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\DOI{10.5802/crchim.450}
\datereceived{2025-12-24}
\daterevised{2026-02-25}
\datererevised{2026-03-23}
\dateaccepted{2026-03-24}
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have declared no affiliation other than their research organizations.}

%\dateposted{2026-04-23}

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\begin{noXML}

\CDRsetmeta{articletype}{research-article}

\title{Investigations on antibacterial, antioxidant, and anticoagulant
activities of natural pectins extracted from Algerian \textit{Citrus
sinensis} peels}

\alttitle{\'{E}valuation des activit\'{e}s antibact\'{e}riennes,
antioxydantes et anticoagulantes de pectines naturelles extraites
d'\'{e}corces de \textit{Citrus sinensis} d'Alg\'{e}rie}

\author{\firstname{Nacer} \lastname{Boudouaia}\CDRorcid{0009-0003-0788-534X}\IsCorresp}
\address{Laboratory of Advanced Materials and Physicochemistry for
Environment and Health, Djillali Liabes University, 22000 Sidi Bel
Abbes, Algeria}
\email[N. Boudouaia]{boudouaia.nacer@gmail.com}

\author{\firstname{Samir} \lastname{Benykhlef}} 
\addressSameAs{1}{Laboratory of Advanced Materials and Physicochemistry for
Environment and Health, Djillali Liabes University, 22000 Sidi Bel
Abbes, Algeria}
\address{Higher School of Applied Sciences of Tlemcen, Algeria}

\author{\firstname{Zohra} \lastname{Bengharez}\CDRorcid{0000-0002-8356-2361}} 
\addressSameAs{1}{Laboratory of Advanced Materials and Physicochemistry for
Environment and Health, Djillali Liabes University, 22000 Sidi Bel
Abbes, Algeria}

\author{\firstname{Mama} \lastname{Merhoum}} 
\address{Department of Pharmacy, Faculty of Medicine, Djillali Liabes
University, Algeria}

\author{\firstname{Amine Ahmed} \lastname{Bendaoudi}} 
\addressSameAs{1}{Laboratory of Advanced Materials and Physicochemistry for
Environment and Health, Djillali Liabes University, 22000 Sidi Bel
Abbes, Algeria}

\author{\firstname{Salah} \lastname{Jellali}\CDRorcid{0000-0002-4095-4154}} 
\address{Center for Environmental Studies and Research, Sultan Qaboos,
University, Muscat, Oman}

\shortrunauthors

\keywords{\kwd{Citrus peel wastes}
\kwd{Pectin extraction}
\kwd{Characterization}
\kwd{Biomedical application}
\kwd{Sustainability}}

\altkeywords{\kwd{D\'{e}chets d'\'{e}corces d'oranges}
\kwd{Extraction de pectine}
\kwd{Caract\'{e}risation}
\kwd{Application biom\'{e}dicale}
\kwd{D\'{e}veloppement durable}}

\begin{abstract}
This work investigates the conversion of orange peel waste into
effective biomaterials. Two pectins were first extracted from these
peels under acidic conditions using citric acid (PCT-1) or hydrochloric
acid (PCT-2), then thoroughly characterized and finally tested for
biomedical applications. The characterization of pectin was carried out
using various analytical techniques including Fourier-transform
infrared spectroscopy, powder X-ray diffraction, scanning electron
microscopy, and differential scanning calorimetry. The biomedical
application of the extracted pectins demonstrates that neither PCT-1
nor PCT-2 have an antibacterial effect on \textit{Pseudomonas
aeruginosa} or \textit{E. coli}. Nevertheless, a significant
antibacterial effect was observed on the \textit{Staphylococcus
epidermidis} strain. This highlights their potential use for the
treatment of some infectious diseases and particularly against
Gram-positive strains. Besides, the extracted pectins exhibit a high
potential use as antioxidants. Although PCT-2 does not display marked
anticoagulant activities, PCT-1 shows an interesting potential to be
valorized as an anticoagulant. All these results show that pectins
extracted from orange peel waste can be valorized as effective
biomaterials with numerous biomedical\break applications.
\end{abstract}

\begin{altabstract}
Ce travail \'{e}tudie la conversion des d\'{e}chets d'\'{e}corces
d'orange en biomat\'{e}riaux efficaces. Deux pectines ont \'{e}t\'{e}
extraites des \'{e}corces d'orange de \textit{Citrus sinensis} {en
milieu acide, \`{a} l'aide d'acide citrique (PCT-1) ou d'acide
chlorhydrique (PCT-2), puis caract\'{e}ris\'{e}es en d\'{e}tail et
enfin test\'{e}es pour des applications biom\'{e}dicales. La
caract\'{e}risation des pectines a \'{e}t\'{e} r\'{e}alis\'{e}e \`{a}
l'aide de diverses techniques analytiques, notamment la spectroscopie
infrarouge \`{a} transform\'{e}e de Fourier, la diffraction des rayons
X sur poudre, la microscopie \'{e}lectronique \`{a} balayage et la
calorim\'{e}trie diff\'{e}rentielle \`{a} balayage. L'application
biom\'{e}dicale des pectines extraites montre que ni PCT-1 ni PCT-2
n'ont d'effet antibact\'{e}rien sur} \textit{Pseudomonas aeruginosa}
{ou} \textit{Escherichia coli}{. N\'{e}anmoins, un effet
antibact\'{e}rien significatif a \'{e}t\'{e} observ\'{e} sur la souche}
\textit{Staphylococcus epidermidis}. Ceci souligne leur potentiel
pour le traitement de certaines maladies infectieuses, et en
particulier contre les souches Gram-positives. De plus, les pectines
extraites pr\'{e}sentent un fort potentiel en tant qu'antioxydants.
Bien que PCT-2 ne pr\'{e}sente pas d'activit\'{e} anticoagulante
marqu\'{e}e, PCT-1 montre un potentiel int\'{e}ressant pour une
valorisation en tant qu'anticoagulant. L'ensemble de ces r\'{e}sultats
d\'{e}montre que les pectines extraites des d\'{e}chets d'\'{e}corces
d'orange peuvent \^{e}tre valoris\'{e}es comme biomat\'{e}riaux
efficaces pour de nombreuses applications biom\'{e}dicales.
\end{altabstract}

%\input{CR-pagedemetas}

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\section{Introduction}\label{sec1}

Rapid urbanization and population growth generate significant amounts
of food waste. This biowaste can contaminate and degrade the
environment and ecosystems. Among food biowaste, citrus peels \mbox{represent}
50\% to 60\% of the fruit mass~\cite{1}. \mbox{Therefore}, it is crucial to
find tailored options for the sustainable management of this biowaste.
Accordingly, several technologies have been investigated. They include
composting, anaerobic digestion, thermochemical conversion, and the
extraction of biomaterials such as pectin~\cite{2}. The last method
promotes the circular economy concept and also improves the
sustainability of food production systems~\cite{3}. 

