International Journal of Biological Macromolecules 82 (2016) 645–652

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International Journal of Biological Macromolecules

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

Extraction, characterization and gelling behavior enhancement of

pectins from the cladodes of Opuntia ficus indica
Khalef Lefsih a,d,∗ , Cédric Delattre b , Guillaume Pierre b , Philippe Michaud b ,
Tejraj M. Aminabhavi c , Farid Dahmoune a , Khodir Madani a
a
Laboratoire de Biomathématiques, Biophysique, Biochimie et Scientométrie, Faculté des Sciences de la Nature et de la Vie, Université de Bejaia,
06000 Bejaia, Algeria
b
Clermont Université, Université Blaise Pascal, Institut Pascal, UMR CNRS 6602 CNRS Polytech Clermont-Ferrand, 24 avenue des Landais, BP 206,
Aubière Cedex F-63174, France
c
Department of Pharmaceutical Engineering and Chemistry, Soniya College of Pharmacy, Dharwad 580 002, India
d
Département de Biochimie et Microbiologie, Faculté des Sciences Biologiques et des Sciences Agronomiques, Université de Tizi ouzou,
15000 Tizi ouzou, Algeria

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

Article history: Total Pectins Fraction (TPF) was extracted at room temperature from dried cladodes of Opuntia ficus indica.
Received 29 July 2015 TPF is constituted of three pectic fractions WSP, CSP and ASP, which are made up of 66.6%, 44.3% and 81.1%
Received in revised form 14 October 2015 (w/w) of galacturonic acid, respectively. The antioxidant ability of TPF increased with the concentration
Accepted 16 October 2015
increasing. It scavenged hydroxyl radical by 90% and chelated 90% of ferrous ions at 5 g/L. FTIR study was
Available online 19 October 2015
carried out. Strong characteristic absorption peaks at 1618 cm−1 assigned to the vibration of COO– group
of galacturonic acid. In the fingerprint region, we noticed three well-defined peaks at 1054, 1085, and
Keywords:
1154 cm−1 characteristic of pectic polysaccharides. TPF are non-gelling pectins. The co-crosslinking of TPF
Pectin
FTIR
with carrageenan was carried out and the gelling behavior was successfully improved. Thermo-sensitive
Antioxidant hydrogel was obtained with 82% of TPF and 18% of carrageenan (w/w).
Gelling behavior © 2015 Elsevier B.V. All rights reserved.
Viscosity
Carrageenan
Co-crosslinking
Opuntia ficus indica

1. Introduction arabinose, galactose, xylose and glucose are usually present in

about 5–10 wt% of galacturonic acid. Functional properties of pectin
Opuntia ficus indica (OFI) is one of the widely cultivated species strongly depend on its structural and compositional parame-
of the genus Opuntia in North Africa that is used for human con- ters, such as galacturonic acid content, methyl-esterification level,
sumption as natural fruits and animal food for its cladodes due to molecular weight distribution and length of galacturonic blocks
its rich nutrient content. It is widely cultivated in the semi-arid [14].
countries to feed goats, sheep and bovines as well as it is well- Commercially, pectin is commonly derived from fruit waste
known as a medicinal plant to cure diarrhea and anti-inflammatory mainly apple and citrus peel. Pectins that are abundant, renewable
diseases. Cladodes of cactus contain important fibers viz., cellu- and biodegradable have the capacity to associate through physical
lose, hemicelluloses and pectins [12]. The major water soluble and chemical interactions with a wide variety of molecules; which
polysaccharides extracted from Opuntia ficus indica cladodes con- would enhance their functionality [15]. In particular, pectins are
sists of pectins [13] of which the main pectin component is a used as thickening additives in foods, cosmetics and pharmaceuti-
central linear backbone chain composed of ␣-d-galacturonic acid cal preparation. Chemical modifications are, in some cases, needed
units linked by (1 → 4) glycosidic bonds. Neutral sugars, rhamnose, to improve their functionality. Fully de-esterified pectin with excel-
lent gelling properties in the presence of calcium was isolated from
fresh nopal cactus pads [13]. For controlled crosslinking of pectin
it is of interest to prolong the life-time over a desired time as
∗ Corresponding author at: Laboratoire de Biomathématiques, Biophysique,
required in many applications, especially as implantable drug deliv-
Biochimie et Scientométrie, Faculté des Sciences de la Nature et de la Vie, Université
ery systems, for preparing stable gels, and membrane devices [16].
de Bejaia, 06000 Bejaia, Algeria.
E-mail address: klefsih@yahoo.fr (K. Lefsih). Intensive research efforts have focused on the mucilage value of OFI

