Vol.4, No.11, 614-619 (2013) Agricultural Sciences
Extraction and characterization of gelling pectin from
the peel of Poncirus trifoliata fruit
Kouassi L. Koffi1, Beda M. Yapo2*, V. Besson3
1Food Research Unit, Polytechnique National Institute of Felix Houphouet Boigny, Abidjan, Côte d’Ivoire
2Subunit of Pedagogy in Biochemistry and Microbiology, University of Jean-Lourougnon Guédé, Daloa, Côte d’Ivoire;
*Corresponding Author: bedamarcel@yahoo.fr
3Food Research and Technology Division, Cargill West Africa, Abidjan, Côte d’Ivoire
Received 6 August 2013; revised 26 September 2013; accepted 18 October 2013
Copyright © 2013 Kouassi L. Koffi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In the framework of searching for new pectin
sources to partially compensate for domestic
and regional demands, the peel (albedo) of the
“non-comestible” fruit of Poncirus trifoliata was
investigated using a relatively simple experi-
ment al design for optimization, in which only the
variable was the extraction pH (1.0, 1.5, and 2.0)
on the basis of our previous studies on diverse
pectin sources. The results showed that the
yield of pectin (7.4% - 19.8%) was strongly in-
fluenced by the extraction pH when the other
parameters, namely the solid to liquid extractant
(S/L) ratio, temperature (T ˚C), and time (t) were
fixed to 1:25 ( w/v), 75˚C, a nd 90 mi n, re spec tiv ely.
Likewise, the galacturonic acid content (GalA:
61.4% - 79.2%), total neutral sugar content (TNS:
9.1% - 22.5%), degree of branching (3.5% -
13.9%), homogalacturonan (HG) to rhamnoga-
lacturonan-I (RG-I) ratio (2.2 - 5.6), degree of me-
thyleste rificatio n (DM: 54 - 77), v i scosity av erage
molecular weight (Mν: 57 - 82 ), and ge lling capac-
ity (GC: 12 4 - 158) were all affe cted by the extrac-
tion pH. The optimum pH for producing pectin
with good yield, quality characteristics (GalA >
65%, DM > 60, Mν > 80 kDa), and gelling capacity
(GC > 150), from the peel of P. trifoliata fruit, was
found to be pH 1.5.
Keywords: Ponciru s trifoli ata; Pectin; Block
Copolymers; Macromolecular Characte ristics;
Gelling Strength
Pectins are natural polysaccharides from all higher
plant cell walls. They are generally composed of
galactopyranosyluronic acid (
-D-GalpA), and three
neutral sugars, namely
-L-rhamnopyranose (
-D-galactopyranose (
-D-Galp), and
ranose (
-L-Araf). The different sugars are linked to one
another in the way that gives rise to pectin structure with
two-to-three block copolymers, viz. homogalacturonan
(HG), type one rhamnogalacturonan (RG-I), and type
two rhamnogalacturonan (RG-II) to a lesser extent. HG
is an 1,4-
-D-GalpA polymer esterified with methyl al-
cohol at C-6 position and sometimes with acetic acid at
O-2 and/or O-3 positions. RG-I is an [1,4)-
-L-Rhap-(1,4] polymer, commonly branched with
-L-arabinan, 1,4-
-D-galactan, and type one arabi-
nogalactan. RG-II is a rhamnose-containing polymer,
which has an oligogalacturonan core branched with four
well-defined side chains [1. However, it remains a mat-
ter of debate if all the block copolymers are covalently
linked to one another, and how they are likely intercon-
nected to one another within pectin structure, due to in-
sufficiency in the availability of compelling and irrefuta-
ble body of structural data. Nevertheless, it is worth un-
derlying that three basic models have been proposed in
the literature [2-4.