{

\advance\baselineskip .27pt

Pectin is a naturally occurring biopolymer that it is abundantly found
in various plant cell walls such as lemon, orange, sugar beet, apple,
etc. It is a heteropolysaccharide compound, formed of linear chains of
${\upalpha}$-($1\,{\rightarrow}\,4$)-linked D-galacturonic acid residues
and combined with small quantities of neutral sugars like L-rhamnose,
D-galactose, L-arabinose, and D-xylose~\cite{4}. The degree of
esterification (DE) of pectin refers to the percentage of carboxyl
groups esterified with methanol and determines its gelling and
functional properties~\cite{5}. Pectins with high DE usually form
rigid gels, while those with low DE form softer gels or act as
thickening agents~\cite{6}. Additionally, the presence of acetyl
groups can also significantly influence the gelling properties of
pectins~\cite{7}.

Due to its unique physicochemical properties, pectin has become the
subject of numerous researches in several application fields~\cite{8},
notably in the food, pharmaceutical and medical industries, material
engineering, and environmental sciences~\cite{9,10,11,12,13,14}. In this context,
several methods have been tested for pectin extraction from plant
sources. They mainly include the use of enzymes~\cite{15},
microwaves~\cite{16}, ultrasound~\cite{17}, subcritical
water~\cite{18}, pressurized carbon dioxide or deionized
water~\cite{19}, and acids~\cite{20,21}. The last method has been
widely tested. It consists in treating the plant material with an acid
to break down the cell walls and release pectin~\cite{22}. Then, the
resulting mixture is filtered and pectin is usually precipitated by
adding alcohol. Pectin extraction yield is dependent on both the plant
peel type and acid used. For instance, in a comparative study, Yu 
et~al.~\cite{23} studied pectin extraction efficiency from potato pulp
using five different acids: nitric (HNO$_{3}$), hydrochloric (HCl),
sulfuric (H$_{2}$SO$_{4}$), acetic (C$_{2}$H$_{4}$O$_{2}$), and citric
(C$_{6}$H$_{8}$O$_{7}$). They found that the highest pectin yield was
observed when using the citric acid. It is important to underline that
in another study~\cite{24}, pectin extraction yield from unripe
Cavendish banana peels by citric acid was evaluated at 11.52\% with a
pH of 1.5. It has been shown that the yield of pectins extracted from
banana and mango peels under optimal conditions using citric acid at pH
2.0 was the highest (12.98\%). In contrast, a lower yield (5.28\%) was
found for extraction with hydrochloric acid (pH 1.5). The yield and
characteristics of acid-extracted pectin also depend on the extraction
parameters used, such as contact time and temperature~\cite{25}.

Currently, the increasing number of foodborne pathogenic bacteria such
as \textit{Pseudomonas \mbox{aeruginosa}}, \textit{E.\ coli}, and
\textit{Staphylococcus aureus} has become a major public health
problem, since it can cause serious foodborne diseases~\cite{26}.
Therefore, it is crucial to develop natural, biodegradable, efficient,
and renewable alternatives. Gao et~al.\ \cite{27} showed a good
antibacterial activity of pectin against the growth of \textit{E.\ coli}
and \textit{S.\ aureus} at minimum inhibitory concentrations (MICs) of
25.0 and 50.0~g/L, respectively. In addition, Di Rong et~al.~\cite{28}
investigated citrus pectin oligosaccharides on anti-adhesion activity
against \textit{E.\ coli} and showed that they could be used as a
reliable antibacterial agent in functional foods. In addition, Tripathi
et~al.\ \cite{29} evaluated the antibacterial and antioxidant activity of
pectin obtained from banana peels. They found a maximum antibacterial
activity against \textit{Staphylococcus aureus} (19.6~mm in the
well-diffusion method). Furthermore, their antioxidant activity study
showed that pectin, up to a concentration of 75~$\upmu$g/mL,
increases the free radical scavenging activity of
2-2-diphenyl-1-picrylhydrazil (DPPH). On the other hand, 
Santana et~al.~\cite{30} showed that sulfated pectin 
extracted from \textit{Citrus
sinensis} has interesting anticoagulant activity. The current study
provides a new contribution to a better understanding of the
antibacterial, antioxidant, and anticoagulant behavior of pectin
obtained by a simple extraction from an abundant and renewable raw
material (orange peel waste). {The main aim of this work is to convert
Algerian citrus peel waste into value-added biomaterial through pectin
extraction using} {a mild organic acid (citric acid) and a strong
mineral acid (hydrochloric acid) to assess their antibacterial,
antioxidant, and anticoagulant effects. This approach will clarify the
trade-off between extraction efficiency and structural integrity, and
it would also address inconsistencies in prior works.} Practically, the
specific objectives are: (i) to study the role of the acid used on
pectin extraction yield, (ii) to fully characterize the extracted
pectins by various analytical techniques, and (iii) to assess the
potential biomedical application of these pectins through the
assessment of their antibacterial, antioxidant, and anticoagulant
\mbox{activities}.\looseness=1 

}

\section{Materials and methods}\label{sec2}

\subsection{Materials collection and preparation}\label{sec21}

The citrus fruit used consists in sweet oranges (\textit{Citrus
sinensis}). They were gathered from an \mbox{agricultural} farm located in the
Sidi Matmar region in the northwestern part of Algeria. The orange
peels were thoroughly washed with distilled water, then cut into small
pieces, and subsequently dried at 50~\textdegree C for 24 h. Finally,
the dried pieces were manually crushed and sieved. The bacteria used in
this work, \textit{Pseudomonas aeruginosa} {(ATCC:27853)},
\textit{Escherichia coli} (ATCC:25922), and \textit{Staphylococcus
epidermidis} ({ATCC:12228)} were provided by the Laboratory of
Microbiology, Hassani Abdelkader Hospital, Sidi Bel Abbes, Algeria.
They were kept at ${+}$4~\textdegree C and renewed every 24 h on
nutrient agar at 37~\textdegree C.