http://dx.doi.org/10.1016/j.ijbiomac.2015.10.046
0141-8130/© 2015 Elsevier B.V. All rights reserved.
646 K. Lefsih et al. / International Journal of Biological Macromolecules 82 (2016) 645–652

cladodes and fruits, but limited information is available as pectin was measured at 510 nm using a Shimadzu UV-1700 spectropho-
composition of the whole cladodes. The main objective of this work tometer (PharmaSpec). The hydroxyl radical inhibition (%) was
is to characterize the extracted pectin from cladodes of OFI in terms calculated using Eq. (2).
of their monosaccharide composition, co-crosslinking and gelling  (A − A ) 
capacity enhancement for further bioassays applications. c s
Hydroxyl radical inhibition(%) = × 100 (2)
Ac
2. Materials and methods where As and Ac are absorbances, of sample and control, at 510 nm
of 0.2 mL of the sample (0–5 g/L) or 0.2 mL of ultra-pure water with
2.1. Plant material 0.4 mL of a solution (v/v) of FeSO4 (5 mM)/H2 O2 (1%).

Cladodes used in this work were harvested in July, 2013 in

2.5. Chelating activity
Kabylia, a region in north Algeria, an area having semi-arid climate
with cold and rainy winter as well as hot and dry summer with tem-
Ionchelating activity of polysaccharide on Fe2+ was measured
peratures ranging between 25 ◦ C and 40 ◦ C. This plant belongs to
as reported previously [20]. A2 mL of the sample was mixed
the Cactaceae family, Opuntia genus and ficus indica species. About
with 3.7 mL of deionized water and reacted with ferrous chlo-
50 kg of fresh cladodes were dried under sun for one month, cut into
ride (5 mmol/L, 0.1 mL) to which 0.2 mL of 5 mmol/L ferrozine
small pieces and were grinded to get fine powder using an indus-
was added, the solution was mixed to measure the absorbance
trial grinder. Finally, a step of sifting is carried out and a powder
at 562 nm employing EDTA as a positive control. A lower level of
of fine granulometry (<125 ␮m) was recovered and preserved in a
absorbance indicated stronger chelating activity. The chelating rate
hermetic bottle.
of polysaccharide on Fe2+ (%) was calculated according:
2.2. Pectins extraction  (A − A )
b s
Chelating rate(%) = × 100 (3)
Ab
Pectic polysaccharides sequentially extracted from 300 g of the
fine powder of grinded and dried cladodes. The water-soluble where Ab is absorbance of the control (deionized water instead of
pectins (WSP) were extracted from water 10% (w/v) at 60 ◦ C under sample) and As is absorbance of the test sample mixed with reaction
stirring (500 rpm) for 1.5 h. The solution was then centrifuged mixture.
at 10,000 g/15 min and the supernatant was neutralized with 5N
NaOH, mixed with three volumes of ethanol (96%), stirred vigor- 2.6. Monosaccharide composition analysis
ously, and left overnight at 4 ◦ C. Next, chelating-soluble pectins
(CSP) were extracted from residue I using an aqueous solution Polysaccharides from cladodes of OFI (10 mg) dissolved in 4 M
of calcium chelating agent (EDTA 0.5%, 80 ◦ C, 1.5 h). After cen- TFA (1 mL) were heated at 100 ◦ C for about 8 hand hydrolysates
trifugation, the supernatant was neutralized with NaOH (5N) and were neutralized with ammonia solution (4 M). Monosaccharide
precipitated with ethanol. Residue II was treated with HCl 0.05 M composition of the polysaccharides was evaluated by High Pressure
at 50 ◦ C for 1 h, from which the residue III and the ASP fraction (acid Anion Exchange Chromatography (HPAEC) on an ICS 3000 (Dionex,
soluble pectins) were obtained. USA) column equipped with pulsed amperometric detection and AS
50 auto-sampler that was assembled with a guard CarboPacTM PA1-
2.3. Anti DPPH radical column (4 mm × 50 mm) and analytical CarboPacTM PA1-column
(4 mm × 250 mm). Samples (10 mg/mL) were filtered using 0.2 ␮m
Antioxidant activities of Total Pectins (TPF) and ascorbic acid membrane filter at the fixed injection volume of 25 ␮L. Before each
were evaluated using 2,2 -diphenyl-1-picrylhydrazyle (DPPH) pro- injection, columns were equilibrated by running for 15 min with
cedures described by [17] as adapted from [18]. Briefly, the fraction 18 mM NaOH. Samples were eluted with 18 mM NaOH for 30 min
was previously dissolved at various concentrations (0–10 g/L) in followed by linear gradient between 0 and 1 M sodium acetate in
ultra-pure water. 1 mL of solution (sample or control) was added 200 mM NaOH for 20 min to elute the acidic monosaccharides. The
into 1 mL of DPPH solution at 0.1 mM in ethanol vigorously stirred column was then washed for 15 min with 200 mM NaOH by keeping
and incubated for 30 min at room temperature (25 ◦ C). Absorbance eluent flow constant at 1 mL/min. Columns were thermostated at
was measured at the max value of 517 nm using Shimadzu UV- 25 ◦ C, results were collected and analyzed with Dionex Chromeleon
1700 spectrophotometer (PharmaSpec). The DPPH inhibition (%) 6.80 software (Sunnyvale, USA).
was calculated using Eq. (1).
  