The main functional property so far known to pectins
is their ability to form gelling systems under specified
conditions [5,6. It is now well known that the pectin HG
domain is responsible for its gelling capability, while the
pectin RG-I region chiefly plays a gel-stabilising role
[6,7. Two different gelling mechanisms have been de-
scribed, depending on the methylesterification degree
(DM) of pectins. Thus, high methylesterified pectins
(DM > 50%) are commonly used to prepare sugar-acid-
mediated gels (HMP-SAG) and low methyl-esterified
pectins (DM 50%) are utilized for the preparation of
calcium-mediated (low calorie) gels. To date, commer-
cial pectins, used for the preparation of different confec-
Copyright © 2013 SciRes. OPEN ACCES S
K. L. Koffi et al. / Agricultural Sciences 4 (2013) 614-619 615
tions and gelling products (such as marmalades, jams,
preserves, and low calorie gels), are principally produced,
in Europe and in the United States of America, from two
pectin-rich sources (lime albedo and apple pomace) un-
der specified industrial extraction conditions: dry raw
material to solvent ratio 1:35 - 1:15 (w/v), water acidi-
fied with HNO3 (or HCl) to pH 1.0 - 3.0, temperature
60˚C - 100˚C, and time 30 - 180 min. Thus, commercial
pectin import in emerging and especially in developing
countries to satisfy their demand represents a high cost
operation with low added values to domestically manu-
factured gelling products. As a result, local food distribu-
tion firms in developing countries such as Côte d’Ivoire
are thus compelled to import ready-to-eat gelling prod-
ucts. To partially remedy this problem, local agricul-
tural byproducts, able to yield pectins with good gelling
properties, are currently being searched for to compen-
sate for the high cost linked with import of commercial
lime and/or apple pectins. In this connection, the present
study reports on the isolation and characterization of pec-
tin with good yield, quality characteristics, and gelling
capacity from the peel (albedo) of the underutilized
Poncirus trifoliata fruit, a locally abundant and hitherto
unexploited pectin source. The Poncirus fruit is a “non-
comestible” fruit, because of its intense bitterness caused
by the presence of poncirin and is so far locally proc-
essed by naturotherapeutists who commonly use the fruit
seeds, juice and essential oil (from the flavedo) as tradi-
tional remedies against malaria and insect (mosquito and
ant) bites.
2.1. Pectin Extraction
Fresh peels (albedo and flavedo) of the fruit of
Poncirus trifoliata were separately collected from a me-
dium-size plant of local producers of Poncirus juice,
seeds and essential oil, in a city named Bacon, located in
the southeastern region of Côte d’Ivoire. The peels were
immediately blanched to prevent enzymatic degradation
of the cell wall pectins, and oven-dried to about 7% final
moisture content. The dried peels were ground in a
hammer mill (Model 912, Winona Attrition Mill Co.,
Winona, MN) to pass through a 12 mm mean diameter
size sieve and kept under moisture-free conditions until
Pectins were extracted only from the albedo using a
relatively simple experimental design for optimization.
On the basis of our previous work on other pectin
sources, the solid to liquid (extractant) ratio (S/L), tem-
perature (T ˚C), and time (t) were invariably fixed to
1:25 (w/v), 75˚C, and 90 min, respectively, and only the
extraction pH was varied from 1.0 to 2.0 per 0.5 unit
interval (1.0, 1.5 and 2.0). The peel was extracted twice
before discarding insoluble fraction. At the end of each
extraction, the slurry was filtered and pectin extract was
rapidly brought to pH 4 for stability. The first and second
extracts were combined, concentrated to desired volume,
and then precipitated in 3 volumes of 95% ethanol at 5˚C
for 2 h. Pectin precipitates were washed (twice) with
70% ethanol, followed by 95% ethanol and dry acetone,
and kept for a while under a fume extractor (for residual
acetone evaporation) and finally oven-dried at 45˚C
overnight and weighed. Extraction of pectins was per-
formed in three independent runs for each pH value.
Dried pectin flakes were finely ground to pass through
60 mesh (0.25 mm) size sifters. Pectin flours were
canned in plastic containers and kept at room tempera-
ture under airless and moisture-free conditions until use.