\subsection{Pectin extraction}\label{sec22}

Pectin extraction from orange peels was carried out with two different
acids, citric and hydrochloric, as follows.

\subsubsection{Extraction by citric acid}\label{sec221}

Pectin extraction by citric acid was performed according to the
following steps~\cite{31}: (i) agitation of 40~g of dried orange peel
waste in 1 L of a 0.1 N citric acid solution at a constant pH of 2 and
temperature of 70~\textdegree C for 40 min, (ii) storing this suspension
for 24~h at room temperature, (iii) recovery of the liquid phase by
centrifugation at 6000 rpm for 10 min, (iv) mixing (1:2 v/v) this
solution with 95\% ethanol at 25~\textdegree C for 24 h to allow the
precipitation and flotation of pectin, (v) filtration of the
precipitated pectin using a B\"{u}chner funnel and washing twice with
ethanol (70\%), and (vi) drying in an oven at 65~\textdegree C for 24 h.
The resulting product was named PCT-1. 

\subsubsection{Extraction by hydrochloric acid}\label{sec222}

Pectin extraction by hydrochloric acid was carried out through the
boiling of 40 g of dried orange peels in 0.8 L of a 0.1 N HCl solution
in a reflux system at 90~\textdegree C for 45 min. After 6 min, the
suspension was placed on ice to stop the hydrolysis reaction. Then, it
was filtered and the filtrate precipitated in ethanol. This filtrate
was washed with 60\%, 80\%, and 98\% ethanol, then centrifuged at 
10\,000 rpm for 20~min and dried at 50~\textdegree C for 2~h, then crushed.
The obtained product was called PCT-2 and used in the experimental
study below. 

For both methods, the pectin extraction yield was evaluated on the
basis of three parallel experiments and the mean values are presented
in this work. The~following equation was used for the calculus of these
yields:
{\begin{equation}\label{eq1}
\mathrm{Yield}~(\%)=\frac{\mbox{Obtained product mass}}
{\mbox{Initial orange peel waste mass}}\times 100 
\end{equation}}\unskip


\subsection{Pectin characterization}\label{sec23} 

\subsubsection{Solubility}\label{sec231}

The solubility of the pectins in different solvents was assessed in
batch mode. The assays consisted in stirring 0.05 g of pectin for 3 h
in 10~mL of ethanol, petroleum ether, ketone, cyclohexane,
dichloromethane, cold water, or hot water. 

\subsubsection{Humidity}\label{sec232}

The humidity (H) of the pectins was evaluated through the drying of
1 g of pectin at 105~\textdegree C for 24~h in a porcelain capsule. The
humidity value is determined as follows:
{\begin{equation}\label{eq2}
\mathrm{H}(\%)=\frac{m_{1}-m_{2}}{m_{1}}
\end{equation}}\unskip
where $m_1$ and $m_2$ are the masses (g) of pectin before and after
drying. 

\subsubsection{Organic matter and ash contents}\label{sec233}

The organic matter (OM) and ash (Cd) contents were assessed according
to the protocol given by Soliman et~al.~\cite{32}. It consists in
placing a porcelain capsule containing 4 g of pectin in a muffle
furnace (Biobase, China) at $550 \pm 15$~\textdegree C for 5 h until a
light gray or whitish color is obtained. Then, the capsule is cooled in
a desiccator until a constant weight. The OM content is calculated as
\mbox{follows}:
{\begin{equation}\label{eq3}
\mathrm{OM}~(\%)=\frac{m_{3}-m_{4}}{m_{3}}\times 100
\end{equation}}\unskip
with $m_3$ and $m_4$ the masses (g) of pectin before and after
carbonization. The ash content (Cd) is deduced as: 
{\begin{equation}\label{eq4}
\mathrm{Cd}~(\%)=100-\mathrm{OM}~(\% )
\end{equation}}\unskip

\subsubsection{Degree of esterification}\label{sec234}

The degree of esterification (DE) of the extracted pectins was assessed
according to the following experimental protocol~\cite{33}: first, 
0.2~g of the dried pectin is moistened with ethanol and dissolved in
20~mL of distilled water, then three drops of phenolphthalein are added
to the sample and titrated by a 0.1~N NaOH solution. The result is
recorded as the initial titration volume once the pink color appears
and the number of free carboxyl groups can be deduced. After that, 
10~ml of a 0.1 N NaOH solution is added to neutralize the polygalacturonic
acid. The sample was capped with a cork and shaken vigorously for 5 h,
then kept at room temperature for 2~h to permit the deesterification of
the pectin. After that, 10~mL of a 0.1 N HCl solution was added to
neutralize the excess of NaOH and the sample was shaken until its pink
color disappears. Then, three drops of phenolphthalein were added to
the sample and titrated with a 0.1 N NaOH solution. The titration
volume was recorded as the final titration volume once a pink color
appears, and the number of esterified carboxyl groups can be deduced.
The DE of pectins is defined as the ratio of the esterified
galacturonic acid to the galacturonic acid groups and calculated as
\mbox{follows}~\cite{26}:
{\begin{eqnarray}
&&\mathrm{DE}~(\%)\nonumber\\
&&\quad=\textstyle\frac{\mathrm{Final~titration~volume~(mL)}}
{\mathrm{Initial~titration~volume~(mL)}\,+\,\mathrm{Final~titration~volume~(mL)}}\nonumber\\
&&\qquad\times\, 100\label{eq5}
\end{eqnarray}}\unskip

All the above parameters were evaluated through triplicate assays and
the mean and standard variation were calculated using Excel 2016
software. 