Asample 2.7. Gas chromatography/mass spectrometry experiments
DPPH inhibition(%) = 1 − × 100 (1)
Acontrol
A 10 mg of polysaccharide was dissolved in 2 M HCl (2 mL),
where Asample and Acontrol are, respectively absorbances at 517 nm heated at 90 ◦ C for 4 h to evaporate under a nitrogen stream.
of 1 mL of the sample (0–5 g/L) and 1 mL of ultra-pure water with Trimethylsilyl-O-glycoside residues were solubilized by adding
1 mL of DPPH at 0.1 mM in ethanol. 500 ␮L of dichloromethane and analyses were carried out by
GC/MS-EI using an Agilent 6890 Series GC system coupled to an
2.4. Anti-hydroxyl radical Agilent 5973 Network Mass Selective Detector. The solutions were
injected into an Agilent HP-1 (30 m, 0.32 mm, 0.25 ␮m) at a Helium
For the adapted hydroxyl radical method [19], the fraction was flow rate of 2.3 mL/min. The helium pressure was adjusted to 8.8 psi
previously dissolved at different concentrations (0–10 g/L) in ultra- at the split ratio of 25:1. The rise in temperature was programmed
pure water. A 0.2 mL of the solution (sample or control) was added for the first step at 100 ◦ C for 3 min, an increment of 8 ◦ C/min up to
into 0.2 mL of an aqueous solution of 5 mM FeSO4 . After stirring, 200 ◦ C for 1 min and then a final increment of 5 ◦ C/min up to 250 ◦ C.
0.2 mL of aqueous solution of H2 O2 at 1% (v/v) was added to the mix- The ionization was performed by Electronic Impact (EI, 70 eV), the
ture solution was stirred and incubated at room temperature. After trap temperature was set at 150 ◦ C and the target ion was fixed at
60 min, 1 mL of ultra-pure water was added and the absorbance 40–800 m/z.
 K. Lefsih et al. / International Journal of Biological Macromolecules 82 (2016) 645–652 647