2.2. Pectin Characterization
2.2.1. Pectin Purification
Prior to characterization, the pectin samples were
treated with a mixture of 1% (v/v) HCl/60% (v/v) etha-
nol (three times), and the remaining pectin fractions were
exhaustively washed with 60% (v/v) ethanol until the
filtrate gave a negative response for chloride ions with
silver nitrate, indicating that no free sugars were present
within the so purified samples. This treatment indeed
aimed at removing free sugars and salts and converting
all the non-methylestrified carboxyl groups of pectin
macromolecules to the free acid (-COOH) form for a
correct titration with 1 N NaOH. Commercial citrus high
methoxy pectin (CCHMP: 95% DM, Sigma-Aldrich Co.,
St. Louis, MO) was used for comparison, especially with
regard to functional (gelling) properties, after enzymatic-
controlled partial desertification to 74% DM (with or-
ange peel PME (P5400) Sigma-Aldrich Co. St. Louis,
MO), which did not significantly affect its sugar compo-
sition and molecular characteristics [7. Pectins were
characterized for the sugar composition, esterification
degree, molecular weight, and gelling capacity.
2.2.2. Pectin Analysis
The GalA content of pectin samples was colorimetri-
cally quantified at 525 nm by a modified sulfamate-
meta-hydroxydiphenyl (MHDP) assay using monoGalA
standard [8. To assess the neutral sugar (NS) content,
pectins were first hydrolyzed with 1 mol·L1 H
(100˚C, 3 h) and freed NS, notably galactose/arabinose
(Megazyme procedure) and rhamnose [9 were quanti-
fied spectrophotometrically at 340 nm using Megazyme
assay kits (Megazyme International Ireland Ldt., Bray,
Co. Wicklow, Ireland). The two NS assays were based on
the quantitative oxidation of galactose/arabinose and
rhamnose to corresponding lactonic derivatives (D-ga-
lactono-(1,4)-lactone for α-L-Arabinose and β-D-Galac-
tose and L-rhamno-(1,4)-lactone for α-L-rhamnose) in
Copyright © 2013 SciRes. OPEN ACCES S
K. L. Koffi et al. / Agricultural Sciences 4 (2013) 614-619
the presence of corresponding dehydrogenases (β-ga-
lactose dehydrogenase (β-GalDH) plus galactose mutaro-
tase (GalM) for α-L-arabinose and β-D-galactose, and L-
rhamnose dehydrogenase for α-L-rhamnose) and the co-
enzyme NAD+, which is stoichiometrically reduced to
NADH with maximum absorbance measured at 340 nm.
D-galactose was quantitatively differentiated from L-
arabinose by reading absorbance at different reaction
times, viz. after 6 min- and 12 min-reaction at room tem-
perature, respectively. L-rhamnose was quantitatively
determined after 1 h-reaction at room temperature. Total
neutral sugar (TNS) was estimated either by the tri-re-
agent (anthrone, orcinol, and MHDP) colorimetric-
H2SO4 assay as reported previously [7 or by calculating
the sum of the amounts of the three NS determined.
The molar ratio of HG to RG-I block copolymers was
roughly estimated using relation 1, reequated from pre-
viously published work [1,10.
  
 
GalA%Rha %
HG RG-I%=1002Rha %Ara %Cal%
The degree of branching (DBr) of pectins rhamnosyl
residues with arabinose- and galactose-containing side
chains was roughly estimated, by Equation (2), as previ-
ously reported [11.
 
DBr100 Rha%Ara%Gal% (2)
It expressed the minimum number of rhamnosyl resi-
dues per 100 arabinosyl and galactosyl residues of pectin,
which should be distinguished from the minimum length
of arabinose- and galactose-containing side chains. The
lower the DBr, the higher the level of branching of the
pectin rhamnosyl residues.
The overall esterification degree of pectins was poten-
tiometrically determined as previously described [12.
The acetylesterification degree (DAc) was colorimetri-
cally measured at 510 nm by the hydroxamic acid assay
using glucose pentaacetate standard [13, and the me-
thylesterification degree (DM) was differentially evalu-
ated. All the measurements were performed in triplicates.
The molecular weight of pectin samples was analysed
by GFC on a high resolution Superdex-200 HR 10/30
column (Amersham Biosciences Corp., NJ). A molecular
weight kit of pullulan standards (Mw 6.0, 10.0, 21.7,
48.8, 113.0, 210.0, 393.0, and 805.0 kDa; Mw wn
1.0-1.2) from American Polymer Standards Corp.
