Preparation and characterization of a quaternary ammonium derivative of pectin

Article abstract of DOI:10.1016/j.carbpol.2012.01.021

Cationic derivatives of pectin were prepared by reacting pectin with 3-chloro-2-hydroxypropyltrimethylammonium chloride (CHPTAC) in presence of sodium hydroxide (NaOH). The chemical structures of derivative were characterized by using elemental analysis, FT-IR, and ¹³C NMR spectroscopy. The results revealed that the degree of substitution (DS) of quaternized pectin (QP) could be controlled by adjusting the molar ratio of CHPTAC to pectin, the molar ratio of NaOH to CHPTAC and reaction temperature occurred during quaternization. The moisture absorption (R_a) and moisture retention (R_h) abilities of QP are closely related to the DS value. With the DS value increasing, R_a and R_h increased. In vitro antimicrobial activity assessment exhibited QP showed pronounced inhibitory effect against the three bacteria (Staphylococcus aureus, Escherichia coli, and Bacillus subtilis). The improved functionalities of the derivative might be explained by its polycationic characteristics.

Full text of DOI:10.1016/j.carbpol.2012.01.021

Carbohydrate Polymers 88 (2012) 707–712
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Preparation and characterization of a quaternary ammonium derivative of pectin
Lihong Fana,∗, Mi Caoa, Song Gaoa, Weiping Wangb, Kai Penga, Chang Tana, Feng Wena,
Shengxiang Taoc,∗∗, Weiguo Xied
a College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, China
b College of Bioengineering, Hubei University of Technology, Wuhan 430068, China
c Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan 430070, China
d The 3rd Hospital of Wuhan, Wuhan 430000, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cationic derivatives of pectin were prepared by reacting pectin with 3-chloro-2-
hydroxypropyltrimethylammonium chloride (CHPTAC) in presence of sodium hydroxide (NaOH).
The chemical structures of derivative were characterized by using elemental analysis, FT-IR, and 13C
NMR spectroscopy. The results revealed that the degree of substitution (DS) of quaternized pectin (QP)
could be controlled by adjusting the molar ratio of CHPTAC to pectin, the molar ratio of NaOH to CHPTAC
and reaction temperature occurred during quaternization. The moisture absorption (Ra) and moisture
retention (Rh) abilities of QP are closely related to the DS value. With the DS value increasing, Ra and Rh
increased. In vitro antimicrobial activity assessment exhibited QP showed pronounced inhibitory effect
against the three bacteria (Staphylococcus aureus, Escherichia coli, and Bacillus subtilis). The improved
functionalities of the derivative might be explained by its polycationic characteristics.
Received 8 November 2011
Received in revised form
22 December 2011
Accepted 7 January 2012
Available online 13 January 2012
Keywords:
Pectin
Quaternization
Moisture absorption
Moisture retention
Antimicrobial activity
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
& Ovodov, 2011; Ralet, Lerouge, & Quéméner, 2009). The degree of
methoxylation (DM) is used to classify the pectins as high methoxyl
In the last few years, considerable attention has been devoted to
the applications of natural plant polysaccharides because of their
unique properties (Crini, 2005; Edgar et al., 2001; Suh & Matthew,
2000). Recently, a large number of studies showed that natural
plant polysaccharides and their derivatives were widely used as
a type of desired biomaterials in many fields, such as drug deliv-
ery, immunization, controlled release, as well as in food application
(Fox, Li, Xu, & Edgar, 2011; Liu, Fishman, & Hicks, 2006; Ramberg,
Nelson, & Sinnott, 2010; Souto-Maior, Reis, Pedreiro, & Cavalcanti,
2010; Velisek & Cejpek, 2005). Pectin is a high-molecular weight,
biocompatible, non-toxic, and anionic natural polysaccharide
extracted from cell walls of higher plants (Zouambia, Moulai-
Mostefa, & Krea, 2009). Chemically it consists of ␣-(l–4)-bond
d-galacturonic acid units, occasionally interrupted by ˛-(1–2)-
bond l-rhamnose units (smooth regions) and branched areas (hairy
regions) represented by rhamnogalacturonan I and rhamnogalac-
turonan II (RGI and RGII) with lateral chains of galactan, arabinan,
arabinogalactan, and other more complicated fragments (Günter
pectins (DM > 50) and low methoxyl pectins (DM < 50) (Tripathi,
Mehrotra, & Dutta, 2010). Pectin is known for a functional ingredi-
ent, gelling/thickening agent, and a stabilizer ingredient in the food
industry due to its ability to form aqueous gels and has been used
in jams and jellies, fruit preparations, fruit drink concentrates, fruit
juice, desserts and fermented dairy products (Canteri-Schemin,
Fertonani, & Waszczynskyj, 2005; Franc¸ oise, Kablan, & Kamenan,
2009). Moreover, characters of excellent gelling properties and
good biocompatibility, as well as biodegradability entitle pectin to
be a novel polymer material, which is employed in pharmaceutical
industry, health promotion and treatment. It has been used poten-
tially as a carrier for drug delivery to the gastrointestinal tract, such
as matrix tablets, gel beads, film-coated dose form (Liu et al., 2006;
Tripathi et al., 2010).
