi­
cochemical and thermal properties of the extracted pectin.
2. Materials and methods
2.1. Conditioning of orange peels for characterization and pectin
extraction
After removing the orange peels from the fruits, they were cleaned
and knife-cut into little pieces. Then, the orange peel pieces were dried
2
International Journal of Biological Macromolecules 263 (2024) 130230
at 40 ◦ C in an oven until consistent weight. The dried peels were ground
into smaller particles using a blender, and the resulting powder was
sieved through a strainer to produce a consistent size of between 0.35
and 1 mm. Before the pectin extraction procedure, the dried orange peel
powder was placed in an airtight, sealable bag and kept in the
refrigerator.
2.2. Characterization of orange peel
2.2.1. Humidity
The orange peels were obtained from a local orange juice store in
Monterrey, N.L. The pulp from the orange peels was removed and
weighed before placing it into an oven at 50 ◦ C until reached constant
weight. After the drying period, the orange peels were weighed again,
and the difference in the weight was calculated to be the humidity of the
sample (Eq. 1).
Wf
Humidity (%) = × 100 (1)
Wi
where Wf and Wi are the final and initial weight of the pectin.
2.2.2. Volatile percentage
The ground and sieved sample without moisture was brought to
950 ◦ C in a muffle for 7 min. After the sample was collocated in a
desiccator until reached room temperature (RT, 22–25 ◦ C), then it was
weighed and the difference in mass was calculated as the volatile’s
percentage [23].
2.2.3. Ash content
To determine the ash content the mass from the calculation of vol­
atiles percentage was used and brought to 575 ◦ C for 2 h. The sample
was weighed at RT and the difference in weight from the volatiles weight
was the ash content of the orange peel sample [24].
2.2.4. Fixed carbon percentage
Fixed carbon is the solid carbon in the biomass that remains after the
devolatilization process. The fixed carbon percentage was determined
using the data from the moisture, volatiles, and ash content of the
sample with Eq. 2.
Fixed carbon (%) = 100 − Humidity (%) − Volatile percentage(%) − Ashes(%)
(2)
2.2.5. Total carbohydrate content (TCC)
For the total carbohydrates assay of orange peels 1 g of orange peel
(particle size <0.35 mm) was weighed and 50 mL of distilled water was
added to a flask and brought to homogenization with a magnetic stirrer
for 1 h at RT. To determine the soluble carbohydrates the protocol for
the total carbohydrate assay described above was followed with the
addition of a centrifugation step at 4500 rpm for 15 min and the su­
pernatant was filtered with a Whatman No. 05. The carbohydrates were
quantified following the protocol of Dubois et al. [25] modified by
López-Legarda et al. [26]. Briefly, 0.3 mL of the samples were placed in a
1.5 mL centrifugation tube (Eppendorf, Hamburg, Germany), and 1 mL
of concentrated sulfuric acid (CTR® Scientific, Monterrey, N.L. Mexico)
was added to each tube, followed by mixing a few seconds in a vortex
and then placed on an ice bath for 5 min. Finally, 0.3 mL of each sample
was transferred to a 96-well plate and measured on a Multiskan SkyHigh
Microplate Spectrophotometer A51119700DPC (Thermo Scientific™
Waltham, MA, USA) at 315 nm. Quantification was carried out using a
standard curve of glucose (Sigma-Aldrich, St. Louis MO, USA)
( )
y = 0.0126x + 0.9145; R2 = 0.9836 . To each sample, three dilutions
were made (1/25, 1/50, and 1/100, v/v) and each dilution and the
original solution were further tested in triplicate.
M. Iñiguez-Moreno et al.
2.2.6. Protein content
The determination of total protein content of the orange peel sample
was determined with the Pierce™ Modified Lowry Protein Assay Kit
(Thermo Scientific™, Rockford, IL, USA). In brief, 40 μL of the standard
and the samples (samples obtained from total and soluble carbohydrates
assay) were pipetted onto a 96-well plate and 200 μL of the Modified
Lowry Reagent was added and mixed, then the plate was covered and
incubated at RT for 10 min. After the incubation period, 20 μL the pre­
pared 1 N Folin-Ciocalteu Reagent (Thermo Scientific™; Rockford, IL,
USA) was added and mixed to be later incubated at RT for 30 min. The
absorbance was measured on a Multiskan SkyHigh Microplate Spectro­
photometer (A51119700DPC, Thermo Scientific™, Rockford, IL, USA) at
750 nm. The standard curve to calculate the protein content was obtained
( )
using bovine serum albumin y = 0.0001x + 0.108; R2 = 0.9908 . All the
samples were measured in triplicate.
2.2.7. Total phenolic content (TPC)
The TPC was determined based on the Folin-Ciocalteu Reagent and
this method. Briefly, 20 μL of the samples from total and soluble car­
bohydrates were collocated in a microplate well, then, 10 μL of Folin-
Cicalteu reagent at 1 N (Thermo Scientific™, Rockford, IL, USA), 60
μL MiliQ Water followed by 110 μL of sodium carbonate at 7.5 % (w/v,
Fisher Chemical, Hampton, NH, USA) were added. The microplate was
mixed and incubated at RT in the dark for 1 h. The absorbance was
measured at 730 nm on a spectrophotometer and TPC was determined
( V1 (mL) + V2 (mL)
according to the standard curve of gallic acid y = 0.0043x +
)
0.0318; R2 = 0.9975 and expressed as milligrams of gallic acid equiv­
alents per gram of dried orange peel (mg GAE/g d.b.) [27]. The test was
carried out in triplicate.
2.2.8. Lipids content
The total lipid content was determined by the modified Sulfo-
Phospho-Vanillin (SPV) [28]. The phosphor-vanillin reagent was pre­
pared with 0.6 g of vanillin reagent (Sigma-Aldrich, St. Louis, MO, USA)
dissolved with 10 mL of absolute ethanol and 90 mL of MiliQ-water. Once
the reagent was dissolved it was brought to 500 mL with concentrated
phosphoric acid (CTR® Scientific, Monterrey, N.L. Mexico). The lipids
were extracted from the orange peel following the procedure. 100 mg of
the orange peel was added into a tube followed by 400 μL of MiliQ-Water,
and 6 mL of a chloroform-methanol mixture (2:1, v/v). After that, it was
mixed and placed in an ultrasonic bath for 10 min, then stirred for 30 min.
Lastly, it was centrifugated at 2000 rpm for 10 min. In a 96-well plate, 5
μL of the sample were added followed by 85 μL of concentrated sulfuric
acid. The plate was incubated at 100 ◦ C for 10 min and then cooled down
in the freezer (− 20 ◦ C) for 5 min. After that, 210 μL of the Phospho-
Vainillin reagent was added and placed in the spectrophotometer where
it was incubated at 37 ◦ C and stirred at medium speed for 15 min. After
that, the absorbance was measured at 530 nm. The estimation of the lipids
content was carried out using a standard curve obtained with extra virgin
( )
olive oil y = 0.0068x + 0.0354; R2 = 0.9634 . The test was carried out
in triplicate.
2.3. Pectin extraction
Citric acid (CTR® Scientific, Monterrey, N.L., Mexico) solutions were
prepared at different pH (2, 3, and 4) for their use as a solvent during the
extraction process. Samples of 5 g of orange peel powder were mixed
with the corresponding solution at a solid-to-liquid ratio of 1:20 (w/v)
[5]. For the extraction, different temperatures (60, 80, and 100 ◦ C) for
30, 60, or 90 min were used according to the Box Behnken Design
(Table 1). The mixture was then allowed to cool to room temperature
before being filtered to extract the soluble pectin-containing filtrate [1].
To allow the pectin to precipitate, a double volume of 95 % (v/v) ethanol
was added to the filtrate, and the mixture was left at 4 ◦ C for a full day.
Following that time, the mixture was filtered through filter paper to
3
International Journal of Biological Macromolecules 263 (2024) 130230
extract the pectin that had developed. After collecting the wet pectin, it
was twice cleaned with 70 % (v/v) ethanol and dried at 50 ◦ C in an oven
until its weight remained constant. The yield of extraction was calcu­
lated based on Eq. 3. All the extractions were repeated once.
Mass of extracted pectin (g)
Yield(%) = × 100 (3)
Mass of dried orange peel (g)
2.4. Pectin characterization
2.4.1. Determination of the degree of esterification (DE)
The estimation of the DE was carried out following the protocol
proposed by Wai et al. [29] and modified by Jafari et al. [17]. Briefly, 2
mL of ethanol was used to soak 100 mg of the dried material, and 20 mL
of carbon dioxide-free water was used to dissolve it at 40 ◦ C. Following
the addition of five drops of phenolphthalein reagent, 0.1 M sodium
hydroxide (V1) was used to titrate the sample. After adding 10 mL of
sodium hydroxide at a concentration of 0.1 M, the sample was left to
stand for 20 min. After adding 10 mL of 0.1 M hydrochloric acid, the
sample was rapidly agitated until the pink hue vanished. Once more, five
drops of phenolphthalein were added, and the mixture was titrated with
0.1 M sodium hydroxide to produce a light pink hue (V2). Eq. 4 was used
to determine the DE.
V2 (mL)
DE(%) = (4)
2.4.2. Determination of equivalent weight (EW) of pectin
For the EW, 0.5 g of dried pectin was soaked in 5 mL ethanol, then 1 g
of sodium chloride and 100 mL of distilled water were added. Once the
mixture was homogenized, six drops of phenol red were added and the
sample was titrated with 0.1 M sodium hydroxide [31]. The titration
point was denoted by the pink color and EW was calculated using Eq. 5.
EW(g/mol) =
WS (1000)
(5)
VNaOH (NNaOH )
where WS is the pectin weight, VNaOH and NNaOH are the volume and
normality of the alkali.
2.4.3. Estimation of methoxyl content (MTC)
After gathering the neutral solution from the EW determination, 25
mL of sodium hydroxide (0.25 M) were added. After giving the mixture a
good stir, it was maintained at 25 ◦ C for 30 min. Then, 0.1 M sodium
hydroxide was used as a titration standard for 25 mL of 0.25 M hydro­
chloric acid [31]. The methoxyl content was calculated using Eq. 6.
VNaOH (NNaOH )3.1
MTC(%) = (6)
WS
2.4.4. Determination of total anhydrouronic acid content (AUA)
The AUA of pectin samples was estimated with Eq. 7.
176(0.1y) 176(0.1z)
AUA(%) = + (7)
WS (10) WS (10)
where y and z are the volume of alkali from EW and MTC, respectively
[31].
2.4.5. Solubility and swelling pectin
Solubility and water absorption power were determined at 20, 50,
and 80 ◦ C. Briefly, solutions of the different pectin samples were pre­
pared at 1 % (w/v). The tubes were maintained at the mentioned tem­
peratures in a water bath for 30 min with manual agitation every 5 min.
The suspension was centrifugated for 15 min at 2120 ×g, the superna­
tant was decanted, and the swollen granules were weighed. Then, the
tubes with the granules were dried at 105 ◦ C until reached constant
weight. Solubility and swelling power were reported as percentages of
M. Iñiguez-Moreno et al. International Journal of Biological Macromolecules 263 (2024) 130230