(Mentor, OH) and homogenous HG standards (Mw 60
and 100 kDa, wn
M 1.0-1.2) [14 with known in-
trinsic viscosity ([
]) and Mw values were used for cali-
bration. To better evaluate the pectin Mν, the so-called
universal calibration technique (UCT) was used by plot-
ting log ([
] Mw) versus the elution volume (Ve) of
standards. Analyses were performed in triplicates.
2.3. Gelling Properties
The gelling capacity (or power) of pectins was evalu-
ated by the determination of the strength of gels prepared
under the following conditions: 65.0% soluble solids
(sucrose), 0.70 wt% pectin, pH 2.3, as previously de-
scribed [12.
2.4. Statistical Analysis
The data were statistically evaluated by the global test
of a single-factor analysis of variance (ANOVA), fol-
lowed by the Bonferroni’s posthoc test for multiple com-
parisons, whenever applicable, using a GraphPad Prism
V.3 software (GraphPad software Inc., San Diego, CA).
Means of different treatments were considered signifi-
cantly different at p < 0.05.
3.1. Extraction Yield of Pectins
The yield of extracted pectins from the peel P.
trifoliata fruit is shown in Table 1. The yield varied from
7.4% to 19.8% as the pH was varied from 1.0 to 2.0, in-
dicating that the yield of pectins was strongly pH-de-
pendent when the other extraction parameters (solid to
liquid ratio (S/L), temperature (T ˚C), and time (t)) were
kept constant. The different pectin yields were signifi-
cantly different from one another (p < 0.05). The yield
(12.6%) obtained at pH 2.0 was lower than the yield
(19.8%) obtained at pH 1.5, suggesting that the yield
tended to increase with increase in the strength of (acid)
extracting agent. However, the yield of pectin substan-
tially decreased as the strength of extractant was further
increased up to pH 1.0. This unexpected drastic drop in
the yield of pectin extracted at pH 1.0 could only be ex-
plained by considerable degradation of pectin polymers,
during solubilization from the cell wall, into smaller oli-
gomers, which were ethanol-soluble, and therefore were
not precipitated in the final sample. This observation is
consistent with various previous studies in which notable
degradation of pectins in hot acid media was reported
[15-17. In the light of the differences observed in yields,
the extraction conditions of S/L (1:25 w/v), T (75˚C), t
(90 min), and pH 1.5 appeared to be the optimum condi-
tions for isolating pectin with good yield from the peel
(albedo) of P. trifoliata fruit.
3.2. Characteristics of Extracted Pectins
3.2.1. Sugar Composition and Block Copoly mers
The sugar composition of the extracted pectins is also
shown in Ta bl e 1 . The galacturonic acid (GalA) content
of pectins ranged from 61.4% to 79.2% as the extraction
pH was varied from pH 1.0 to pH 2.0. The pectin GalA
amounts were significantly different from one another (p
Copyright © 2013 SciRes. OPEN ACCES S
K. L. Koffi et al. / Agricultural Sciences 4 (2013) 614-619 617
Table 1. Sugar composition and molecular characteristics of
extracted pectins from Poncirus peel.