\subsubsection{Morphology and structure} \label{sec235}

The morphology of the extracted PCT-1 and PCT-2 was determined through
scanning electronic microscopy (SEM, brand:~Carl Zeiss, model:~Sigma
300~VP) coupled with an energy dispersive X-ray (EDX) detector capable
of elemental surface analysis. Moreover, in order to study their
crystalline structure, the pectins were examined using an X-ray
diffraction (XRD) apparatus (Bruker model D8 ADVANCE). The diffraction
data was recorded in a 2${\theta}$ range of 5\textdegree~to
40\textdegree~at a scan rate of 1\textdegree/min. The X'Pert High score
program was used for the analysis. The International Center for
Diffraction Data (ICDD) database {was used} to identify the crystalline
peaks.

\subsubsection{Surface chemistry}\label{sec236}

The richness in functional groups of the extracted pectins was assessed
using a Fourier-transform infrared (FTIR) spectrometer (Bruker alpha-P)
equipped with an ATR (attenuated total reflectance) diamond crystal.
The infrared spectra of the pectin samples were obtained over a
400--4000~cm$^{-1}$ wavenumber range. 

\subsubsection{Thermal properties}\label{sec237}

Thermal analysis of the extracted pectins was carried out using a
differential scanning calorimetry (DSC) device (DSC-NETZSCH DSC-214
polyma). During this analysis, 10 mg of the sample was placed in the
crucible and heated for 45 min at a rate of 10~\textdegree C/min. 

\subsection{Antibacterial activity}\label{sec24}

The antibacterial action of the extracted pectins was studied using the
disk diffusion method and the bacteria \textit{Pseudomonas aeruginosa,
Escherichia coli}, and \textit{Staphylococcus epidermidis} were chosen
for this study~\cite{34}. For this purpose, pure colonies were
collected, isolated, and stored at 4~\textdegree C. This method is
particularly suitable for studying the action of antibiotics on the
growth of bacteria; it allows determining their antibiograms (data not
shown), which reflect the specific sensitivity of different bacterial
species to given antibiotics. Petri dishes containing an already
solidified suitable agar medium were inoculated with the tested
microbial strain~\cite{34}. The antibiotic discs were then placed on
the surface of the agar; the antibiotics used in these tests were:
fusidic acid, vancomycin, spiramycin, ampicillin, oxacillin,
norfloxacin, cefotaxime, doxycycline, and cefixime. The bacterial
strains were stored at 4~\textdegree C, and these strains were renewed
every 24 h on nutrient agar at 37~\textdegree C. The most commonly used
medium for antibacterial susceptibility testing is Mueller Hinton Agar
(MHA)~\cite{35}. Using a sterilized (Bunsen burner) Pasteur pipette, a
few pure colonies were collected and isolated in a test tube containing
9~mL of saline solution and then thoroughly vortexed. The resulting
bacterial solution was inoculated over the entire surface of the
culture medium using a swab in each Petri dish.

Furthermore, the antibacterial action of PCT-1 and PCT-2 was assessed
at different concentrations (5, 2.5, 1.66, and 1.25~mg/mL) against the
selected bacteria on MHA culture medium, then incubated at
37~\textdegree C for 24 h. Whatman paper discs of diameter 6~mm were
prepared and sterilized in the autoclave at 120~\textdegree C for 15
min. The discs were removed using sterilized forceps, and then soaked
with the PCT solutions of each concentration for 30 s. Using sterile
forceps, five discs at different concentrations were placed in each
Petri dish containing a bacterium to be tested and then incubated at
37~\textdegree C in the oven for 24 h. The reading was carried out by
measuring the diameter of the inhibition zone around the two tested
pectins PCT-1 and PCT-2~\cite{28}.

\subsection{Antioxidant activity} \label{sec25}

Unlike most free radicals, DPPH$\bullet$ is stable in solution; it
cannot dimerize, due to steric hindrance around the nitrogen atom
carrying the free electron. It exhibits a characteristic absorbance in
the 512--517 nm range. The purple color disappears rapidly after
reduction of DPPH to diphenylpicrylhydrazine by a compound with
anti-radical properties, resulting in discoloration. The color
intensity is proportional to the proton-donating capacity of
antioxidants present in the medium~\cite{29}. The antiradical
scavenging activity of the extracted pectins was studied by measuring
the retention power of the DPPH$\bullet$ radical according to the
protocol described by Qian et~al.~\cite{36}. However, this radical
can be reduced by a hydrogen transfer from the various antioxidants
found in the reaction medium. Pectin (0.5, 1, 2, and 4~mg/mL) was mixed
with DPPH (0.2~mmol/L) in ethanol (0.5~mL) and incubated at
37~\textdegree C for 35 min. After 2 h, the optical density (OD) of the
samples was determined by a UV spectrophotometer (Shimadzu UV-2401PC)
at a wavelength of 517 nm. These results were compared to those from
blank tests prepared by replacing the sample solution with anhydrous
ethanol. The antiradical power is deduced as\break follows:
{\begin{equation}\label{eq6}
Y_{1}~(\%)=\frac{A_{\mathrm{control}}-A_{\mathrm{sample}}}{A_{\mathrm{control}}}
\times 100
\end{equation}}\unskip
with $Y_1$ (\%) the reduction percentage of the free radical
(DPPH$\bullet$), $A_{\mathrm{control}}$ the absorbance of the control
sample, and $A_{\mathrm{sample}}$ the absorbance of the sample. The
reaction can be summarized in the form below, where (AH)$_n$ represents
a compound capable of donating a hydrogen to the (DPPH$\bullet$)
radical (purple) to transform it into diphenylpicrylhydrazine
(yellow)~\cite{37}.
{\begin{equation*}
\text{DPPH}\bullet +(\mathrm{AH})_{n}\rightarrow 
\text{DPPH}-\mathrm{H}+(\mathrm{AH})_{n-1}\mathrm{A}\bullet
\end{equation*}}\unskip

\subsection{Anticoagulant activity}\label{sec26}

The anticoagulant activity of the pectin samples was evaluated in vitro
through the activated partial thromboplastin time (APTT) and
prothrombin time (PT), with a saline solution as negative control and
heparin sodium as positive control, according to the experimental
protocol given by Souza et~al.\ \cite{38}. The APTT and PT of the
pectins were measured through the analysis of 100~${\upmu}$L of
citrated normal human plasma mixed with 50~${\upmu}$L of PCT-1 or
PCT-2 incubated at 37~\textdegree C for 15 min by a specific automated
\mbox{device}.