2.8. Degree of methylation (DM) 110

100

Scavenging effect (%)

From [21], it is inferred that the ratio of the area of the
90
band at 1736 cm−1 (corresponding to the number of esterified
80
carboxylic groups) over the sum of the areas of the bands at
1735 cm−1 and 1618 cm−1 (corresponding to the number of total 70
carboxylic groups) should be proportional to the degree of meth- 60
ylation (DM) as given by: 50
 A1735
 40
DM = + 0.107 × 100 (4)
(A1735 + A1618 ) 30
20
2.9. Solid FTIR spectroscopy
10
A
The polysaccharide fractions were analyzed using Fourier trans- 0
form infrared spectrophotometer. The dried polysaccharides were 0 2 4 6 8 10 12
ground with spectroscopically pure potassium bromide (KBr) pow-
Concentration (g/L)
der and pressed into pellets (150 mg of dried KBr and 1 mg of
lyophilized samples) and spectra were recorded in the transmission
mode at room temperature (mid-infrared region, 4000–600 cm−1 ) 110
using a Nicolet spectrometer coupled with the personnel computer
100
loaded with OMNIC software. A total of 40 scans were measured
90
with a resolution of 4 cm−1 .

Scavenging effect (%)

80
2.10. Viscosity analysis and polymer blend 70
60
The gelling behavior of the native pectin and saponified pectin 50
was studied by monitoring the evolution of the viscosity over 40
time. Viscosity measurements were performed by a SNB-1 dig-
30
ital viscometer (Princeton instruments). TPF (10 g/100 mL) were
20
saponified with NaOH (0.1N) for 24 h at 4 ◦ C under vigorous stir-
ring. Then the samples were neutralized with HCl (3N), precipitated 10 B
with two volumes 96% ethanol and dried in oven at 40 ◦ C. 0
Polymer blends were prepared with TPF and different pro- 0 2 4 6 8 10 12
portions of ␬-carrageenan (10%, 12%, 14%, 16%, 18%, and 20%)
(Sigma–Aldrich). Viscosity measurements were performed for each Concentration (g/L)
TPF/carrageenan blend over time.
Fig. 1. Scavenging effects on (A) hydroxyl radical and (B) DPPH radical for (䊉): TPF
Finally, the effect of pH (2, 4, 6 and 8) and temperature (5 ◦ C, and, (䊏): ascorbic acid.
15 C, 25 ◦ C, 35 ◦ C and 45 ◦ C), on the viscosity of pectin/carrageenan
◦

(18%) blend were monitored.

3. Results and discussion

110
3.1. Antioxidant and chelating activities
100
The scavenging abilities of TPF increased with increasing con-
90
centration reaching a plateau of 85 ± 2% at concentrations higher
than 2 g/L. TPF showed high antioxidant activity as it scavenged 80
Chelating effect (%)

hydroxyl radical by 90% at 5 g/L, by 70% at 0.5 g/L (Fig. 1A), while
70
it scavenged DPPH radicals by ≈90% at 2.25 g/L and by 85% at 1 g/L
(Fig. 1B). These values are very close to those given by the scaveng- 60
ing activity of the ascorbic acid used as a positive control. The TPF
50
showed a good chelating ability compared to the chelating effect
of synthetic metal chelator (EDTA). The TPF chelated 90% of fer- 40
rous ions at 5 g/L and 85% and 55% at 2.2 g/L and 1 g/L, respectively
30
(Fig. 2).
20
3.2. Monosaccharide composition and DM 10

In their work, [22] have shown that cladodes of OFI are mainly 0
constituted of carbohydrates and that cladode’s polysaccharides 0 2 4 6 8 10 12
are mostly made up of pectins. Similarly, glucose and galacturonic Concentration (g/L)
acid are the main sugars of Opuntia cladodes [12]. Neutral sugars
are from the highly branched chains of galactan and arabinan [23]. Fig. 2. Chelating effects of (䊉): TPF and, (䊏): EDTA.
In this work, the monosaccharide composition obtained by HPAEC
of the three soluble fractions is shown in Table 1. The mass yields
648 K. Lefsih et al. / International Journal of Biological Macromolecules 82 (2016) 645–652