pH 1.0 pH 1.5 pH 2.0
Yield (%) 7.4 ± 1.2a 19.8 ± 2.5b 12.6 ± 1.6c
GalA (% w/w) 61.4 ± 2.7a 79.2 ± 3.1b 67.2 ± 2.5c 85.2 ± 2.7d
Rha (% w/w) 0.3 ± 0.1a 1.7 ± 0.2b 2.6 ± 0.8c 0.9 ± 0.2d
Ara (% w/w) 3.1 ± 0.4a 4.3 ± 0.8a 7.6 ± 1.4b 2.2 ± 0.5a
Gal (% w/w) 5.7 ± 1.2a 8.2 ± 1.1a 12.5 ± 1.7c 5.1 ± 1.2a
TNS (% w/w) 9.1 ± 0.8a 14.2 ± 0.9b 22.7 ± 1.5c 8.2 ± 0.7a
Rha/GalA 0.6/100 2.5/100 4.6/100 1.3/100
DBr (%) 3.5 ± 0.4a 13.9 ± 1.4b 13.4 ± 1.6b 12.7 ± 1.2b
HG (mol%) 84.7 ± 1.3a 80.5 ± 2.1b 68.5 ± 3.4c 88.7 ± 1.5a
RG-I (mol%) 15.3 ± 1.1a 19.5 ± 1.7b 31.5 ± 1.2c 11.3 ± 1.6a
HG/RG (%) 5.6 ± 1.2a 4.1 ± 0.9a 2.2 ± 0.3b 7.9 ± 1.1a
DM 54 ± 6a 77 ± 3b 63 ± 2c 74 ± 1c
DAc Tr 4 ± 1 7 ± 2 Tr
](mL/g) 201 ± 8a 327 ± 6b 281 ± 5c 378 ± 7d
Mv (kDa) 57 ± 15a 82 ± 5b 68 ± 6c 89 ±3b
Gelling power
(˚sag) 124 ± 2a 158 ± 3b 142 ± 2c 179 ± 5d
Data are expressed as mean ± SD (n = 3). Mean values in the same line with
different letters are significantly different (p < 0.05). Tr: trace < 0.05%; PTP:
Poncirus trifoliata pectin. CCHMP: Commercial citrus high methoxy pec-
< 0.05). The highest amount of GalA was obtained at pH
1.5 and the lowest at pH 1.0. This is likely to support the
hypothesis that higher degradation of isolated pectin
polymers into shorter oligomers occurred at pH 1.0 than
at the remainder pH values. In general, the GalA content
of pectin (PTP) from the peel of P. trifoliata fruit was
less great, compared to commercial citrus high methoxy
pectin (CCHMP).
The three individual NS, quantified in all the PTP iso-
lates, were galactose (Gal: 5.7% - 12.5%), arabinose
(Ara: 3.1% - 7.6%), and rhamnose (Rha: 0.3% - 2.6%),
and the amount of total neutral sugar (TNS), calculated
as the sum of the three NS, was in the range of 9.1% -
22.7%. Also, evaluation of TNS by the tri-reagent
method revealed appreciable amounts of NS other than
the three mentioned above (probably xylose and glucose
from neighbouring polysaccharides such as xyloglucans)
within the pH 2.0-isolate, but not in the other two iso-
lates. Hence, all the pectin samples, with the exception of
the pH 2.0-isolate were highly purified. The amounts of
TNS in all the PTP isolates were significantly different (p
< 0.05) from one another, though some individual NS
were present in similar amounts. PTP isolated at pH 1.0
had the lowest NS content (9.1%) and PTP isolated at pH
2.0 had the highest NS content (22.5%). This showed
that pectin extraction at pH 1.0 indeed resulted in sub-
stantial degradation of solubilized pectin macromole-
cules, mainly in their NS-containing RG-I regions. This
was further confirmed by the observation that the pH
1.0-PTP isolate had a much lower DBr value (3.5%),
compared with the other two PTP samples (DBr 13.5%
- 13.9%) and even with CCHMP (12.7%) to which it
had a similar TNS content. Rapid degradation of the pec-
tin NS (especially furanosyl residues) in hot acid media,
due to high lability and sensitivity to acid, is a well-
known and documented phenomenon [15-17. The dif-
ference in DBr between the PTP isolated at pH 1.0 and
CCHMP, to which it had similar TNS content (8.2% -
9.1%), was mainly accounted for by discrepancy in the
amount of rhamnose, which was three-time greater in
latter pectin sample than in the former. This suggested
that the rhamnosyl residues of the RG-I block copoly-
mers of PTP might be scarcely branched with NS side
chains within the cell wall, thereby rendering them more
accessible and sensitive to acid cleavage. In general, ga-
lactose appeared to be the major NS in all the pectin
samples, followed by arabinose, indicating that neutral
branches were dominantly (arabino) galactans.
In all the pectin samples, the amount of HG was >50%
(68.5% - 84.7%), indicating that this block copolymer
was predominant over RG-I within the pectin structure,
assuming that no free (non-interlinked) polymer stretches
were present. It is, indeed, known that peels (albedo) of
mature fruits from the Citrus genus such as orange, lime,
lemon, and grapefruit (of the Rutaceae family) are pectin
HG-rich sources as also found here for the peel of mature
fruit from the Poncirus genus. The HG to RG-I ratio var-
ied from 2.2 (in pH 2.0-isolate) to 5.6 (in pH 1.0-isolate),
suggesting the presence of at least two-to-six HG per one
RG-I block copolymers.