Physical or chemical modification of pectin can lead to new
products with significantly functional properties. Previously, sev-
eral pectin derivatives were prepared by crosslinking (Souto-Maior
et al., 2010), amidation (Mishra, Datt, Pal, & Banthia, 2008), thi-
olation (Perera, Hombach, & Bernkop-Schnurch, 2010), sulfation
(Cipriani et al., 2009) and then their properties and application were
assessed. The applications of pectin have been also extended greatly
from food and food additives to various fields, such as drug delivery
(Mishra et al., 2008; Souto-Maior et al., 2010), antithrombotic agent
(Cipriani et al., 2009), and mucoadhesive (Sharma & Ahuja, 2011).
∗
Corresponding author. Tel.: +86 27 62773634; fax: +86 27 87859019.
Corresponding author. Tel.: +1 3886136267.
E-mail addresses: lihongfan2000@hotmail.com (L. Fan),
∗∗
zn-taoshengxiang@163.com (S. Tao).
0144-8617/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2012.01.021

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L. Fan et al. / Carbohydrate Polymers 88 (2012) 707–712
It is reported that quaternization is an efficient means of impart-
ing new functional properties to polysaccharides (Geresh, Dawadi,
& Arad, 2000). So far, quaternization has been widely applied to
a variety of polysaccharides, such as chitosan (Yevlampieva et al.,
2011), konjac glucomannan (Tian, Wu, Liu, & Xie, 2010), starch (Xu,
Cao, Wu, & Wang, 2006), alginate (Kim, Kim, & Rhee, 2010), and
cellulose (Niidei et al., 2010). However, quaternization of pectin is
little reported. Therefore, in this study, a quaternary ammonium
derivative of pectin was prepared by reacting with CHPTAC.
In the previous reports (Badawy, 2010; Kim et al., 2010;
Sajomsang, Gonil, & Tantayanon, 2009), the antimicrobial abil-
ity of natural polysaccharides is improved by quaternization. As
a polymer having quaternary ammonium salt group, QP possess
potential antimicrobial properties. Hyaluronic acid (HA), an impor-
tant functional ingredient in cosmetics, is unique for its excellent
moisture-absorption and moisture-retention abilities, but the total
amount is limited, and the price is high. The results showed QP
could replace HA for its better moisture-absorption and moisture-
retention abilities.
temperature to obtain nine quaternized pectin derivatives (coded
as QPs).
2.3. Characterization of the products
Nitrogen contents (N %) of QPs were measured with an elemen-
tal analyzer (CHN-O-Rapid, Foss Hera us GmbH, Hanau, Germany).
The DS value of QP was determined by nitrogen contents and cal-
culated according to the following equation:
176 × N%
DS =
(14 − 151.5 × N%)
FT-IR spectra of QP samples and pectin were performed with a
Nicolet 170SX (USA) Fourier transform infrared spectrometer. The
test specimens were prepared by the KBr-disk method.