Table 1 Table 3
HPAEC determination of monosaccharide composition of pectic fractions extracted Degree of methylation and yield of extraction of WSP, CSP and ASP.
from the cladodes of Opuntia ficus indica.
Fraction Yield from dry mater (%, w/w) Galacturonic acid (%, w/w) DM (%)
Constituents Mass yield (%w/w)
WSP 5.75 66.6 30.6
WSP ASP CSP CSP 0.21 81.1 33.5
ASP 0.11 44.3 28
Galactose 11.4 40.2 8.2
Glucose 6.9 7.3 1.9
Galacturonic acid 66.6 44.3 81.1
Arabinose 9.2 0.8 4.1 showed that pectins resulting from the pulp of cactus cladodes are
Xylose / / 0.5 low-methylated as opposed to those extracted from the walls of
Rhamnose 2.1 6.6 / the cladodes are high-methylated.
Mannose 3.8 0.8 4.0

Sum 100 100 99.8 3.3. FTIR analysis

FTIR spectra of pectin fractions (CSP, WSP and ASP) are shown
Table 2
CG-MS determination of monosaccharide composition of the whole cladode and in Fig. 3. The results were analyzed in three characteristic regions:
residue III. O H stretching bands envelope (3200–3600 cm−1 ); C H (methyl)
stretching bands (2800–3000 cm−1 ) and the fingerprint region
Constituents Mass yield (%, w/w)
envelope (700–1800) cm−1 [30]. A broad absorption band at
Whole cladode Residue III 3434 cm−1 due to stretching frequency of O H group and a band
Galactose 31.8 45.3 around 2850–2919 cm−1 are attributed to the C H stretching vibra-
Glucose 25.1 37.2 tion [31]. The Fingerprint region of the FTIR spectra for these three
Galacturonic Acid 23.2 7.9
pectin fractions show practically identical parts with three bands
Arabinose 18.8 8.4
Xylose 1.1 1.4 that are characteristics of pectin polysaccharides at 1054, 1085
Rhamnose / / and 1154 cm−1 , assigned to C OH, C C and C O vibra-
Mannose / / tion mode, respectively [13,30,32]. Pectins belong to the class of
Sum 100 100.02 carboxypolysaccharides, which differ from the neutral polysaccha-
rides, with an intense band in the region 1750–35 cm−1 related
to the vibration of esterified carboxyl group and in the regions of
(%, w/w) of galacturonic acid in these fractions are 66.6%, 44.3% and 1400–1450 cm−1 and 1600–1650 for free carboxyl group [33]. The
81.06% for WSP, ASP and CSP, respectively suggesting the presence absence of a band at 1750 cm−1 might be because it is covered by
of an important amount of pectin polymers. The major pectin frac- a strong peak at 1618 cm−1 (asymmetrical COO− stretching vibra-
tion referred here as water-soluble pectin (WSP) represents 5.14% tion). In the ASP spectrum, the absence of a band at 1415 cm−1
of dry cladode weight. It also contains 11.4% of galactose, 9.2% of present in WSP and CSP, spectra indicate that it is either over-
arabinose and 6.9% of glucose resulting from the ramified chains of lapped with a strong band at 1318 cm−1 or due to the absence of
arabinogalactan and contaminating glucan. The presence of 6.6% of O-acetyl-ester group.
rhamnose and 44.3% of galacturonic acid in ASP fraction suggests The most obvious difference between WSP, CSP and ASP spec-
the occurrence of rhamnogalacturonan backbone for acid soluble tra is the presence of well resolved peaks in ASP spectrum at
pectin with homogalactan branched chains (40.2% of galactose and 1318 cm−1 , assigned to C H vibration [1] in haired zones which
0.8% of arabinose). The CSP fraction represents 0.21% of dry weight are liberated by acid treatment, and galactose absorption peak at
and is mostly constituted of homogalacturonan pectin type (81.1% 782 cm−1 [2]. This is in conformity with the monosaccharide com-
of galacturonic acid and does not contain rhamnose residue). This position of these fractions.
high content of galacturonic acid is in accordance with the results We can notice well-defined peaks of C H methyl stretching
of [24] who studied the structural characteristics of pectin polysac- around 2850–2919 cm−1 for CSP fraction, which may be dependent