Table 1
Box-Behnken Design with experimental and predicted yield and DE of pectin extracted from orange peels.
Run Temperature (◦ C) Time (min) pH Experimental values Predicted values

Yield (%) Degree of esterification (%) Yield (%) Degree of esterification (%)

1 60 30 3 5.11 ± 0.25a 84.46 ± 5.78a 5.14 82.80

2 100 30 3 9.22 ± 0.85d 69.60 ± 3.20cd 8.89 68.45
3 60 90 3 3.66 ± 0.44a 73.98 ± 4.52bc 3.99 75.13
4 100 90 3 10.17 ± 0.54d 62.84 ± 3.49d 10.14 64.49
5 60 60 2 22.77 ± 4.02c 34.76 ± 3.44e 22.45 36.95
6 100 60 2 31.20 ± 3.96a 28.93 ± 1.46e 31.25 30.63
7 60 60 4 3.26 ± 0.30a 90.86 ± 1.73a 3.20 89.16
8 100 60 4 3.99 ± 0.09a 72.70 ± 3.16bc 4.30 70.50
9 80 30 2 25.49 ± 0.18bc 33.32 ± 5.45e 25.77 32.77
10 80 90 2 26.77 ± 2.86b 31.66 ± 1.80e 26.75 28.30
11 80 30 4 3.57 ± 0.03a 76.80 ± 4.77b 3.59 80.15
12 80 90 4 3.00 ± 0.08a 72.46 ± 0.61bc 2.72 73.00
13 80 60 3 3.81 ± 0.21a 70.08 ± 1.28bc 3.82 70.76
14 80 60 3 3.81 ± 0.33a 70.65 ± 2.21bc 3.82 70.76
15 80 60 3 3.84 ± 0.13 71.56 ± 1.75bc 3.82 70.76

Each value represents the mean of two tests ± standard deviation. Values in the same column followed by different lower-case letters are significantly different ac­
cording to Fisher’s LSD test at p < 0.05.

solubilized pectin and grams of absorbed water per gram of pectin and performed using the three samples of pectin obtained in this study, and a
calculated with Eq. 8 and 9, respectively. sample of commercial citrus pectin was used for data comparison.
Weight of dried granules (g)
Solubility (%) = × 100 (8) 2.4.9. Morphological analysis of the orange peel pectin
Weight of the sample (g)
Scanning Electron Microscopy (SEM, Zeiss, EVO MA25,
Overcoached, Germany) was used for the morphological studies of
pectin extracted from orange peels. Before the analyses, the samples

Weight of swollen granules (g) − Weight of the dried granules(g)

Swelling power = (9)
Weight of the sample(g)

were immobilized in a double-sided carbon tape and covered with gold

for 5 min. Pectin was observed with an accelerating voltage of 6.0 kV
under a high vacuum and with a magnification of 500×.
2.4.6. Analysis of functional groups
To determine the structural properties of pectin using Fourier
2.5. Dual-objective optimization pectin extraction process
Transform Infrared Spectroscopy (FTIR) analysis. The spectra were ob­
tained using an FTIR Spectrometer (PerkinElmer, Waltham, MA, USA) in
A Box-Behnken Design was used to optimize the yield and DE of the
the range of 4000 to 500 cm− 1 with 64 scans and a resolution of 4 cm− 1.
pectin. The experimental design consisted of 15 experiments with three
The test was carried out using the optimal points for yield, DE, dual-
central points for the estimation of pure error. The second-order poly­
response point, and low methoxyl citrus pectin (29–33 %, Food grade
nomial (Eq. 10) was fitted to the experimental data using a non-linear
E-440; Mi Granero, Cholula, Pue., Mexico) as a control in all the tests.
regression technique, which also allowed for the identification of
pertinent model terms. The quadratic response model can be defined as
2.4.7. Antioxidant properties of the extracted pectin
follows taking into account all of the linear terms, square terms, and
The antioxidant properties of pectin were assessed in terms of free
interaction items:
radical scavenging capacity using 2,2-Diphenyl-1-picrylhydrazil (DPPH)
[32] and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ∑
k ∑
k ∑∑
k

(ABTS) [33] assays. Each sample was analyzed in triplicate and a cali­ Y = β0 + β j xj + βjj x2j + βij xi xj + ei (10)
bration curve was used with Trolox (6-hydroxy-2,5,7,8-tetramethyl­
j=1 j=1 i <j=2

chroman-2-carboxylic acid) y = − 3.5966x + 0.6481, R2 = 0.9976 and
y = − 5.4473x + 0.6421; R2 = 0.9975 to DPPH and ABTS, respectively.
The results were expressed in mg of Trolox equivalents per gram of dry
residue (mg TE/g pectin).
2.4.8. Thermal analyses of pectin
For the test, approximately 6 mg of the sample were heated in an
8000 thermogravimetric analyzer (PerkinElmer, Waltham, MS, USA)
from 25 to 600 ◦ C at a rate of 5 ◦ C/min under a nitrogen atmosphere (40
mL/min) [34]. The software Pyris™ (PerkinElmer, Waltham, MS, USA)
was used to analyze the data, and the derivative thermogravimetric
(DTG) curve was obtained directly from the TGA curve. The test was
where Y is the response; xi and xj are variables (i and j range from 1 to k);
β0 is the model intercept coefficient; βj , βjj , and βij are interaction co­
efficients of linear, quadratic, and second-order terms, respectively; k is
the number of independent parameters (k = 2 in this study); andei is the
error [35].
2.6. Statistical analysis
Using least squares to fit quadratic response surfaces and regression
models, statistical analysis of variance (ANOVA) was used to examine
data from BBD (yield and DE). With general linear regression algo­
rithms, which describe and predict the response of the variables under