The 13C NMR spectrum of the QP sample was recorded on Bruker
AMX-500 NMR spectrometer at an ambient temperature. The sam-
ple was dissolved in D2O. Tetramethylsilane (TMS) was used as
internal standard. Chemical shifts were expressed in ı (ppm) rela-
tive to the resonance.
The purpose of this work was to prepare a pectin derivative by
quaternization. The results reported in this article may contribute
to finding the application of QP in pharmaceutical, packaging,
preservatives, and cosmetic fields.
2.4. Moisture absorption and retention test (Chen, Du, & Zeng,
2002)
2. Experimental
2.1. Materials
Prior to the moisture-absorption testing, the samples were dried
over P2O5 in vacuo for 24 h. The water-absorption ability was eval-
uated by the percentage of weight increase of dry sample (Ra):
CHPTAC was purchased from Guofeng fine chemical Co. Ltd.,
Shandong, China and it was used as etherifying reagents without
further purification. Pectin (DM > 50) was purchased from Huilong
pectin Corp., Quzhou, China. The content of galacturonic is 90.5%.
And the molecular weight calculated from the static light scattering
method (Fan et al., 2011) was about 12.8 × 104. All other reagents
were of analytical grade and were used without further purification.
(Wn − W0)
Ra (%) = 100 ×
W0
W0 and Wn are the weights of sample before and after putting it
into a saturated (NH4)2SO4 desiccator (81% relative humidity) and
in a saturated Na2CO3 desiccator (43% relative humidity) at 20 ◦C
after 48 h of the test.
2.2. Synthesis of the derivative
In a typical reaction procedure, pectin (5 g) was dissolved
in deionized water (250 mL), and then various molar ratio of
NaOH/CHPTAC aqueous solution was dropped into the pectin
solution through pressure-equalizing dropping funnel. After that,
magnetic stirring was continuous for 17 h at certain temperature.
The solution was neutralized with aqueous hydrochloric acid and
then purified by dialysis through a 10 000–8000 molecular weight
cut-off dialysis tubing for three days. The dialyzed product was
finally freeze-dried with lyophilizer to obtain the purified pectin
derivative. The dried samples were stored in vacuum desiccators
over P2O5 for further analysis. According to Table 1, Experiments
were conducted under different reaction conditions, such as the
molar ratio of CHPTAC/pectin, the molar ratio of NaOH/CHPTAC, and
In the moisture-retention test, wet samples were prepared by
adding 10% water to each sample. The moisture-retention ability
was evaluated by the percentage of residual water of wet sample
(Rh):
Hn
Rh (%) = 100 ×
H0
H0 and Hn are the weights of water in sample before and after
putting in the silica gel desiccator at 20 ◦C 48 h of the test.
2.5. Antimicrobial activity assessments
Table 1
In vitro antimicrobial activity of pectin and QP was assessed
against three strains of bacteria (Staphylococcus aureus, Escherichia
coli, and Bacillus subtilis) and three fungal strains (Aspergillus niger,
Mucor sp., and Rhizopus sp.) by zone of inhibition. The strains were
kept at 4 ◦C for the antimicrobial tests. Petri dishes were inoculated
with 0.1 ml test bacteria solutions and fungal solutions, and acti-
vated respectively at 37 ◦C and at 28 ◦C for 24 h. The filter paper
discs disinfected of 6 mm in diameter was impregnated with ster-
ile distilled water and saturated with pectin and QP, followed by
lying on the agar plates. The agar plates of bacteria were incubated
at 37 ◦C for 24 h. At the same time, the agar plates of fungus were
incubated at 28 ◦C for 24 h. Blank sterile filter paper discs and sterile
water were used as control in this study.
Conditions and results of quaternary ammonium derivatives of pectin.
Sample ID
Molar ratioa
Molar ratiob
Temp (◦C)
Nitrogen (%)
DS
QP-1
QP-2
QP-3
QP-4
QP-5
QP-6
QP-7
QP-8
QP-9
4
5
5
5
5
6
5
7
5
1.2
1.0
1.2
1.2
1.2
1.2
1.1
1.2
1.2
50
50
40
50
60
50
50
50
70
2.54
2.90
3.08
3.74
4.56
4.64
6.19
6.25
6.36
0.44
0.53
0.58
0.79
1.13
1.17
2.36
2.43
2.56
a
The molar ratio of CHPTAC/pectin.