3.2.2. Esterification Degree
The degree of methylesterification (DM) of extracted
pectins varied from 54 to 77 (Table 1), indicating that the
pectin polymers within the albedo of the mature fruit of P.
trifoliata are highly methylesterified. The highest value
of DM was obtained at pH 1.5 and the lowest at pH 1.0.
This may be explained by degradation of pectin ester-
groups under more severe extraction conditions. In all
cases, the degree of acetylesterification (DAc) was rather
low (<10%). Pectin from the citrus genus is also natu-
rally of a low DAc. In the light of these results, the ex-
traction conditions of S/L (1:25 w/v), T (75˚C), t (90
min), and pH 1.5 proved to be the best for isolating pec-
tin with high GalA content (>65%) and DM (>60%),
Copyright © 2013 SciRes. OPEN ACCES S
K. L. Koffi et al. / Agricultural Sciences 4 (2013) 614-619
from the peel of P. trifoliata fruit, two of the quality
characteristics that need to be fulfilled for possible mass
production and marketing.
3.2.3. Macromolecular Features
The intrinsic viscosity ([
]) of PTP varied from 201 to
327 mL/g (Table 1), with the pH 1.0-PTP and pH 1.5-
PTP isolates possessing the lowest and highest intrinsic
viscosities, respectively (p < 0.05). Nevertheless, these
intrinsic viscosities were great enough, suggesting that
PTP macromolecules probably had an overall extended
rod-like conformation, as imposed by the dominance of
HG over NS-ramified RG-I block copolymers, rather
than a compact sphere-like conformation. Likewise, the
viscosity-average molecular weight (Mν) of PTP varied
from 57 to 82 kDa (Tabl e 1), with the pH 1.0-PTP and
pH 1.5-PTP isolates exhibiting the lowest and highest
values, respectively (p < 0.05). Both results definitively
substantiated the fact that the extraction condition using
pH 1.0 induced higher degradation of pectin polymers
than the remainder, and that the extraction condition us-
ing pH 1.5 was more suitable for producing PTP with a
Mν comparable to that of CCHMP having a similar DM.
3.3. Gelling Properties of Extracted Pectins
The gelling capacity of PTP varied from 124 to 158
(Ta bl e 1). The pH 1.0-PTP and pH 1.5-PTP isolates dis-
played the lowest and highest values, respectively. This
difference could be attributed to discrepancy in GalA
content, DM, and Mν. The gelling capability of pectin is
(one of) the most important factor(s) for labeling as a
food additive and international marketing.
To date, only citrus (lime) peel and apple pomace are
used for the production of commercial pectins in Western
countries, mainly because citrus and apple pectins pos-
sess high gelling strengths [5,6 in addition for the two
raw materials to being available in large quantities. How-
ever, new sources such as mango and yellow passion
fruit rinds are more and more domestically used in dif-
ferent emerging and developing countries such as India,
Brazil, and South Africa to a lesser extent to partially
solve the problem of high cost pectin import-low added
value to locally manufactured gelling products. Our re-
sults showed that pectin with good yield, quality charac-
teristics and gelling capacity (amply comparable to the
benchmark citrus pectin) can be produced from the peel
of underutilized Poncirus trifoliata fruit.
This study shows that Poncirus peel is a pectin-rich
source. About 20% pectin isolate, with a high galactu-
ronic acid content (>65%), methylesterification degree
(>60%), viscosity-average molecular weight (>80 kDa),
and good gelling capability (>150) can be produced un-
der the optimum extraction conditions of 1:25-solid to
solvent ratio, 75˚C-temperature, 90 min-time, and pH 1.5.
Our future investigation should mainly focus on the
rheological (viscoelastic) properties of the pH 1.5-pectin
isolate to find out the optimum conditions (temperature,
time, and velocity) of gelation for possible small-size
industrial production to satisfy the increasing indoors and
outdoors demands at a reasonable cost.
We are indebted to Cargill West Africa for some financial support.
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