4
M. Iñiguez-Moreno et al.
various extraction settings, the quadratic polynomial equation or model
for each response was created. The criteria for each term’s and its
interaction terms’ (second order) significance are established by the
conventional method at the 5 % level (or significant at p ≤ 0.05). Using
Statgraphics Centurion 16.1.15 (StatPoint Technologies, Inc. Warren­
ton, VA, USA) software, a one-way analysis of variance (ANOVA) was
used to examine the esterification degree and yield. To compare means,
the Fisher post-hoc least significant difference test (p ≤ 0.05) was
employed.
2.7. Model evaluation
To validate the adequacy of the model/equation, the optimal points
established to reach i) yield of 30 %, ii) DE of 50 %, and iii) optimization
of both parameters (yield and DE) were included. The goodness of the fit
of the quadratic models was determined using the coefficient of deter­
mination (R2, Eq. 11), F-test, and lack of fit test.
MSE
R2 = 1 − (11)
MST
where MSE is the mean square error and MST is the mean total sum of
squares.
3. Results
3.1. Proximate analyses of the dried orange peels
The proximal analysis allowed us to establish the percentages of the
main components of the orange peel. Peels had a moisture content of
11.76 ± 0.11 %, which is similar to other reports (9.8 ± 0.2 %) about
the characterization of dried peels [36]. The soluble carbohydrates and
TCC represented 50.45 ± 2.48 and 70.60 ± 1.56 % of the dried weight
of the orange peel, respectively; in agreement with previous reports
(TCC 52.90 and 70.19 %) [37,38]. The TCC and dietary fiber in orange
peels provide structural support and contribute to their texture. Pectin,
cellulose, and hemicellulose are the main carbohydrates in orange peels;
however, simple sugars like glucose, fructose, and sucrose also are
present in this material [2]. Whereas the estimated ashes content was
0.09 ± 0.00 %, the value of ashes in orange peel ranges from 0.052 to
7.8 % [2,37,38]. The volatile components represent 87.26 ± 1.53 %,
this percentage is attributed to the loss of organic components under the
assessed conditions, including terpenes, aldehydes, alcohols, esters, and
ketones that are the main components of essential oil [4]. By using the
data of moisture content, ashes, and volatile compounds it was possible
to determine the fixed carbon in the dried orange peels (0.89 ± 0.03 %);
which agreed with a previous study (0.69 %) [2]. The fixed carbon
content is a relevant parameter for determining the energy content and
combustion characteristics of solid fuels such as coal. It helps to assess
the suitability of the fuel for various applications such as power gener­
ation and industrial processes [2,37,38].
On the other hand, the lipid content of orange peels was 5.35 ± 0.09
% (w/w), which is between the reported range (0.80 to 10 %, w/w)
[39,40]. Phenolic compounds are a group of secondary metabolites that
are widely distributed in plants and have been associated with various
health benefits due to their antioxidant properties [41]. The TPC of the
orange peels in this study was 36.75 ± 1.77 mg GAE/g d.b. The TCP
reported in the literature to dried orange peels ranged from 0.67 to
26.88 mg GAE/g d.b. [37,41,42]. The value of the assessed parameter in
the proximal analysis of materials such as orange peels depends on
several factors such as variety, maturity, growing conditions, season of
collection, geographical zone cultivation, postharvest handling, method,
and conditions used for its extraction [41,42].
5
International Journal of Biological Macromolecules 263 (2024) 130230
3.2. Effect of extraction conditions on pectin yield
The yield of pectin obtained from orange peels ranged from 3.26 to
31.21 % (Table 1). These results agree with the previous reports about
pectin extraction by the thermal process using organic and inorganic
acids. Duwee et al. [5], Fakayode et al. [4], and Tovar et al. [8] obtained
yields ranging from 0.61 to 58.39 %, 12.93 to 29.05 %, and 10.12 to
29.37 %, respectively. The variation in the yield is related to the values
of the parameters and solvent used during the extraction process. pH and
temperature showed the most pronounced influence on the extraction
yield of pectin compared to the time factor. These findings are in
agreement with previous reports for pectin extraction from orange peels
[4,5], guava fruit pomace [43], durian [15], and watermelon [44] rinds.
As temperature increases promotes pectin solubility leading to an
increment in the extraction rate [4]. Besides, high temperatures increase
the solvent diffusivity into the cell tissues, increasing the mass transfer
of pectin into the solvent [27,45]. Besides, the parallel increment of the
yield with the temperature is related to the conversion of protopectin
(insoluble) into pectin (soluble) [46]. However, beyond the optimal
temperature, pectin yield decreases due to degradation processes that
result in pectin with a smaller molecular size that cannot be precipitated
with ethanol [4]. At the assessed parameters in this study, increasing
extraction temperature at low pH leads to a corresponding increase in
pectin yield (Fig. 1). Whereas at pH 3 and 30 min an increment of 40 ◦ C
only increases ~4 % the pectin yield (Table 1). Similar behavior was
previously reported in studies for the optimization of pectin extraction
from carrot pomace [17], orange [5], and melon peels [16]. Low pH
means an increase in acid concentration (H+), and high acidity increases
the extraction yield of the various pectin and protopectin species. In
neutral sugars, the splitting of glycosidic bonds is more sensitive to pH
than the linkage between two galacturonic acids, which leads to the
degradation of the side chains [47]. Hence, the higher yield of pectin at
low pH and high temperature owing to low pH could promote the hy­
drolysis of protopectin increasing the extraction rate [4]. The hydrolysis
reaction, which resulted in a loss of charge in the carboxylate groups by
changing the hydrated carboxylate groups into the hydrated carboxylic
acid group, inhibits the repulsion of the polysaccharide molecules at
high H+ concentrations [4]. As a result, the decreasing repulsion be­
tween the polysaccharide molecules led to better gelling behavior of
pectin and thus improved the efficiency of the pectin precipitation [5].
In this study, the extraction time has not had a significative effect on the
pectin yield (Table 2), this agrees with previous studies [5,48]. Never­
theless, in several studies, the increment in time and temperature had
shown a linear effect in the increment of the pectin yield [4,15]. How­
ever, it has been reported that long extraction processes lead to levels off
or decrements in the rate yield rate [4,49] because the concentration
gradient is reduced and the solution becomes more viscous [50].
3.3. Effect of extraction conditions on DE of pectin
In addition to the yield, the effect of temperature, time, and pH on
the DE of pectin also was assessed. The DE of pectin refers to the extent
of esterification of the carboxyl groups present in the pectin molecule.
The esterification of pectin occurs through the methylation of some
carboxyl groups, resulting in the formation of methyl ester groups and is
expressed as the percentage of esterified carboxyl groups relative to the
total number of carboxyl groups in the pectin molecule [5]. Pectin with a
high DE value has a higher percentage of esterified carboxyl groups
(HMP ≥ 50 %), while pectin with a low DE value has a lower percentage
(LMP < 50 %) [4,51]. In this study the DE for the extracted pectin
ranged from 28 to 90 % (Table 1), hence depending on the extraction
conditions the DE varies allowing the obtention of LMP or HMP. It has
been reported that DE is affected by solvent type, pH, and extraction
time [5]. The higher DE obtained with a combination of the parameters
set at low or medium values (60 ◦ C/60 min/pH 4) showed reductions at
hard conditions, being particularly affected by pH reduction (p < 0.05).
M. Iñiguez-Moreno et al.
Fig. 1. Principal effect plots and observed versus predicted values for yield and degree of esterification (DE) of pectin. The effect of the assessed parameters on the
response values A) yield and B) DE of the obtained pectin from orange peels acquired from the Box Behnken Design and Response Methodology Surface. The observed
responses against the predicted counterparts are expressed in percentages obtained from Eq. 12 and 13 to C) yield and D) DE, respectively. Dots represent the
experimental data, whereas the predicted data are represented by a straight line.
For the DE all the assessed factors had a significant effect (Table 2),
being pH the factor with the higher effect on the DE; but was inversely to
yield, as the pH increased the DE decreased (Fig. 1). Fig. 2 shows that the
interaction between pH and temperature had a higher effect on the DE
reduction. Similar behavior in the relation of these variables was pre­
viously reported during the extraction of pectin from orange peels [5]
and watermelon rind waste [48]. The agreement between these studies
suggests that severe conditions could speed up the de-esterification of
polygalacturonic chains at lower pH and higher temperatures for a
longer time, decreasing the pectin DE [13]. Hence exists a positive
relation between the increment of pH and DE of pectin owing to the
limited concentration of H+ in the surrounding media. Instead, DE
showed a negative relation with the temperature increment (Fig. 3). This
might be because there is not enough thermal energy to hydrolyze the
ester bonds in the pectin structure at lower temperatures, which pre­
vents pectin from being de-esterified [52].
3.4. RSM for yield and DE optimization
BBD is an autonomous, rotatable quadratic design in which the
variable combinations are the variable’s edges and midpoints [35].
Citric acid was used to optimize the pectin extraction process through
the application of BBD and RSM. A total of 15 experimental runs were
conducted under various extraction conditions, yielding experimental
6
International Journal of Biological Macromolecules 263 (2024) 130230
and projected values for DE and yield (Table 1). Depending on the
experimental setup, yield, and DE values varied from 3.0 to 31.20 % and
28.93 to 90.86 %, respectively. An analysis of the experimental data
using general linear regression and an ANOVA was produced. Eq. 12 and
13 represent the quadratic order polynomial model for pectin yield and
DE, respectively. The quadratic models represented the relationship
between the independent variables (A: temperature in ◦ C; B: time in min;
and C: pH) and responses.
Yield (%) = 129.409 − 0.411073A − 0.207951B − 60.3712C + 0.00477292A2
+ 0.001AB − 0.0963125BC + 0.00146019B2 − 0.0154583BC
+ 9.57542C2
(12)
DE (%) = − 110.99 − 0.984969A − 0.0674792B + 136.031C
+ 0.00651563A2 + 0.00155AB − 0.154312BC − 0.000720833B2
− 0.0222917BC − 16.555C2
(13)
The sign of the models’ regression coefficient may be used to forecast
the influence of independent variables on the answers. Regression co­
efficients with positive signs suggest that the linked variable has a
positive impact on the answer, whereas those with negative signs may
have the opposite effect [53]. Besides, the pH had a stronger effect on
M. Iñiguez-Moreno et al. International Journal of Biological Macromolecules 263 (2024) 130230