The molar ratio of NaOH/CHPTAC.
b

L. Fan et al. / Carbohydrate Polymers 88 (2012) 707–712
709
Scheme 1. Reaction scheme for the quaternization of pectin with CHPTAC.
3. Results and discussion
the molar ratio of CHPTAC/pectin from 4 to 7, the molar ratio of
NaOH/CHPTAC from 1.0 to 1.2 and the reaction temperature from
40 ◦C to 70 ◦C.
3.1. Synthesis of the derivative
Scheme 1 illustrates the quaternization of pectin by using CHP-
TAC as etherifying agent. Under the alkaline conditions, epoxide
is produced in situ from CHPTAC. QP is then formed through
reaction between the pectin and the epoxide. The reaction con-
ditions for quaternization of pectin are summarized in Table 1. The
content of nitrogen and the degree of quaternization (expressed
as DS value) under various reaction conditions are shown in
Table 1. As can be seen, the N content and DS value of the
quaternized derivatives increased with increasing molar ratio of
CHPTAC to pectin, as well as the reaction temperature. Further-
more, the optimal molar ratio of NaOH/CHPTAC is 1.1, which is the
same as quaternization of konjac glucomannan (Yu, Huang, Ying, &
Xiao, 2007). The reasons might be as follows: first, the lower molar
ratio of NaOH/CHPTAC could make the first step reaction incom-
plete and the epoxide is produced insufficiently. Second, the higher
concentration of sodium hydroxide solution can hydrolyze CHPTAC
and produce the diol. Obviously, quaternized pectin derivatives
with the DS values of 0.44–2.56 could be obtained by adjusting
3.2. Characterization of the derivative
Fig. 1 shows the FT-IR spectra of the native citrus pectin and
the quaternized pectin. The native pectin showed a broad band
at about 3419 cm−1, assigned to stretching vibration modes of
O–H groups. The bands at 2937 and 1051 cm−1 were assigned to
stretching vibration of –CH2– groups. Three peaks at 1747, 1636,
and 1443 cm−1 in the carboxylic groups region were assigned to
the C O stretching of methyl-esterified carboxyl groups, to anti-
symmetric and symmetric stretching modes of COO−, respectively
(Kurita, Fujiwara, & Yamazaki, 2008; Monfregola, Leone, Vittoria,
Amodeo, & De Luca, 2011; Pappas et al., 2004). In the case of QP, the
broad band at 3399 cm−1 was assigned for O–H stretching vibration.
The peak of 2938 and 1057 cm−1 referenced as stretching vibration
peaks of –CH2– group. The peak of 1747 cm−1 of pectin disappeared
due to removal of the ester group from the backbones of pectin dur-
ing reaction. Meanwhile, the peak of 1611 cm−1 of QP was assigned
to stretching vibrations of the carboxyl group. The most striking

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L. Fan et al. / Carbohydrate Polymers 88 (2012) 707–712
Table 2
Moisture-absorption and retention property of QP with different DS values.
Sample ID
DS
Ra (%) (dry sample)
Rh (%) (wet sample)
RH 81%
RH 43%
Silica gel
Pectin
QP-1
QP-2
QP-3
QP-4
QP-5
QP-6
QP-7
QP-8
QP-9
HA
/
23.45
40.81
51.99
54.51
62.30
81.02
82.21
87.37
91.45
100.35
56.2
11.74
36.45
41.30
42.84
50.26
55.61
57.44
59.00
60.88
66.38
24.8
45.48
48.31
50.24
51.49
55.37
58.42
59.16
64.34
67.68
70.78
72.8
0.44
0.53
0.58
0.79
1.13
1.17
2.36
2.43
2.56
/
QP, quaternized pectin; HA, hyaluronic acid; RH, relative humidity.