charides obtained from OFI. Very low quantities of rhamnose and on the presence of traces of lipids in this fraction. It is highlighted
xylose are in accordance with those reported by [13] and corrobo- by the disappearance of these peaks by degreasing treatment of CSP
rate the earlier findings [25]. This low yield of rhamnose was also by toluene-ethanol (40:60, v/v) mixture.
explained [24] to confirm that Opuntia is rich in pectin polysaccha- For a more explicit study of the vibrations in the area of
rides. 400–900 cm−1 , we have carried out microfiltration of the fraction
The monosaccharide composition obtained by CG-MS from the WSP against deionized water on a membrane of 0.2 ␮ thickness.
final insoluble fraction (Residue III) and that of the whole cladode The microfiltration gave a fraction WSP-HM, with a high molec-
is presented in Table 2. By comparing the results of both the frac- ular weight and a second fraction WSP-LM with low molecular
tions, we can see the enrichment of the final residue with glucose weight. The spectral analysis by FTIR of fractions WSP, WSP-HM and
and galactose i.e., 45.3% and 37.2%, respectively, suggesting a high WSP-LM is shown in Fig. 4, wherein the vibrations in the around
content of the final residue in cellulose and hemicellulose. 400–900 cm−1 are in the shaded part. We observe a broad band
The highest rates of galactose are found in the final residue and in in this part of the spectrum for WSP-LM, whereas it is hardly per-
the ASP fraction, suggesting that the chains of galactan are related to ceptible in the spectra of WSP and WSP-HM. These vibrations are
the insoluble parietal polysaccharides and are released by the acid essentially due to monosaccharide and oligosaccharide molecules
treatment as was suggested before [26,27]. In general, the overall [34], except the peak at 690 cm−1 that is assigned to the stretching
monosaccharide composition of Opuntia cladodes is in concurrence vibration of C-Br formed during the preparation pallets in KBr.
with those of the earlier reports [23,25,28].
Table 3 shows the results of polysaccharide extraction yield 3.4. Spectral subtraction
for each pectic fraction as well as its rate in galacturonic acid
and its degree of methylation. The results of DM show that three The most straightforward method of analysis for complex spec-
pectin fractions belong to the family of low-methylated pectins tra is Difference Spectroscopy (Spectral Subtraction) that was
(DM < 50%). Our results are in agreement with those of [29], which carried out by simply subtracting the infrared spectrum of one
 K. Lefsih et al. / International Journal of Biological Macromolecules 82 (2016) 645–652 649

Fig. 3. FTIR spectra of CSP, WSP and ASP.

Fig. 4. FTIR spectra of WSP, WSP-HM and WSP-LM.

component of the system from the combined spectrum to leave

the spectrum of other component. If the interaction between com-
ponents results in the change of spectral properties of either one
or both the components, these changes will be observed in the dif-
ference spectra which manifest via the appearance of positive or
negative peaks in the spectrum. Spectral subtraction may be used
for the data collected for solutions and solids [35]. Since the three
extracted pectic fractions result sequentially from the same sam-
ple of whole cladode (WC), therefore the technique of FTIR spectral
subtraction enables us to check the quality and purity of each frac-
tion compared to the whole cladode sample (WC).
Fig. 5 shows the fingerprint region of WC spectrum as well as
the subtracted spectra of each fraction from that of WC. Subtraction
of WSP spectrum from that of WC give the subtraction spectrum Fig. 5. Subtraction spectra, [A]: WSP from WC; [B]: CSP from WC; [C]: ASP from WC
as shown in Fig. 5A, where only two peaks are observed in the [WC = Whole Cladode].