Table 2 3.5. Model validation

Analysis of variance (ANOVA) for yield and degree of esterification of pectin.
Yield (%) By using both models the set optimal conditions were 100 ◦ C/30
min/pH 2.48 (Table 3). This point and the optimal points to yield and DE
Source Sum of squares Mean square F-value p-value
of pectin were used to confirm the model’s performance (Table 3). All
A 97.8616 97.8616 39.98 0.0000 parameters were inside the design parameter ranges of the BBD points.
B 0.01156 0.01156 0.00 0.9460
C 2134.900 2134.9 872.24 0.0000
As a result, there was a great agreement between the predicted and
AA 26.9163 26.9163 11.00 0.0041 experimental values of yield and DE of pectin obtained with the two
AB 2.88 2.88 1.18 0.2932 established models (Eq. 12 and 13), since experimental values showed
AC 29.6835 29.6835 12.13 0.0029 deviations with deviations below 10 % of the predicted value. RSM
BB 12.7535 12.7535 5.21 0.0356
predicted the optimization of both variables with a desirability of 0.86.
BC 1.72051 1.72051 0.70 0.4134
CC 677.085 677.085 276.63 0.0000 It demonstrates that the models selected can accurately forecast the
Lack of fit 1.1687 0.3896 0.16 0.9223 yield and DE of pectin obtained with citric acid from orange peels using
Pure error 41.6093 2.4476 traditional thermal processes. Previously it was reported a yield of 35.20
Cor total 2998.96 ± 0.39 % of pectin with a DE of 45.43 ± 0.39 % under optimum con­
R2 0.9857
2
R -adj 0.9793
ditions (90 ◦ C/66 min/pH 2.19) using conventional extraction with
Degree of esterification (%) citric acid [5]. Hence that model enables the extraction of LMP, whereas
A 624.625 624.625 62.19 0.0000 in this study HMP was obtained but with a lower yield. It is very
B 135.083 135.083 13.45 0.0019 important to define the desirable properties or applications of the
C 8477.81 8477.81 844.07 0.0000
extracted pectin, due to the production of LMP it will be linked to a
AA 50.1603 50.1603 4.99 0.0391
AB 6.9192 6.9192 0.69 0.4180 higher yield.
AC 76.1995 76.1995 7.59 0.0135
BB 3.1080 3.1080 0.31 0.5853 3.6. Pectin characterization
BC 3.57781 3.57781 0.36 0.5585
CC 2023.89 2023.89 201.50 0.0000
Lack of fit 93.3679 31.1226 3.10 0.0545
3.6.1. Physicochemical properties of pectin
Pure error 170.748 10.044 The physicochemical properties of the extracted and commercial
Cor total 11,724.9 pectin samples are shown in Tables 3 and 4. The DE of the samples
R2 0.9774 ranged between 33.28 and 52.75 % depending on the extraction con­
2
R -adj 0.9673
ditions (Table 3; the factors that affect the DE value were explained in
A: Temperature, B: Time, C: pH, p-value < 0.05 indicates model terms are Section 3.3). However, the DE only indicates the ratio between the
significant. carboxyl groups esterified with methanol and the free carboxyl groups,
while the MTC refers to the number of methoxyl groups in a sample.
the yield and DE of pectin. Yield increased as the pH was reduced, Therefore, the DE and MTC should be evaluated together, because DE
instead, DE decreased with the pH increment (Fig. 1). Whereas even does not reflect the amount of methyl esterification, especially if the
when the time extraction did not have a significant effect, its effect on content of galacturonic acid is low [4]. MTC of pectin ranged from 2.23
the yield was higher by the interaction with the pH (Fig. 2). In the case of to 5.27, being higher than pectin extracted using the optimal conditions
the DE, the interaction of time extraction with pH and temperature did to DE (Table 4). The MTC of pectin typically ranges from 0.2 to 12 %
not have a significant effect (Table 2). Even this, a shorter extraction depending on the extraction process and source [54]. Values lower than
period and a higher solvent pH or temperature resulted in a higher 7 % suggest a low ester characteristic, indicating that it is good in terms
pectin DE. Reduced extraction time allowed for a shorter time for pectin of quality [55]. Pectin with low MTC will generate a thermo-irreversible
de-esterification, which decreased the frequency of de-esterification gel that will remain gelled even at temperatures that would typically
reactions occurring [52]. cause it to melt [4]. The AUA of the assessed pectin ranged from 63 to 77
The correlation coefficient (R2) and the adjusted correlation coeffi­ % (Table 4). These values agree with previous AUA reports on pectin
cient (R2adj ) values indicated that both models were highly significant. from citrus peels [4,54]. These values indicate a high purity degree,
Furthermore, the model satisfactorily matches the experimental data owing to it having been established that pectin is composed of at least
collected for yield and DE of the extracted pectin from orange peel, as 65 % of galacturonic acid [56,57]. The ability of pectin to form gel
indicated by the lack-of-fit test being insignificant (Table 2). Fig. 1 depends on the amount of AUA present. The high value suggests that the
shows the observed values vs. predicted values using the polynomial extracted pectin contains a minimal amount of protein [4,56].
models for yield and DE (Eq. 12 and 13). On the other hand, EW is crucial in defining how pectin behaves
The response surface curves can be used to identify the ideal value of when it comes to function. Pectin’s ability to form gels is roughly related
each variable for a predicted maximum yield and DE, as well as to to its corresponding weight. Greater gel formation is caused by high EW.
illustrate how the variables interact. Fig. 2 and 3 show the predictive In this study, a higher EW was obtained for pectin obtained under
mathematical model visualized as a three-dimensional response graph. optimal conditions to DE, whereas the pectin obtained under optimal
These graphs show the response one variable is fixed at the middle- conditions to maximize the yield showed a lower EW (Table 4). This
assessed level: A) temperature: 80 ◦ C; B) pH: 3; C) time: 60 min and indicates that harsh conditions during the extraction process promote
the interaction of the other two variables A) time and pH, B) tempera­ low EW and DE in pectin. Even this, the obtained values in this study are
ture and time, and C) temperature and pH on the yield (Fig. 2) and DE higher than the reported pectin from 599.74 orange peel (extraction
(Fig. 3). The tested parameters demonstrated their impact on the yield conditions 93.07 ◦ C/117.00 min/pH 1.609) [4], 386.45 lime (extraction
and DE of the pectin. The optimum parameters for a yield of 30 % and conditions 70 ◦ C/60 min/pH 3.2), and 253.70 lemon (extraction con­
DE of 50 % were at 96.73 ◦ C/68.30 min/pH 2.01 and 86.36 ◦ C/64.84 ditions 60 ◦ C/60 min/pH 3.2) [54]. Low EW causes the pectin to
min/pH 2.41, respectively. At the same time, the optimal conditions for partially degrade more quickly, which is not desirable.
both responses were settled at 100 ◦ C/30 min/pH 2.48 (Table 3).
3.6.2. Solubility and swelling power
Pectin solubility refers to the ability of this polymer to dissolve or
disperse in water or other solvents. Owing to pectin being insoluble in
organic solvents, the test was carried out in distilled water. Solubility is

7
M. Iñiguez-Moreno et al. International Journal of Biological Macromolecules 263 (2024) 130230

Fig. 2. Response surface plots to yield. Response surface plots representing the effects of A) time and pH, B) temperature and time, and C) temperature and pH on the
yield of pectin with fixed values of the remaining parameter at A) temperature: 80 ◦ C; B) pH: 3; C) time: 60 min.

critical in various food applications, particularly in the production of
jams, jellies, fruit fillings, gummy candies, and edible coatings. The
solubility of pectin extracted by the different conditions increased with
temperature increment (Fig. 4) and agreed with previous reports
[43,58]. Pectin is a polyelectrolyte and a weak organic acid due to the
presence of carboxylic acid groups. During water pectin solubilization,
carboxylic acid groups are dissociated making the molecules negatively
charged. These events promote polymer solubility, reducing the inter­
molecular association [58].
Swelling power refers to the ability of a material to absorb and hold
water. When pectin comes in contact with water or other aqueous so­
lutions, it can hydrate and form a gel-like structure, which leads to an
increase in its volume and viscosity. The ability of the extracted pectin in
the present study was significantly lower than that observed in the
commercial polymer. Whereas the pectin obtained optimal yield and DE
showed similar behavior in the measure of this parameter. It is worth
recalling that the extracted pectin swelling power increased one time
with the increment of the temperature from 20 to 80 ◦ C (Fig. 4). This
behavior was reported and attributed to the H-bonds in the pectin
sample being broken due to the increase in temperature, thereby
increasing the swelling. The parallel increment of temperature and
swelling value of the pectin could also be caused by the increase in
thermal mobility of the polymer molecules within the pectin. Pectin’s
ability to swell and form a gel is influenced by factors such as DE. For
instance, LMP (commercial pectin DE 18.67 ± 1.45 %) tends to have a
higher swelling power compared to HMP, owing to the requirements of
this last one to develop a gel [10,11]. The swelling power of pectin plays
a crucial role in the functionality of pectin as a gelling agent, thickener,

8
M. Iñiguez-Moreno et al. International Journal of Biological Macromolecules 263 (2024) 130230

Fig. 3. Response surface plots to the degree of esterification. Response surface plots representing the effects of A) time and pH, B) temperature and time, and C)
temperature and pH on the degree of esterification of pectin with fixed values of the remaining parameter at A) temperature: 80 ◦ C; B) pH: 3; C) time: 60 min.