in Table 2. Obviously, the moisture absorption and retention abil-
ities of QP depend on the DS value. With an increase in DS, the
moisture absorption and retention abilities of QP increased accord-
ingly. And all QPs showed better Ra and Rh than pectin, indicating
that the introduction of –N+(CH3)3 group is a convenient and effec-
tive method to enhance both moisture absorption and retention
abilities for pectin. The group has obvious charges, which are sim-
ilar to those of HA, and the increased moisture absorption and
retention abilities may be largely due to the charged group (Yang
et al., 2010). QPs with the DS value from 0.79 to 2.56 showed bet-
ter Ra than that of HA. And when the DS value is more than 2.0,
QP showed good Rh. Therefore, QP has potential to be used as a
moisture absorption and retention ingredient.
Fig. 1. FT-IR spectra of the native citrus pectin and the quaternized pectin (QP).
difference between native pectin and QPs positioned at 1480 cm−1
,
which corresponded to the methyl groups of ammonium. More-
over, the peak of QCs positioned at 1415 cm−1 was referenced as
the C–N stretching vibration (Pal, Mal, & Singh, 2005). FT-IR spec-
tra have given an evidence of the introduction of the quaternary
ammonium salt group on the pectin backbone.
The 13C NMR spectrum of QP in D2O at 25 ◦C is shown in Fig. 2.
The peaks were assigned according to the native citrus pectin
(Synytsya et al., 2004; Zhao et al., 2006) and the quaternary ammo-
nium derivatives of konjac glucomannan (Yu et al., 2007), cellulose
(Song, Sun, & Zhang, 2008), and starch (Pi-xin et al., 2009). The
peak at lower field (175.5 ppm) was assigned to C6 of COOH group.
The chemical shift at 99.0 ppm attributed to C1. Unsubstituted
and substituted C2 and C3 appeared between 68.2 and 68.8 ppm.
The peak at 77.9 ppm corresponded to C4, while C5 appeared at
71.4 ppm which was overlapped by the chemical shift of C7. The
signals at 63.7 and 66.5 ppm were believed to belong to the car-
bons of C8 and C9, respectively. A relatively sharp high intensity
signal at 54.2 ppm was attributed to a quaternary ammonium N-
methyl carbon nucleus (C10). Therefore, the results of 13C NMR
spectrum further proved the successful synthesis of quaternized
pectin.
3.4. Antimicrobial activity
The capabilities of pectin, QP-3, and QP-7 in inhibiting the
growth of the tested microbes are listed in Table 3. Obviously,
pectin and QPs are both devoid of antifungal activity, whereas they
showed inhibitory effect against the three bacteria investigated.
As shown in Fig. 3, the higher zone of inhibition was recorded
at QP-7 than QP-3 for S. aureus and E. coli. However, it is oppo-
site for B. subtilis. This result implies that high DS value causes
gain of antimicrobial activity for S. aureus and E. coli. While, with
an increase in DS value, the antimicrobial activity for B. subtilis is
decreased.
Although pectin showed inhibitory effect against the three bac-
teria, the water-solubility and pH value that is far from the human
physiological pH value limited its application. The antimicrobial
mechanism of the QP on test bacteria and fungus is probably com-
plex and still not resolved. In the case of antibacterial mechanism
against S. aureus and E. coli, it is generally believed that the pos-
itive charge of the quaternized group resulted in a polycationic
structure absorbed onto the negatively charged cell surface of bac-
teria led to great alteration of the structure of outer membrane
3.3. Moisture absorption and retention properties
The moisture absorption and retention properties of QPs which
were examined and compared with hyaluronic acid (HA) are shown
Table 3
Antibacterial and antifungal activity of natural pectin and quaternized pectin.
Microorganisms
Antibacterial and antifungal activity
QP-3
QP-7
Pectin
Control
Bacteria
S. aureus
E. coli
B. subtilis
Fungi
+
+
+
+
+
+
+
+
+
−
−
−
A. niger
Mucor sp.
Rhizopus sp.
−
−
−
−
−
−
−
−
−
−
−
−
Fig. 2. 13C NMR spectrum of quaternized pectin.