positive region. On the other hand, the peak at 1318 cm−1 that is
characteristics of cellulosic substances (insoluble in water) as well parietal polysaccharides. Otherwise, all the peaks allotted to the
as, the peak at 782 cm−1 is attributed to galactose confirm that the pectic substances are in the negative area, which proves that
ramified chains of galactans are not easily extractable. This suggests extraction of water-soluble pectins was effective. The connec-
that the ramified chains of galactans are in connection with the tion of sidechains of pectins to other cell wall materials such as
650 K. Lefsih et al. / International Journal of Biological Macromolecules 82 (2016) 645–652

Fig. 6. Subtraction spectra, [A]: WSP from RIII; [B]: CSP from RIII; [C]: ASP from RIII.

hemicelluloses and cellulose was reported in other works [3–5]. In 0.5

fact, the arabinogalactan sidechains of pectins may act as bridges
to connect the rhamnogalacturonan backbone of pectins and the 0.45
other cell wall materials such as cellulose and hemicellulosic
0.4
xyloglucans [3].
Difference between WC and CSP is shown in the subtraction
Viscosity Pa.S

0.35
spectrum (Fig. 5B); it is noticed that the major part of the spectrum
is in the negative area confirming that CSP fraction was effectively 0.3
extracted and is independent of the parietal polysaccharides. This
can be explained by the fact that chelating soluble pectins belong 0.25
to the mucilage. In addition, mucilage polysaccharides in Opun-
tia do not seem to be chemically associated, either covalently or 0.2
otherwise, to the structural cell-wall pectins [14].
0.15
Subtraction spectrum (Fig. 5C) illustrates that only the acid
pectins are majority in ASP fraction as proved by the presence of a
0.1
peak at 1650 cm−1 , which is characteristics of the COOH group in 0 1 2 3 4 5
the negative area. The occurrence of a peak at 1320 cm−1 in the neg- Time (hours)
ative area highlights the poor ASP fraction in the cellulosics, which
may reflect the association of acid pectin with parietal polysaccha- Fig. 7. Viscosity of TPF 5% (w/v) (䊉) and saponified TPF 5% (w/v) (䊏).
rides.
The results of spectral subtraction of the three pectic fractions Fig. 7 demonstrated the effect of saponification on the viscos-
from the final residue (RIII) give the spectra A, B, C of Fig. 6. All the ity of the TPF. In fact, saponification causes physical and chemical
three spectra are entirely in the negative area, which implies that changes which tend to increase the solubility of the pectin, thereby
RIII does not only contain traces of the components present in the decreasing the local crystallizations that promote increase in vis-
fractions WSP, CSP, ASP, proving that RIII is completely exhausted cosity or gelation [37]. Gelation consists in the association of the
in pectins present in the fractions WSP, CSP and ASP. polygalacturonate chains by forming the junction zones [38].
The effect of divalent ions (Ca++ ) on the gelling of the TPF is illus-
trated in Fig. 8. In fact, there is an increase in viscosity, reaching the
3.5. Viscosity measurement of TPF and TPF/Carrageenan blend maximum values for concentrations of Ca2+ from 40 mM to 80 mM;
beyond this range, there is a significant drop in viscosity. These low
Pectins from various sources do not have the same capacity for concentrations of Ca++ are dependent of the weak dissociation of
gelling since the ability of pectins to form a gel depends on its carboxylic groups, due to low pH, reducing the binding probability
degree of polymerization, the degree of esterification, degree of [39]. It is very important to note that despite the increase in vis-
branching [36]. cosity, in the presence of Ca++ , there is no gel formation, although
 K. Lefsih et al. / International Journal of Biological Macromolecules 82 (2016) 645–652 651