It is worth mentioning that antibacterial, antioxidant, and
anticoagulant activities were assessed in triplicate. The mean values
and the standard variation of the experimental data are given in the
corresponding figures.

\section{Results and discussion}\label{sec3}

\subsection{Pectin extraction yields} \label{sec31}

The pectin extraction yields with citric acid (PCT-1) and hydrochloric
acid (PCT-2) were determined according to the experimental protocol
given in Section~\ref{sec222} at 6.3\% and 4.7\%, respectively
(Table~\ref{tab1}). 

\begin{table*}
\caption{\label{tab1}Main physicochemical parameters of the extracted
pectins}
\begin{tabular}{cccccc}
\thead
Biomaterials & Extraction process & Yield (\%) & H (\%) & Cd (\%) & DE (\%) \\
\endthead
PCT-1 & Citric acid  & 
6.3 ${\pm}$ 0.21 & 
4.4 ${\pm}$ 0.26 & 
3.5 ${\pm}$ 0.32 & 
40.0 ${\pm}$ 2.10 \\
PCT-2 & Hydrochloric acid & 
4.7 ${\pm}$ 0.20 & 
3.6 ${\pm}$ 0.21 & 
4.6 ${\pm}$ 0.28 & 
57.0 ${\pm}$ 3.56
\botline
\end{tabular}
\vspace*{3pt}
\end{table*}

The higher extraction yield observed with citric acid is mainly due to
its larger extraction time (overnight). Moreover, the cleavage action
of hydrochloric acid on pectin's glycoside and ester bonds leads to a
reduction in pectin extraction yield. A similar trend was observed by
Chan et~al.~\cite{39} when investigating pectin extraction by these
same acids from cocoa husks. Our results are also in agreement with
those reported by Nateghi et~al.~\cite{40} who reported that citric
acid has a greater pectin extraction ability than hydrochloric acid and
sulfuric acid due to the chelating characteristic of citric acid. On
the other hand, Maran et~al.~\cite{41} obtained a higher pectin
extraction yield (9.0\%) when using citric acid assisted with
ultrasound for pectin extraction from an industrial waste (\textit{Musa
balbisiana}). It is worth mentioning that pectin extraction yields are
very dependent on the citrus variety studied. Comparable yields were
reported in previous published works~\cite{42}.

\subsection{Characterization of pectins}\label{sec32}

\subsubsection{Solubility, humidity, organic matter, and ash
contents}\label{sec321}

Solubility tests showed that both extracted pectins are perfectly
soluble in hot and cold water. This finding may be attributed to
pectin's richness in hydrophilic groups (i.e., hydroxyl OH, carboxyl
COOH). Moreover, ionization of the carboxyl groups into negatively
charged carboxylate ions can enhance pectin solubility in water through
electrostatic interaction with water molecules. A similar outcome was
reported for pectins extracted from sweet oranges (\textit{Citrus
sinensis} L) by citric acid~\cite{43}. However, PCT-1 and PCT-2 were
completely insoluble in all the organic solvents tested (ethanol,
petroleum ether, ketone, cyclohexane, dichloromethane). This result is
in agreement with those reported by W\"{u}rfel et~al.~\cite{44}. Pure
white powdered pectin dissolves in water with a 1:20 (w/v) ratio to
form a negatively charged viscous colloidal solution, but it is
insoluble in organic solvents such as ether and acetone. {Generally,
the solubility of pectin in water is linked to its long chains of
galacturonic acid and richness in hydrophilic carboxyl and hydroxyl
groups, which easily form hydrogen bonds with water.} The longer the
polygalacturonic acid chain, the lower the pectin solubility in water.
As shown in Table~\ref{tab1}, PCT-1 had a higher humidity than PCT-2.
This finding can be explained by the characteristics of the chelator
present in citric acid~\cite{43}. Moreover, the ash contents of PCT-1
and PCT-2 were evaluated at $3.5 \pm 0.32\%$ and 
$4.6 \pm 0.28\%$, respectively. Akhtar et~al.~\cite{45} investigated the
extraction of pectin from orange and lime peels using hydrochloric,
sulfuric, nitric, and citric acids. The pectin extracted from orange
and lime peels had ash contents of $4.26\% \pm 0.12\%$ and 
$2.41\% \pm 0.07\%$, respectively, which are
consistent with our results. Mamiru
et~al.~\cite{46} found higher ash contents for pectins extracted from
watermelon peels and suggested that ash content variation is primarily
affected by both the feedstock type and the extraction conditions.
Comparable values were reported for pectin extracted from orange peels
using the acid extraction and a Box-Behnken design~\cite{47}. It is
important to underline that the ash content of pectins is usually
dependent on the mineral composition of the raw\unskip\break material. For instance,
due to the high content in mineral salts and heavy metals in dragon
fruit peels (\textit{Hylocereus polyrhizus}), the ash contents of the
related extracted pectins under three different extraction conditions
were higher than our measured value (7\% to 11\%)~\cite{48}. 

\begin{figure*}
\vspace*{-4pt}
\includegraphics{fig01}
\vspace*{-6pt}
\caption{\label{fig1}SEM micrographs of PCT-1 (left) and PCT-2 (right).}
\vspace*{-4pt}
\end{figure*}

\subsubsection{Degree of esterification}\label{sec322}
The degree of esterification (DE) of pectin has significant
implications for its functional properties in various applications,
such as food and pharmaceuticals~\cite{49}. Pectins with a DE above
50\% are designated high-methoxyl pectins (HMPs), while those with a DE
under 50\% are designated low methoxyl pectins (LMPs). Such a
difference in the DE value is due to the distribution of methyl ester
groups along the polygalacturonic acid chain, the molecular weight, and
the type and amount of neutral sugars bound to the pectin
molecules~\cite{50}. The DE of the extracted pectins is highly
dependent on the extracting acid used. Indeed, the DE of PCT-2
extracted with hydrochloric acid was evaluated at 57\%, which makes
PCT-2 a HMP (Table~\ref{tab1}). This value was obtained at an acidic pH
of 3.3 with a short extraction time (45~min), conditions favorable to
the preparation of sugar-rich products~\cite{51}. However, the DE
of PCT-1 extracted using citric acid is much lower (40\%) and
indicates a LMP
(Table~\ref{tab1}). A similar trend was reported by 
Mansor et~al.~\cite{52} who show that the DE of pectins depends on both
the type of extracting acid and its interactions with the citrus
matrix.