Table 3
Validation of the quadratic models.
Optimum conditions Experimental conditions Experimental values Predicted values

Temperature (◦ C) Time (min) pH Yield (%) Degree of esterification (%) Yield (%) Degree of esterification (%)

Yield 96.73 68.31 2.01 27.75 ± 0.67a 33.28 ± 2.87b 30.01 30.44
Degree of esterification 86.36 64.85 2.41 13.87 ± 0.43c 48.19 ± 3.14a 15.42 49.90
Dual response 100.00 30.00 2.48 20.82 ± 0.62b 52.75 ± 2.98a 18.18 53.36

Each value represents the mean of two tests ± standard deviation. Values in the same column followed by different lower-case letters are significantly different ac­
cording to Fisher’s LSD test at p < 0.05.

and stabilizer in food products [58]. Owing to their low values for water
absorption the pectin extracted in this research is suitable for applica­
tions for example in the development of edible coatings.
3.6.3. Functional groups analyses
The origin of the plant and the extraction technique have an impact
on the dependence of the pectin structure. This variability in the
structure affects the physicochemical and functional properties of the
pectin, hence its application in the food industry will change [59]. FTIR
analyses enable the identification of any potential modifications to the
pectin structure during its extraction (Fig. 4). Pectin, and other carbo­
hydrates, have strong valence vibrations of hydroxyl groups (O–H). In
the pectin case belonging to the pyranose ring, major absorptions in the
3200–3600 cm− 1 (3389 cm− 1) spectral range of optimal point for yield

9
M. Iñiguez-Moreno et al. International Journal of Biological Macromolecules 263 (2024) 130230

Table 4 was calculated. Samples of pectin displayed a three-step classical ther­

Characteristics of the pectin extracted under optimal conditions. mal decomposition. According to previous reports, the first stage
Pectin Equivalent Methoxyl AUA Antioxidant (25–150 ◦ C) corresponds to the loss of moisture and the physical and
weight content (%) properties chemical-bound water by the pectin molecule [68], corresponding to a
(%) (mg Equivalent weight loss of about 12 %. It could be explained by the hygroscopic
Trolox/g Pectin)
properties of this hydrocolloid [69]. The second step, between 150 and
ABTS DPPH 250 ◦ C, showed a weight loss of around 28 to 32 % of the weight and it
Optimum 390.63 ± 3.29 ± 63.71 0.531 ± 0.393 was related to polymer chain decomposition attributable to saccharide
conditions for 2.67d 0.08c ± 0.005cA ± rings scission [34,69]. The breakdown of carbonaceous material was
yield 1.38b 0.102aB linked to the third weight loss (250–350 ◦ C) and was observed in the
Optimum 2380.95 ± 5.27 ± 77.31 0.764 ± 0.294
four samples, corresponding to around 18 % of the initial weight. In this
conditions for 28.47a 0.36b ± 0.021aA ±
the degree of 4.78a 0.030aB step, organic compounds like CO, CO2, CH4, and volatile compounds are
esterification released [70]. Finally, the pectin obtained under the dual response
Dual-response 1219.51 ± 2.23 ± 67.10 0.653 ± 0.267 model showed a lower residual mass (19.82 %) in comparison with the
optimization 17.89c 0.21d ± 0.008bA ± other samples in the experiment, in which the percentage ranged from
point 3.17b 0.033aB
Commercial 2072.65 ± 11.59 ± 66.70 0.243 ± 0.183
26.64 to 30.30 % (Fig. 5A). The lower residual mass can be related to the
pectin 21.15b 0.98a ± 0.006dA ± DE of each sample, HMP could have a lower interaction with minerals in
2.67b 0.035bB the raw material during the extraction process [10,11]. The thermal
Each value represents the mean of two tests ± standard deviation. Values in the
decomposition of the pectin samples was in agreement with the com­
same column followed by different lower-case letters are significantly different mercial pectin and with the results obtained in other studies [34,70,71].
according to Fisher’s LSD test at p < 0.05. To ABTS and DPPH, the values in the
same row followed by different capital case letters are significantly different 3.6.5. Antioxidant properties of pectin
according to Fisher’s LSD test at p < 0.05. AUA: Anhydrouronic acid. Since high TPC and lipids, which include essential oil, are considered
potential antioxidants [4,41], the antioxidant properties of the pectin
and dual-response pectin samples [5,15]. This vibration is caused by were assessed by DPPH and ABTS. As shown in Table 4 the antioxidant
O–H stretching, the bands are shifted toward lower frequency, and activity values were higher by using ABTS tests than DPPH (measured in
absorption in pectin is obtained using the optimal parameters to DE and mg TE/g pectin). This finding may have indicated that the antioxidant
commercial pectin, which implies a decrement of the hydroxyl pectin activity of orange pectin is mostly mediated by the radical scavenging of
groups [60]. Bands in the 3000–2800 cm− 1 frequency range are related the radical cation ABTS•+. This agrees with previous reports about the
to C–H stretching vibrations of CH3, CH2, and CH groups, in this study antioxidant properties of pectin from Riang pod husk (0.95 ± 0.05 and
the vibration was observed around 2932 cm− 1 in all the samples 0.63 ± 0.01 mmol TE/g pectin to ABTS DPPH, respectively) pod powder
[59,61]. However, assigning the C–H stretching modes of methyl and (0.16 ± 0.01 and 0.24 ± 0.08 mmol TE/g pectin to ABTS DPPH,
methylene groups of the galacturonic acid can be difficult because the respectively) [72]. Whereas, it has been demonstrated that the presence
bands are easily overlapped by a broad O–H band, as happened in this of hydroxyl and acetylated monosaccharide units in a polymer could
study. Similar behavior was previously reported for pectin obtained donate a proton to reduce the radical and increase the antioxidant ac­
from apple pomace [59], citrus peel pectin [62], and commercial pectin tivity [73]. Besides, among the polymers that can be found in plant cells,
[63]. The H-bonds interactions can shift CO and O–H stretching fre­ pectin and xylan are the two with the higher ability to bind a hydroxyl
quencies to lower numbers, suggesting that pectin’ structures are sta­ radical [74]. The antioxidant capacity of the pectin is strongly affected
bilized by the intra- and intermolecular H-bonds [59,64]. The peak at by its DE, LMP tends to have a better capability in radical scavenging
1735 cm− 1 corresponds to the C– –O stretching vibration of the methyl- because, at pH ranging from 2.8 to 4, the carboxylic groups of gal­
esterified carboxyl group (COOCH3). Instead, the stretching vibration of acturonic acid units are negatively charged [75]. Finally, the difference
free carboxyl groups (COOH) of galacturonic acid was observed around between the values of the antioxidant properties estimated by the tests
1645 cm− 1 [15,65]. These bands are correlated to the degree of could be linked to the fact ABTS•+ is more sensible to antioxidants,
methylation [60]. In this study, as increased the DE, the ester carbonyl hence it reacts faster than that of DPPH• [76]. Hence, pectin obtained
groups’ intensity and band area increased, whereas the carboxylate with citric acid by conventional extraction could be an alternative in the
stretching band’s intensity reduced. This behavior was previously re­ formulation of foods or products that require a thickening agent and
ported in the commercial pectin [66]. At 1426 cm− 1, all the samples oxidation stability.
showed the vibration attributable to carboxylate groups. Around 1360
appeared the ring stretched vibrations. The bands at 1226 cm− 1 are 3.6.6. Morphological analysis of extracted pectin
assigned to O–H and side-chain (-CH3CO) vibrations, respectively SEM microstructure of pectin extracted by extraction with citric acid
[15,65]. Side-chain vibrations are stronger in the pectin obtained under under the optimal conditions is shown in Fig. 5. As illustrated, no
the optimal yield and dual response parameters than in the other two changes were appreciated between the samples obtained under the
samples. Whereas commercial pectin showed a slight displacement of different conditions (Table 3). Pectin samples showed a homogeneous
this signal at a lower frequency (Fig. 4). The band at 1150 cm− 1 was structure with a smooth surface with some long cracks in their surface.
linked to asymmetric C-O-C stretching vibration attributable to -O-CH3 These observations are in agreement with previous reports [77]. Het­
[6,67]. The strong spectra vibration at 1013 cm− 1 of pectin samples erogeneous architecture with irregular and rough surfaces with compact
obtained in this study is related to the C–O stretching vibrations [5,15]. and flaky shapes has been reported in pectin extracted by supercritical
The vibrations of the C-OH bridges are mostly responsible for the bands fluid extraction, this is caused by the larger concentration of neutral
at 920 cm− 1 characteristics in the polymers [15,59]. The bands between sugar sides in their structure [77,78].
1300 and 800 cm− 1 are identified as in the “fingerprint” zone in the
polysaccharides [15]. FTIR spectra showed that the obtained pectin was 4. Conclusion
in an esterified form.
The use of BBD and RSM allowed to establishment of the dual-
3.6.4. Thermal analyses objective optimization parameters for the extraction of HMP by the
Using TGA, the weight loss that occurred during the heating process thermal process using citric acid as solvent at 86.36 ◦ C/64.84 min/pH
2.41 with <10 % error. The extraction process variables were found to

10
M. Iñiguez-Moreno et al. International Journal of Biological Macromolecules 263 (2024) 130230

Fig. 4. Solubility, swelling power, and Fourier Transform Infrared spectra of the extracted pectin. Pectin obtained to optimal yield —, degree of esterification —,
dual response — conditions, and commercial pectin —.