0.4 8
0.35 10%
7
0.3
12%
Viscosity Pa.S

0.25 6

0.2

Viscosity Pa.S
5 14%
0.15
4
0.1 16%
0.05 3
0 18%
0 50 100 150 200 2
Concentration of Ca++ (mM)
1 20%
Fig. 8. Effect of Ca++ on the viscosity of TPF.
0
TPF are low methylated. This can be explained by a high degree of 0 50 100 150 200
branching of these pectins. This means that despite the low degree Time (mn)
of methylation, polygalacturonate zones, which may interact with
Ca++ are very limited due to the high degree of branching. The effect Fig. 9. Effect of carrageenan ratio (10% = 0.11 g/20 mL; 12% = 0.13 g/20 mL;
14% = 0.16 g/20 mL; 16% = 0.2 g/20 mL; 18% = 0.22 g/20 mL; 20% = 0.25 g/20 mL) on
of branching on pectin gelation may be more relevant in HM pectin, the viscosity of TPF (1 g/20 mL).
as intermolecular association by hydrogen bonding is the primary
gelation mechanism. In LM pectin, the interactions between car-
1.4
boxyl groups of pectin and divalent ions are more important for
gelation than intermolecular interactions, thus the effect of branch-
ing may not be pronounced in this case [6]. Two contradictory 1.2 pH:2
arguments have been issued for the role of sidechains of pectins
pH:4
in gelling. Selvendran [7] stated that the arabinose and galactose 1
sidechains of pectins could give a positive contribution to gelling pH:6
Viscosity Pa.S
by holding water molecules within the gel framework. In contrast,
0.8
BeMiller [8] stated that the sidechains of pectins might tend to limit pH:8
the extent of interchain association, and thus formation of junction
zones for gelling may be inhibited. Matthew et al. [9] reported that 0.6 pH:10
sugar beet pectins treated with an enzyme prepared by Aspergillus
niger significantly improved gelation. This was due to the com- 0.4
bined effects of deacetylation, demethoxylation and the significant
reduction in arabinose residues of sidechains.
0.2
Pectic molecules in solution are highly hydrated and the total
negative charge depends on the dissociation of carboxylic func-
tional groups. The decrease of hydration is accomplished by the 0
addition of carrageenan, which plays the role of water scavenger; 0 50 100 150 200
on the other hand, it can form as copolymer with pectin, a three-
dimensional physical network promoting gelation. The presence of
Time (mn)
carrageenan increases the viscosity and to induce a good gelation. Fig. 10. Effect of pH on the viscosity of TPF (82%)/carrageenan (18%) blend.
Carrageenan plays a potential role in the formation of gel, in Fig. 9.
The minimum ratio of carrageenan giving a perfectly solid gel is 4.5
18%.
The effect of pH and temperature on the mixing viscosity TPF 4
(82%)/Carrageenan (18%) is illustrated in Figs. 10 and 11, respec-
tively. It is clearly seen in Fig. 10 that the viscosity of the hydrogel is 3.5
5°C
not very sensitive to changes in pH closer to neutrality. The increase
3
in viscosity with elevated pH is related to the increase of the disso- 15°c
Viscosity Pa.S

ciation rate of the carboxyl groups (COO– ) of pectin and ester sulfate 2.5
groups (OSO3 – ) of carrageenan which promote interaction with the 25°c
divalent ions present in the medium. Conversely, decreasing the pH 2
decreases the dissociation of the previous ionizable groups, which 35°C
induces an increase in the hydration of the TPF and carrageenan, as 1.5
well as their solubility, which results in a decrease in viscosity. 45°C
1
The viscosity of pectin/carrageenan is very influenced and sen-
sitive to the temperature. More the temperature is low, more the 0.5
viscosity is increased (Fig. 11). The sol–gel transition appears, in the
case of pectins, during cooling. Our results are in conformity with 0
previous findings [10,11], they attributed the decrease in viscosity 0 20 40 60 80 100
to the depolymerization of pectin with increasing temperature. Time (mn)
The most important result here is that the gelation
of TPF/Carrageenan blend is thermo-reversible. At cooling Fig. 11. Effect of temperature on the viscosity of TPF (82%)/carrageenan (18%) blend.
652 K. Lefsih et al. / International Journal of Biological Macromolecules 82 (2016) 645–652

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