‘−’: no inhibition; ‘+’: inhibition.

L. Fan et al. / Carbohydrate Polymers 88 (2012) 707–712
711
Fig. 3. Effect of natural and quaternized pectin on growth of three bacteria strains.
which caused release of major proportion of proteinaceous mate-
rial from the cell. Cytoplasmic membrane is disrupted followed by
the binding of quaternized pectin, and then releasing cytoplasmic
constituents, such as K+ ions, DNA and RNA, ultimately leading
to bacterial death (Jiang, Wang, Yu, & Chen, 2005; Sajomsang,
Tantayanon, Tangpasuthadol, & Daly (2009b); Sun, Du, Fan, Chen, &
Yang, 2006). Thus, high DS value causes gain of antimicrobial activ-
ity for S. aureus and E. coli. Although B. subtilis and S. aureus are
both Gram-positive bacteria, the effect on the antimicrobial activ-
ity of the QPs against them is different. The facts indicated that the
different bacterium which is used has a great effect on the results
of antimicrobial activity of the QPs. This is highly related to the
structure and physiological activity of various bacteria. In addition,
there maybe not only the effect of quaternized group of QPs but
also another antimicrobial mechanism against B. subtilis, which is
not totally the same as S. aureus and E. coli. However, most works
(Dizman, Elasri, & Mathias, 2006; Lei, Yang, Jia, & Zhang, 2010) of
antimicrobial activity were aimed at S. aureus and E. coli. The study
of antimicrobial mechanism against B. subtilis was less reported.
Thus, further research is still needed. As regards antifungal mech-
anism, it might be due to direct interference of nutrilite synthesis
or blocking of fissiparism. Furthermore, major component of cell
walls of bacteria is peptidoglycan and teichoicacid or lipoprotein
and lipopolysaccharide, whereas major constituent of cell walls of
fungi is chitin and glucan. The difference of inhibiting effect of QP
on growth of bacteria and fungi tested may be due to the difference
of structure and component of cell (Yu et al., 2007). More work is
needed to confirm this hypothesis.
Water-soluble QPs with DS value of 0.44–2.56 could be obtained
by adjusting the molar ratio of CHPTAC to pectin from 4 to 7, the
molar ratio of NaOH to CHPTAC from 1.0 to 1.2, and the reaction
temperature from 40 to 70 ◦C. The QP was evaluated in terms of
moisture absorption, moisture retention, and antibacterial activity
test. The results indicated that Ra and Rh of QP are closely related to
the DS value. With the DS value increasing, the Ra and Rh increased.
And all QPs showed better Ra and Rh than pectin. Furthermore, QPs
with the DS value from 0.79 to 2.56 showed better Ra than that
of HA. When the DS value is more than 2.0, QP showed good Rh.
In vitro antimicrobial activity assessments exhibited QP showed
pronounced inhibitory effect against the three bacteria (S. aureus,
E. coli, and B. subtilis). Therefore, the QP is promising derivative of
pectin which applied in pharmaceutical, packaging, preservatives,
and cosmetic fields.
Acknowledgments
The work was supported by the National Natural Science
Foundation of China (Foundation No. 51173143, No. 50973088),
Technology Innovation Foundation for SMEs of Ministry of Science
of China (Foundation No. 11C26214202642, No. 11C26214212743),
Supply and Demand Docking Program of Science and Technol-
ogy of Wuhan (Foundation No. 201150124013), Doctor Subject
Foundation of the Ministry of Education of China (Foundation
No. 200804971074), Wuhan Science and Technology Develop-
ment (Foundation No. 201060623262), Key Research Project
of Health Department of Hubei Province (Foundation No.
JX4B54), Wuhan Academic Leaders Program (Foundation No.
200851430480), and Independent Innovation Research Founda-
tion of Wuhan University of Technology (Foundation No. 2010-IV
-070).
4. Conclusions
Cationic derivatives of pectin were successfully synthesized
by reacting pectin with CHPTAC in NaOH aqueous solutions.

712
L. Fan et al. / Carbohydrate Polymers 88 (2012) 707–712
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