11
M. Iñiguez-Moreno et al. International Journal of Biological Macromolecules 263 (2024) 130230

Fig. 5. Thermogravimetric analysis and micrographs obtained by Scanning Electron Microscopy of pectin samples. A) Thermogravimetric and derivative ther­
mogravimetric (DTG) curves and micrographs of pectin samples obtained under the established conditions to A) optimal yield —, B) degree of esterification —, C)
dual response — (yield and DE), and D) commercial pectin —.

12
M. Iñiguez-Moreno et al.
have an opposing effect on pectin yield and DE. The HMP extraction
from orange peels was therefore better adapted to the optimum condi­
tions (DE 50 %). The findings of this investigation confirmed that pec­
tin’s DE value, which was one of the crucial characteristics influencing
its gelling abilities, is strongly affected by extraction conditions. During
the process, the DE showed an inverse relation with the yield. As the
yield increases due to hard conditions, these conditions also promote the
decrement in DE values. The solubility of the different pectin samples
ranged from 40 to 90 % depending on the temperature, pH, and time;
besides, the low swelling values enable the use of this polymer for the
development of stable edible coatings at high relative humilities. The
model obtained for the optimal conditions considering the dual response
can be applied to the production of thermal stable pectin in the food
industry as a gelling agent, stabilizer, thickener, and texturizer in a wide
range of food, pharmaceutical, and healthcare products that addition
require oxidation stability. The valorization of agro-waste such as citrus
wastes without using harmful solvents to obtain polymers or active
compounds is a reliable alternative to promote agro-industrial systems’
circular economy and sustainability.
Funding
This research was funded by Tecnologico of Monterrey Challenge-
Based Research Funding Program 2022, grant number: I025-
IAMSM005-C3-T1-T (Development of smart edible coating for the
preservation of berries).
CRediT authorship contribution statement
Maricarmen Iñiguez-Moreno: Conceptualization, Data curation,
Visualization, Writing – original draft. José Juan Pablo Pizaña-Ara­
nda: Methodology, Visualization. Diana Ramírez-Gamboa: Method­
ology, Writing – original draft. Claudia Angélica Ramírez-Herrera:
Methodology, Writing – review & editing. Rafael G. Araújo: Concep­
tualization, Data curation. Elda A. Flores-Contreras: Methodology.
Hafiz M.N. Iqbal: Writing – review & editing. Roberto Parra-Saldívar:
Funding acquisition, Project administration. Elda M. Melchor-Martí­
nez: Project administration, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
The data are contained within the article.
Acknowledgments
The authors would like to gratefully acknowledge the Latin American
and Caribbean Water Center from Tecnologico de Monterrey for the
laboratory facilities. This work was also partially supported by Consejo
Nacional de Ciencia y Tecnología (CONACYT) under Sistema Nacional
de Investigadores (SNI) program awarded to Maricarmen Iñiguez-Mor­
eno (CVU: 513991), Elda A. Flores-Contreras (CVU: 631205), Hafiz M.N.
Iqbal (CVU: 735340), Elda M. Melchor-Martínez (CVU: 230784), Rafael
G. Araújo (CVU: 714118), Roberto Parra-Saldívar (CVU: 35753). Dr.
Iñiguez-Moreno would like to thank to “L’Oréal-UNESCO-AMC-CON­
ALMEX-2023 For Women in Science Fellowship”. Graphical abstract
was created by Biorender using premium version.
13
International Journal of Biological Macromolecules 263 (2024) 130230
References
[1] S. Suri, A. Singh, P.K. Nema, Current applications of citrus fruit processing waste: a
scientific outlook, Applied Food Research 2 (2022) 100050, https://doi.org/
10.1016/j.afres.2022.100050.
[2] J. Ayala, G. Montero, M.A. Coronado, C. García, M.A. Curiel-Alvarez, J.A. León, C.
A. Sagaste, D.G. Montes, Characterization of orange peel waste and valorization to
obtain reducing sugars, Molecules 26 (2021) 1348.
[3] Food and Agriculture Organization of the United Nations (FAOSTAT), Cultivos.
http://www.fao.org/faostat/es/#data/QC, 2023. (Accessed 10 May 2023).
[4] O.A. Fakayode, K.E. Abobi, Optimization of oil and pectin extraction from orange
(Citrus sinensis) peels: a response surface approach, J Anal Sci Technol 9 (2018)
20, https://doi.org/10.1186/s40543-018-0151-3.
[5] Y.S. Duwee, P.L. Kiew, W.M. Yeoh, Multi-objective optimization of pectin
extraction from orange peel via response surface methodology: yield and degree of
esterification, J. Food Meas. Charact. 16 (2022) 1710–1724, https://doi.org/
10.1007/s11694-022-01305-5.
[6] P.H.F. Pereira, T.Í.S. Oliveira, M.F. Rosa, F.L. Cavalcante, G.K. Moates, N. Wellner,
K.W. Waldron, H.M.C. Azeredo, Pectin extraction from pomegranate peels with
citric acid, Int. J. Biol. Macromol. 88 (2016) 373–379, https://doi.org/10.1016/j.
ijbiomac.2016.03.074.
[7] E. Tsouko, S. Maina, D. Ladakis, I.K. Kookos, A. Koutinas, Integrated biorefinery
development for the extraction of value-added components and bacterial cellulose
production from orange peel waste streams, Renew. Energy 160 (2020) 944–954,
https://doi.org/10.1016/j.renene.2020.05.108.
[8] A.K. Tovar, L.A. Godínez, F. Espejel, R.M. Ramírez-Zamora, I. Robles, Optimization
of the integral valorization process for orange peel waste using a design of
experiments approach: production of high-quality pectin and activated carbon,
Waste Manag. 85 (2019) 202–213, https://doi.org/10.1016/j.
wasman.2018.12.029.
[9] D. Mohnen, Pectin structure and biosynthesis, Curr. Opin. Plant Biol. 11 (2008)
266–277, https://doi.org/10.1016/j.pbi.2008.03.006.
[10] B. De Cindio, D. Gabriele, F.R. Lupi, Pectin: properties determination and uses, in:
Encyclopedia of Food and Health, 1st ed., Elsevier Ltd., 2015: pp. 294–300. doi:
https://doi.org/10.1016/B978-0-12-384947-2.00531-6.
[11] P. Sharma, K. Osama, S. Varjani, A. Farooqui, K. Younis, Microwave-assisted
valorization and characterization of Citrus limetta peel waste into pectin as a
perspective food additive, J. Food Sci. Technol. 60 (2023) 1284–1293, https://doi.
org/10.1007/s13197-023-05672-9.
[12] C. Löfgren, S. Guillotin, H. Evenbratt, H. Schols, A.M. Hermansson, Effects of
calcium, pH, and blockiness on kinetic rheological behavior and microstructure of
HM pectin gels, Biomacromolecules 6 (2005) 646–652, https://doi.org/10.1021/
bm049619+.
[13] B. Pasandide, F. Khodaiyan, Z. Mousavi, S.S. Hosseini, Pectin extraction from
citron peel: optimization by Box–Behnken response surface design, Food Sci.
Biotechnol. 27 (2018) 997–1005, https://doi.org/10.1007/s10068-018-0365-6.
[14] S. Singhal, N.R. Swami Hulle, Citrus pectins: structural properties, extraction
methods, modifications and applications in food systems – a review, Applied Food
Research 2 (2022) 100215, https://doi.org/10.1016/j.afres.2022.100215.
[15] S.H. Jong, N. Abdullah, N. Muhammad, Optimization of low-methoxyl pectin
extraction from durian rinds and its physicochemical characterization,
Carbohydrate Polymer Technologies and Applications 5 (2023) 100263, https://
doi.org/10.1016/j.carpta.2022.100263.
[16] Z. Raji, F. Khodaiyan, K. Rezaei, H. Kiani, S.S. Hosseini, Extraction optimization
and physicochemical properties of pectin from melon peel, Int. J. Biol. Macromol.