On the other hand, the reduction in carboxylate functions is
responsible for the diminishing repulsive forces of the
polysaccharides, which favors pectin gelation and results in more
precipitated pectin at lower pH values. This finding is in agreement
with those reported for pectins extracted from apple \mbox{pomace} and sugar
beet pulp~\cite{49,53}. It is important to mention that the DE
highly influences the functional properties of pectins. Indeed, high
DEs usually result in more rigid gels~\cite{54}, whereas lower DEs lead
to softer gels or even to pectin dissolution. This property is crucial
in various food applications, such as in the production of jams,
jellies, and fruit-based desserts, where the desired texture and
stability depend on the DE of the pectin used~\cite{55}. Additionally,
the DE affects the interactions of pectin with other molecules (e.g.,
sugars, proteins) which significantly impact the overall texture and
stability of food products~\cite{54}.

\vspace*{-4pt}

\subsubsection{Morphology and structure} \label{sec323}

Figure~\ref{fig1} shows the SEM images of both PCT-1 and PCT-2. These
images indicate that the small pectin particles exhibit the same rough
and irregular morphological characteristics with no coherent porous
surface. However, differences were observed when changing the image
acquisition and resolution, as shown in Figure~\ref{fig1}. On the other
hand, these particles are able to disperse in solutions thus improving
the solubility and intermolecular interactions through H-bonds and
dipole formation~\cite{56}.


The XRD analyses show that PCT-1 and PCT-2 exhibit only a weak
characteristic diffraction peak at 2$\theta$ around  21\textdegree~and
another small peak at 2$\theta$  of 6\textdegree~for PCT-2,
as shown in Figure~\ref{fig2}. This result
shows that both pectins have a semi-crystalline nature. This finding is
in agreement with that reported for pectins extracted from apple
waste~\cite{57}.

\begin{figure}
\includegraphics{fig02}
\caption{\label{fig2}XRD
patterns of PCT-1 and PCT-2.}
\end{figure}

This can be attributed to the variation in their molecular weights due
to the use of different extraction conditions. Similar observations
have been reported by previous studies on pectin extracted from black
carrot pomace, fruit peels, citrus fruits~\cite{56,57,58},
sunflowers~\cite{59}, and sweet lemon~\cite{60}.

\subsubsection{Surface chemistry}\label{sec324}

The FTIR spectra of both extracted pectins are shown in
Figure~\ref{fig3}. The wave number range of 850 and 1250~cm$^{-1}$
corresponds to the ``fingerprint'' region of carbohydrates~\cite{61}.
In this spectral region, and for both PCT-1 and PCT-2, the
characteristic peaks observed at 920, 1016, 1090, and  1130~cm$^{-1}$
confirm the typical profile of polygalacturonic acid~\cite{62}.

\begin{figure}
\includegraphics{fig03}
\caption{\label{fig3}FTIR spectra of PCT-1 and PCT-2.}
\end{figure}

Moreover, other specific functional groups are present in both
extracted pectins. They include a broad peak at 3700--3000~cm$^{-1}$
corresponding to O--H stretching due to hydrogen bonds in galacturonic
acid~\cite{62}. Peaks at 2954 and 2941~cm$^{-1}$ for PCT-1 and PCT-2,
respectively, can be attributed to C--H bond stretching~\cite{62}.
Peaks at 1730 and 1620~cm$^{-1}$ correspond to esterified and free
carboxyls, respectively. Bands at 1100--1020~cm$^{-1}$ can be imputed
to C--O--C stretching vibrations and confirm the presence of pyranoses
in the structure of both pectins. Peaks around 1000--1030~cm$^{-1}$
correspond to the \mbox{C--O} stretching vibrations of ether or ester
groups.

The asymmetric stretching observed around 1643--1626~cm$^{-1}$ can be
imputed to carbohydrate functions, and the bands in the
1300--800~cm$^{-1}$ region correspond to the main carbohydrate chemical
groups in galacturonic acid~\cite{62}. Finally, the peaks observed at
920--820~cm$^{-1}$ refer to the absorption of D-glucopyranosyl and
${\upalpha}$-D-mannopyranose, \mbox{respectively}.

\subsubsection{Thermal behavior}\label{sec325}

The thermograms (Figure~\ref{fig4}) show endothermic peaks at 89.0 and
77.7~\textdegree C for PCT-1 and PCT-2, respectively. These peaks
correspond to residual water retention due to existing hydrogen bonds
between the galacturonic acid units~\cite{58}. Moreover, exothermic
peaks are recorded at 260 and 253~\textdegree C for PCT-1 and PCT-2,
respectively; they are related to the thermal degradation of polymers
and pectin~\cite{58}. A close value (240~\textdegree C) was reported by
Aldemir et~al.~\cite{50} for pectin extracted from~crab apple peels.

\begin{figure*}
\includegraphics{fig04}
\vspace*{-4pt}
\caption{\label{fig4}Differential scanning calorimetry thermograms of
PCT-1 (left) and PCT-2 (right).}
\vspace*{-2pt}
\end{figure*}

In addition, PCT-1 has a higher degradation temperature than PCT-2.
This can be explained by the different operating conditions used during
the two extraction processes. Moreover, PCT-1 exhibits greater changes
than PCT-2 during the heating process (Figure~\ref{fig4}). This finding
indicates that PCT-1 has a higher thermal stability than PCT-2. Similar
trends were observed by Ezzati et~al.~\cite{59} and Rahmani
et~al.~\cite{60} when studying pectin extraction from sunflowers and
sweet lemon, respectively. 