98 (2017) 709–716, https://doi.org/10.1016/j.ijbiomac.2017.01.146.
[17] F. Jafari, F. Khodaiyan, H. Kiani, S.S. Hosseini, Pectin from carrot pomace:
optimization of extraction and physicochemical properties, Carbohydr. Polym. 157
(2017) 1315–1322, https://doi.org/10.1016/j.carbpol.2016.11.013.
[18] L.B.S. Filho, R.C. Coelho, E.C. Muniz, H. de S. Barbosa, Optimization of pectin
extraction using response surface methodology: A bibliometric analysis,
Carbohydrate Polymer Technologies and Applications 4 (2022) 100229. doi:https
://doi.org/10.1016/j.carpta.2022.100229.
[19] A.B. Kute, D. Mohapatra, N. Kotwaliwale, S.K. Giri, B.P. Sawant, Characterization
of pectin extracted from orange peel powder using microwave-assisted and acid
extraction methods, Agric. Res. 9 (2020) 241–248, https://doi.org/10.1007/
s40003-019-00419-5.
[20] N.H. Hamidon, D.N.A. Zaidel, Effect of extraction conditions on pectin yield
extracted from sweet potato peels residues using hydrochloric acid, Chem. Eng.
Trans. 56 (2017) 979–984, https://doi.org/10.3303/CET1756164.
[21] H. Zhu, Y. Xu, G. Qi, S. Wang, H. Wang, Modeling the combined effect of high
hydrostatic pressure and mild heat on the sub-lethal injury of Listeria
monocytogenes by Box–Behnken design, J. Food Process Eng. 43 (2020) 1–8,
https://doi.org/10.1111/jfpe.13480.
[22] S.Y. Chien, S. Sheen, C.H. Sommers, L.Y. Sheen, Modeling the inactivation of
intestinal pathogenic Escherichia coli O157:H7 and uropathogenic E. coli in ground
chicken by high pressure processing and thymol, Front Microbiol 7 (2016) 920.
doi:https://doi.org/10.3389/fmicb.2016.00920.
[23] ASTM E872-82, Standard Test Method for Volatile Matter in the Analysis of
Particulate Wood Fuels E872 - 82, ASTM International 82 (2019) 14–16.
[24] ASTM E830-87, Standard Test Method for Ash in the Analysis Sample of Refuse-
Derived Fuel, 87 (2004) 1–2.
[25] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method
for determination of sugars and related substances, Anal. Chem. 28 (1956)
350–356, https://doi.org/10.1021/ac60111a017.
M. Iñiguez-Moreno et al.
[26] X. López-Legarda, C. Arboleda-Echavarría, R. Parra-Saldívar, M. Rostro-Alanis, J.
F. Alzate, J.A. Villa-Pulgarín, F. Segura-Sánchez, Biotechnological production,
characterization and in vitro antitumor activity of polysaccharides from a native
strain of Lentinus crinitus, Int. J. Biol. Macromol. 164 (2020) 3133–3144, https://
doi.org/10.1016/j.ijbiomac.2020.08.191.
[27] S.S. Hosseini, F. Khodaiyan, M. Kazemi, Z. Najari, Optimization and
characterization of pectin extracted from sour orange peel by ultrasound assisted
method, Int. J. Biol. Macromol. 125 (2019) 621–629, https://doi.org/10.1016/j.
ijbiomac.2018.12.096.
[28] J. Folch, M. Lees, G.H. Sloane Stanley, A simple method for the isolation and
purification of total lipides from animal tissues, J. Biol. Chem. 226 (1957)
497–509, https://doi.org/10.1016/s0021-9258(18)64849-5.
[29] W.W. Wai, A.F.M. Alkarkhi, A.M. Easa, Effect of extraction conditions on yield and
degree of esterification of durian rind pectin: an experimental design, Food
Bioprod. Process. 88 (2010) 209–214, https://doi.org/10.1016/j.fbp.2010.01.010.
[31] S.D. Yadav, N.S. Bankar, N.N. Waghmare, D.C. Shete, Extraction and
characterization of pectin from sweet lime, in: 4th International Conference on
Multidisciplinary Research & Practice, 2017: pp. 58–63.
[32] W. Brand-Williams, M.E. Cuvelier, C. Berset, Use of a Free Radical Method to
Evaluate Antioxidant Activity, 1995.
[33] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans,
Antioxidant Activity Applying an Improved ABTS Radical Cation Decolorization
Assay, 1999.
[34] I.A. Brion-Espinoza, M. Iñiguez-Moreno, J.A. Ragazzo-Sánchez, J.C. Barros-
Castillo, C. Calderón-Chiu, M. Calderón-Santoyo, Edible pectin film added with
peptides from jackfruit leaves obtained by high-hydrostatic pressure and pepsin
hydrolysis, Food Chem X 12 (2021) 100170 Contents. doi:https://doi.org/10.10
16/j.fochx.2021.100170.
[35] J. Prakash, M. S. Manikandan, K. Thirugnanasambandham, C.N. Vigna, R. Dinesh,
Box-Behnken design based statistical modeling for ultrasound-assisted extraction of
corn silk polysaccharide, Carbohydr Polym 92 (2013) 604–611. doi:https://doi.
org/10.1016/j.carbpol.2012.09.020.
[36] F. Bigi, E. Maurizzi, H. Haghighi, H.W. Siesler, F. Licciardello, A. Pulvirenti, Waste
orange peels as a source of cellulose nanocrystals and their use for the development
of nanocomposite films, Foods 12 (2023) 960, https://doi.org/10.3390/
foods12091902.
[37] R.A. Magda, A.M. Awad, K.A. Selim, Evaluation of mandarin and navel orange
peels as natural sources of antioxidant in biscuits, Alexandria Journal of Food
Science and Technology 5 (2008) 75–82. doi:10.21608/ajfs.2008.19647.
[38] S. Gotmare, J. Gade, Orange Peel: A Potential Source of Phytochemical
Compounds, Int J Chemtech Res (2018) 3–7. doi:10.20902/ijctr.2018.110229.
[39] N. M’hiri, I. Ioannou, M. Ghoul, N. Mihoubi Boudhrioua, Proximate chemical
composition of orange peel and variation of phenols and antioxidant activity
during convective air drying, Journal of New Sciences (2015) 881–890.
[40] F. Adewale, C. Children, F. Adewumi, J. Jonathan, Phytochemical constituents and
proximate analysis of orange peel (Citrus fruit), Journal of Advanced Botany and
Zoology 1 (2014) 3–5. doi:10.15297/jabz.v1i3.02.
[41] N. M’hiri, I. Ioannou, N. Mihoubi Boudhrioua, M. Ghoul, Effect of different
operating conditions on the extraction of phenolic compounds in orange peel, Food
Bioprod. Process. 96 (2015) 161–170, https://doi.org/10.1016/j.fbp.2015.07.010.
[42] V. Goulas, G.A. Manganaris, Exploring the phytochemical content and the
antioxidant potential of Citrus fruits grown in Cyprus, Food Chem. 131 (2012)
39–47, https://doi.org/10.1016/j.foodchem.2011.08.007.
[43] M.M. Kamal, M. Akhtaruzzaman, T. Sharmin, M. Rahman, S.C. Mondal,
Optimization of extraction parameters for pectin from guava pomace using
response surface methodology, J Agric Food Res 11 (2023) 100530, https://doi.
org/10.1016/j.jafr.2023.100530.
[44] D. Mamiru, G. Gonfa, Extraction and characterization of pectin from watermelon
rind using acetic acid, Heliyon 9 (2023) e13525, https://doi.org/10.1016/j.
heliyon.2023.e13525.
[45] N.H. Hamidon, D.N. Abang Zaidel, Y.M. Mohd Jusoh, Optimization of pectin
extraction from sweet potato peels using citric acid and its emulsifying properties,
Recent Pat. Food Nutr. Agric. 11 (2020) 202–210, https://doi.org/10.2174/
2212798411666200207102051.
[46] T.A. Thu Dao, H.K. Webb, F. Malherbe, Optimization of pectin extraction from fruit
peels by response surface method: conventional versus microwave-assisted
heating, Food Hydrocoll. 113 (2021) 106475, https://doi.org/10.1016/j.
foodhyd.2020.106475.
[47] P. Putnik, D. Bursać Kovačević, A. Režek Jambrak, F.J. Barba, G. Cravotto,
A. Binello, J.M. Lorenzo, A. Shpigelman, Innovative “green” and novel strategies
for the extraction of bioactive added value compounds from citrus wastes - a
review, Molecules 22 (2017) 680, https://doi.org/10.3390/molecules22050680.
[48] D.A. Méndez, M.J. Fabra, L. Gómez-Mascaraque, A. López-Rubio, A. Martinez-
Abad, Modelling the extraction of pectin towards the valorisation of watermelon
rind waste, Foods 10 (2021) 738. doi:https://doi.org/10.3390/foods10040738.