\subsubsection{Antibacterial action}\label{sec326}

The antibacterial action of PCT-1 and PCT-2 on \textit{Pseudomonas
aeruginosa}, \textit{E.\ coli}, and \textit{Staphylococcus epidermidis} has been
assessed in vitro according to the experimental protocol given in
Section~\ref{sec24}. Results (data not shown) indicate that neither PCT-1 nor
PCT-2 has an antibacterial effect on \textit{Pseudomonas aeruginosa} or
\textit{E.\ coli}. Both pectins exhibit significant antibacterial
potential against \textit{Staphylococcus epidermis}. Indeed, for a
content of 1~mg/mL, the zone of inhibition was evaluated at 11 and
9.5~mm for PCT-2 and PCT-1, respectively. This result highlights the
effect of the acid extraction method used and of the pectin's
properties (humidity H, ash content {Cd}, and degree
of esterification DE). The high resistance level of these
Gram-negative bacteria to the extracted pectins can be attributed to
the complexity of their cell envelope, which contains double
membranes~\cite{63}. It is important to underline that antibacterial
activity is determined not only by the number of free OH groups, but
also by the overall structure of the molecule; molecular mass and
monosaccharide composition can influence its ability to reach and
interact with the bacterial membrane. In general, a lower DE, or a
higher number of free hydroxyl groups, is associated with stronger
antibacterial activity. This is because free OH groups can interact
more easily with bacterial membranes, disrupting their structure and
function, leading to increased antibacterial activity. They are more
reactive and can interact with bacterial cellular components. Moreover,
free OH groups can form hydrogen bonds with phospholipids and proteins
in the bacterial cell membrane, increasing its permeability and leading
to cell death~\cite{63,64}. In contrast to that, a \mbox{significant}
antibacterial effect was observed on the \textit{Staphylococcus
epidermidis} strain (Figure~\ref{fig5}a). Indeed, PCT-1 and PCT-2 show
a concentration-dependent inhibitory effect against
\textit{Staphylococcus epidermidis} strain (Figure~\ref{fig5}a). Pectin
suspensions of 1~mg/mL showed a significant reduction in the growth
curve of \textit{Staphylococcus epidermidis} compared to the lowest
concentration group of 1/6~mg/mL. In contrast, neither PCT showed an
antibacterial effect at 1/8~mg/mL. Likewise, Abdelgawad
et~al.~\cite{63} observed a concentration-dependent inhibitory effect
of pectin against two bacterial strains (\textit{E.\ faecalis} and
\textit{F.\ nucleatum}). This significant antibacterial activity can be
explained by a higher sensitivity of \textit{Staphylococcus
epidermidis,} as a Gram-positive bacteria with a simple membrane
structure, to the action of the pectins. Pectins easily act on their
cell wall leading to a significant change in cell
permeability~\cite{64}.\looseness=1

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}(a) Illustration of the antibacterial action of
PCT-1 and PCT-2 against \textit{Staphylococcus epidermidis}, (b) DPPH
radical scavenging activity of PCT-1 and PCT-2, (c) Anticoagulant
activity of PCT-1 and PCT-2 fractions.}
\end{figure*}

\vspace*{2pt}

\subsubsection{Antioxidant activity}\label{sec327}

\vspace*{2pt}

The antioxidant activity of PCT-1 and PCT-2 was evaluated based on the
experimental protocol given in Section~\ref{sec25}. Results
(Figure~\ref{fig5}b) show that both pectins exhibit a significant
DPPH$\bullet$ scavenging capacity. Moreover, this effect is highly
dependent on the pectin concentration used (Figure~\ref{fig5}b).
Indeed, for an initial concentration of 0.5~mg/mL, the DPPH scavenging
capacity was evaluated at 13.8\% and 12.0\% for PCT-1 and PCT-2,
respectively. Increasing pectin concentration to 4~mg/L allowed this
capacity to reach more than 36.3\% and 26.0\% for PCT-1 and PCT-2,
respectively. This result is imputed to the presence of more free
hydroxyl groups in the polysaccharide structure~\cite{64}. Moreover,
PCT-1 has a better antioxidant activity than PCT-2
(Figure~\ref{fig5}b). This may be linked to its lower DE (see
Table~\ref{tab1}). Indeed, polysaccharides with low DE values have more
\mbox{terminal} reducing hydroxyl groups and more effectiveness in scavenging
free radicals. Relatively high antioxidant capacities were also
reported for pectins extracted using citric acid~\cite{65,66}. It is
worth mentioning that the antioxidant capacity of polysaccharides is
mainly affected by electron- or hydrogen-donating abilities. The higher
DPPH$\bullet$ scavenging capacity of PCT-1 compared to PCT-2 may be
attributed to its higher total sugar content, making it the better
hydrogen donor~\cite{65}.

\begin{sidewaystable*}[p!]
\vspone
\caption{\label{tab2}Antibacterial, antioxidant, and anticoagulant
activities of different polysaccharides compared with our pectins}
\fontsize{8.3}{10.2}\selectfont\tabcolsep2.6pt
\begin{tabular}{cccccccccccc}
\thead
\xmorerows{1}{Origin} &  \multicolumn{5}{c}{Antibacterial activity} & 
\multicolumn{3}{c}{Antioxidant activity} & 
\multicolumn{3}{c}{Anticoagulant effect} \\\cline{2-6}\cline{7-9}\cline{10-12}
& Polysaccharides &  {Strain} & \parbox[t]{1cm}{\centering  Inhibition
zone} &  {Notes} &  {Reference} &  \parbox[t]{1.4cm}{\centering 
DPPH$\bullet$ scavenging capacity}\vspace*{2pt}
&  Notes &  Reference &  {Effect}  & {Notes} & {Reference} \\
\endthead
\parbox[t]{1.5cm}{\centering  Plant polysaccharides} &
\parbox[t]{1.5cm}{\centering Cellulose composites} & 
\parbox[t]{1.5cm}{\centering \textit{S.\ epidermis}} & {Variable} &
\parbox[t]{1.5cm}{\centering Often combined with chitosan oils} & 
\cite{69} &  {Very low} & \parbox[t]{1.5cm}{\centering Increases only
with added antioxidant compounds} &  \cite{69} & 
\parbox[t]{1.5cm}{\centering None (native)} & 
\parbox[t]{2cm}{\centering  Modified cellulose sulfate shows
antioxidant effect}  & \cite{75}\vspace*{5pt} \\