[49] E.G. Maxwell, N.J. Belshaw, K.W. Waldron, V.J. Morris, Pectin - an emerging new
bioactive food polysaccharide, Trends Food Sci. Technol. 24 (2012) 64–73, https://
doi.org/10.1016/j.tifs.2011.11.002.
[50] B.M.V. Gama, C.E.D. Silva, L.M.O. Da Silva, A.K. de S. Abud, Extraction and
characterization of pectin from citric waste, Chem Eng Trans 44 (2015) 259–264.
doi:https://doi.org/10.3303/CET1544044.
14
International Journal of Biological Macromolecules 263 (2024) 130230
[51] D. Gawkowska, J. Cybulska, A. Zdunek, Structure-related gelling of pectins and
linking with other natural compounds: a review, Polymers (Basel) 10 (2018) 762,
https://doi.org/10.3390/polym10070762.
[52] S. Sangheetha, D.C.K. Illeperuma, A.N. Navaratne, C. Jayasinghe, Effect of pH,
temperature and time combinations on yield and degree of esterification of mango
peel pectin: a Box-Behnken design based statistical modelling, Tropical
Agricultural Research 30 (2018) 1–12, https://doi.org/10.4038/tar.v30i2.8304.
[53] N.A.A.R. Zahari, G.H. Chong, L.C. Abdullah, B.L. Chua, Ultrasonic-assisted
extraction (UAE) process on thymol concentration from Plectranthus amboinicus
leaves: kinetic modeling and optimization, Processes 8 (2020) 322, https://doi.
org/10.3390/pr8030322.
[54] P. Kanmani, Extraction and analysis of pectin from citrus peels: augmenting the
yield from citrus limon using statistical experimental design, Iranica Journal of
Energy and Environment 5 (2014) 303–312, https://doi.org/10.5829/idosi.
ijee.2014.05.03.10.
[55] B.M. Yapo, K.L. Koffi, Extraction and characterization of highly gelling low
methoxy pectin from cashew apple pomace, Foods 3 (2014) 1–12, https://doi.org/
10.3390/foods3010001.
[56] L.H. Reichembach, C.L. de Oliveira Petkowicz, Extraction and characterization of a
pectin from coffee (Coffea arabica L.) pulp with gelling properties, Carbohydr
Polym 245 (2020) 116473. doi:https://doi.org/10.1016/j.carbpol.2020.116473.
[57] A.N. Grassino, M. Brnčić, D. Vikić-Topić, S. Roca, M. Dent, S.R. Brnčić, Ultrasound
assisted extraction and characterization of pectin from tomato waste, Food Chem.
198 (2016) 93–100, https://doi.org/10.1016/j.foodchem.2015.11.095.
[58] J.M. Joel, J.T. Barminas, E.Y. Riki, J.M. Yelwa, F. Edeh, Extraction and
characterization of hydrocolloid pectin from goron tula (Azanza garckeana) fruit,
World Sci News 101 (2018) 157–171.
[59] A. Kozioł, K. Środa-Pomianek, A. Górniak, A. Wikiera, K. Cyprych, M. Malik,
Structural determination of pectins by spectroscopy methods, Coatings 12 (2022)
546, https://doi.org/10.3390/coatings12040546.
[60] L. Monfregola, M. Leone, V. Vittoria, P. Amodeo, S. De Luca, Chemical
modification of pectin: environmental friendly process for new potential material
development, Polym. Chem. 2 (2011) 800–804, https://doi.org/10.1039/
c0py00341g.
[61] S. Zhao, F. Yang, Y. Liu, D. Sun, Z. Xiu, X. Ma, Y. Zhang, G. Sun, Study of chemical
characteristics, gelation properties and biological application of calcium pectate
prepared using apple or citrus pectin, Int. J. Biol. Macromol. 109 (2018) 180–187,
https://doi.org/10.1016/j.ijbiomac.2017.12.082.
[62] Y. Zouambia, N. Moulai-Mostefa, M. Krea, Structural characterization and surface
activity of hydrophobically functionalized extracted pectins, Carbohydr. Polym. 78
(2009) 841–846, https://doi.org/10.1016/j.carbpol.2009.07.007.
[63] S. Lal, V. Kumar, S. Arora, Eco-friendly synthesis of biodegradable and high
strength ternary blend films of PVA/starch/pectin: mechanical, thermal and
biodegradation studies, Polym. Polym. Compos. 29 (2021) 1505–1514, https://doi.
org/10.1177/0967391120972881.
[64] N. Rekik, N. Issaoui, B. Oujia, M.J. Wójcik, Theoretical IR spectral density of H-
bond in liquid phase: combined effects of anharmonicities, Fermi resonances,
direct and indirect relaxations, J. Mol. Liq. 141 (2008) 104–109, https://doi.org/
10.1016/j.molliq.2007.10.009.
[65] Y. Yang, Z. Wang, D. Hu, K. Xiao, J.Y. Wu, Efficient extraction of pectin from sisal
waste by combined enzymatic and ultrasonic process, Food Hydrocoll. 79 (2018)
189–196, https://doi.org/10.1016/j.foodhyd.2017.11.051.
[66] A. Gnanasambandam, R. Proctor, Determination of pectin degree of esterification
by diffuse reflectance, Food Chem. 68 (2000) 327–332.
[67] N.M. Rasnani, H. Mirhosseini, B.S. Bin Baharin, C.P. Tan, Effect of pH on
physicochemical properties and stability of sodium caseinate-pectin stabilized
emulsion, J Food Agric Environ 9 (2011) 129–135.
[68] R.S. Giri, B. Mandal, Boc-Val-Val-OMe (Aβ39-40) and Boc-Ile-Ala-OMe (Aβ41-42)
crystallize in a parallel β-sheet arrangement but generate a different morphology,
CrystEngComm 20 (2018) 4441–4448, https://doi.org/10.1039/c8ce00097b.
[69] E. Skwarek, O. Goncharuk, D. Sternik, W. Janusz, K. Gdula, V.M. Gun’ko,
Synthesis, structural, and adsorption properties and thermal stability of
nanohydroxyapatite/polysaccharide composites, Nanoscale Res Lett 12 (2017)
155. doi:https://doi.org/10.1186/s11671-017-1911-5.
[70] M.R. Suhasini, K.M. Rajeshwari, S. Bindya, A.B. Hemavathi, P.M. Vishwanath,
A. Syed, R. Eswaramoorthy, R.G. Amachawadi, C. Shivamallu, V.K. Chattu, S.
S. Majani, S.P. Kollur, Pectin/PVA and Pectin-MgO/PVA Films: Preparation,
Characterization and Biodegradation Studies, Heliyon 9, 2023, https://doi.org/
10.1016/j.heliyon.2023.e15792.
[71] M. Calderón-Santoyo, M. Iñiguez-Moreno, J.A. Ragazzo-sánchez,
Microencapsulation of citral and its antifungal activity into pectin films,
Biointerface Res Appl Chem 12 (2022) 7488–7502.
[72] C. Buathongjan, K. Israkarn, W. Sangwan, T. Outrequin, C. Gamonpilas, P.
Methacanon, Studies on chemical composition, rheological and antioxidant
properties of pectin isolated from Riang (Parkia timoriana (DC.) Merr.) pod, Int J
Biol Macromol 164 (2020) 4575–4582. doi:https://doi.org/10.1016/j.ijbiomac.20
20.09.079.
[73] B. Yang, M. Zhao, K.N. Prasad, G. Jiang, Y. Jiang, Effect of methylation on the
structure and radical scavenging activity of polysaccharides from longan
(Dimocarpus longan Lour.) fruit pericarp, Food Chem. 118 (2010) 364–368,
https://doi.org/10.1016/j.foodchem.2009.04.128.
M. Iñiguez-Moreno et al.
[74] J.B. Pristov, A. Mitrović, I. Spasojević, A comparative study of antioxidative
activities of cell-wall polysaccharides, Carbohydr. Res. 346 (2011) 2255–2259,
https://doi.org/10.1016/j.carres.2011.07.015.
[75] M. Celus, L. Salvia-Trujillo, C. Kyomugasho, I. Maes, A.M. Van Loey, T. Grauwet,
M.E. Hendrickx, Structurally modified pectin for targeted lipid antioxidant
capacity in linseed/sunflower oil-in-water emulsions, Food Chem. 241 (2018)
86–96, https://doi.org/10.1016/j.foodchem.2017.08.056.
[76] R.L. Prior, X. Wu, K. Schaich, Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements, J. Agric.
Food Chem. 53 (2005) 4290–4302, https://doi.org/10.1021/jf0502698.
15
International Journal of Biological Macromolecules 263 (2024) 130230
[77] 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.
[78] N. Wathoni, C. Yuan Shan, W. Yi Shan, T. Rostinawati, R.B. Indradi, R. Pratiwi, M.
Muchtaridi, Characterization and antioxidant activity of pectin from Indonesian
mangosteen (Garcinia mangostana L.) rind, Heliyon 5 (2019) e02299. doi:https://
doi.org/10.1016/j.heliyon.2019.e02299.