& {Pectin} & \parbox[t]{1.5cm}{\centering\textit{S.\ epidermis}} &
{Variable} & \parbox[t]{1.5cm}{\centering Moderate activity alone} &
\cite{69} &  \parbox[t]{1.5cm}{\centering Moderate to higher depending
on form} & \parbox[t]{1.5cm}{\centering Increases with concentration} &
\cite{72} &  {Weak} & \parbox[t]{1.5cm}{\centering Slight prolongation 
PT/ATPP} & \cite{76}\vspace*{5pt} \\

\parbox[t]{1.5cm}{\centering Animal  polysaccharides} & 
\parbox[t]{1.5cm}{\centering Chitosan composites} & 
\parbox[t]{1.5cm}{\centering\textit{S.\ epidermis}} & 
\parbox[t]{1.5cm}{\centering Variable  (often measurable)} &
\parbox[t]{1.5cm}{\centering Depends on formulation} & \cite{70} & 
\parbox[t]{1.5cm}{\centering High  to medium} &
\parbox[t]{1.8cm}{\centering Higher when modified with phenolic
compounds} &  \cite{69} & {Weak} & \parbox[t]{1.5cm}{\centering
Sulfated chitosan} & \cite{77}\vspace*{5pt} \\

{Marine Algae}  &   {Alginate} &
\parbox[t]{1.5cm}{\centering\textit{S.\ epidermis}} & {Low/None} &
\parbox[t]{1.5cm}{\centering Often combined} &  \cite{69} &
\parbox[t]{1.5cm}{\centering Low  to moderate} &
\parbox[t]{1.5cm}{\centering Weak activity} & \cite{73} & {Minimal}  & 
\parbox[t]{1.5cm}{\centering Sulfated alginate extracts} &
\cite{78}\vspace*{5pt} \\

 &   {Agar} & \parbox[t]{1.5cm}{\centering\textit{S.\ epidermis}} & {No
effect} & \parbox[t]{1.5cm}{\centering No activity alone} & \cite{71} &
Low  & \parbox[t]{1.5cm}{\centering  Enhanced in blends with
antioxidants} & \cite{74} &  \parbox[t]{1cm}{\centering Very limited} &
Not pure agar & \cite{79}\vspace*{5pt} \\

\parbox[t]{1.8cm}{\centering Algerian \textit{Citrus sinensis} Peels} &
\parbox[t]{1.5cm}{\centering  Pectin extracted} &
\parbox[t]{1.5cm}{\centering\textit{S.\ epidermis}} &  {Significant} &
{Moderate} & {This study} & {Significant}  &
\parbox[t]{1.5cm}{\centering  Dependent on concentration} &  {This
study} & {Interesting} & \parbox[t]{2cm}{\centering  Comparable to a
sulfated animal polysaccharide}  & {This study}\vspace*{2pt} 

\botline
\end{tabular}
\end{sidewaystable*}

\subsubsection{Anticoagulant activity}\label{sec328}

The anticoagulant activity of PCT-1 and PCT-2 was evaluated as
described in Section~\ref{sec26}. Experimental results (Figure~\ref{fig5}c)
show the PT values for PCT-1 (15.3 s) and PCT-2 (14.9 s) as well as the
APTT values for PCT-1 (33.4~s) and PCT-2 (31.9~s).

These values are comparable to those of heparin (a sulfated
polysaccharide of animal origin) indicating an interesting potential
use for our pectins. Likewise, Chaouch et~al.~\cite{67} developed two
sulfated pectins. They showed that their extracted pectins exhibited a
strong anticoagulant activity and could be considered as potential
alternatives of heparin. Furthermore, Bae et~al.\  assessed the
anticoagulant activity of commercial pectin and found PTs and APTTs of
22--38~s and 10--14~s, respectively~\cite{68}. Further research is
crucial to determine whether our pectin can be used safely and
effectively as a substitute for heparin, particularly in the context of
PT/APTT testing in normal plasma.

Table~\ref{tab2} gives a comparison of the antibacterial, antioxidant,
and anticoagulant effects of our pectins with those of common
polysaccharides from plants, animals, and marine algae.

\section{Conclusion} 

The aim of this work is to turn abundant agricultural waste (local
Algerian orange peels) into an effective biomaterial. Two pectins were
extracted using citric (PCT-1) and hydrochloric acid (PCT-2). Their
thorough characterization shows that both pectins have interesting
thermal and physicochemical properties. PCT-1 is more stable. It showed
a better antibacterial activity with a concentration-dependent
inhibitory effect against \textit{Staphylococcus epidermidis} but had
no antibacterial effect on \textit{Pseudomonas aeruginosa} or
\textit{E.~coli}. PCT-1 also exhibits greater antioxidant activity than
PCT-2. The prothrombin and activated partial thromboplastin time values
of PCT-1 (15.3 and 33.4 s) and PCT-2 (14.9 and 31.9 s) demonstrated
that the extracted pectins could be considered as a potential
alternative to heparin. Future work will be undertaken to optimize the
extraction process using other methods (i.e., microwave, ultrasound),
to evaluate the antimicrobial capacity of the extracted pectins on
other pathogenic bacteria, and the antioxidant and anticoagulant
activities under a wider range of experimental conditions. 

\section*{Acknowledgments}

The authors would like to express their gratitude to the Directorate
General of Scientific Research and Technological Development (DGRSDT),
Laboratory of Microbiology, Hassani Abdelkader Hospital, Sidi Bel Abbes
and the Ministry of Higher Education and Scientific Research (MESRS),
Algeria, for their support to this research work.

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