Food Hydrocolloids 33 (2013) 58e65

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Highly acetylated pectin from cacao pod husks (Theobroma cacao L.)
forms gel
Lcia C. Vriesmann, Carmen L.O. Petkowicz*
Universidade Federal do Paran, Departamento de Bioqumica e Biologia Molecular, CP 19046, 81531-980 Curitiba-PR, Brazil

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

Article history: A pectin (OP) obtained from cacao pod husk with a high acetyl content, which is a structural feature that
Received 27 September 2012 could disturb the pectins gel formation, was able to form gels at low pH and a high sucrose content. Pectin
Accepted 20 February 2013 gels (1.32% GalA equivalent, w/w) were prepared at pH 2.5e3.3 in the presence of 60% sucrose (w/w).
Rheological analyses were performed to determine the optimal pH for further studies. Next, the OP
Keywords: samples were prepared at pH 2.7 in concentrations ranging from 0.33 to 1.98% GalA (w/w) with 60% sucrose
Pectin
(w/w) and subjected to rheological analysis. Dynamic oscillatory experiments at 25
C indicated the
High acetyl
presence of gels for all of the analysed concentrations. Measurements of the elastic (G0 ) and viscous (G00 )
Cacao pod husks
Theobroma cacao
moduli at 25
C also indicated that increasing the pectin concentration resulted in stronger gels. Rotational
Gel experiments revealed a shear-thinning behaviour in which the apparent viscosities of the samples
Rheology increased as the concentration increased. Although the OP had a high degree of acetylation, this pectin was
able to form gels, which suggests its potential for use as a gelling and thickening additive.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Cacao pod husks (Theobroma cacao L.), which are considered an
undesirable waste product of the cocoa industry, have been
investigated as a new source of pectins. Pectins have been isolated
from cacao pod husks by aqueous (Vriesmann, Amboni, &
Petkowicz, 2011) and acid extractions (Vriesmann, Telo, &
Petkowicz, 2011). Experimental design and statistical approaches
using response surface methodology were employed to optimise
the nitric-acid-mediated pectins extraction from the cacao pod
husks to improve the pectin yield and uronic acid content
(Vriesmann, Telo et al., 2011). The optimised pectin (OP) was
composed of a homogalacturonan (degree of esterication, DE
56.6  1.4%) with rhamnogalacturonan insertions possessing side
chains primarily containing galactose. The polymers were highly
acetylated (degree of acetylation, DA 17.1  0.5%) indicating a
chemical pattern that differs from that of commercial pectins.
In addition to the temperature, pH, pectin concentration and
presence of co-solutes and ions, pectin gelation is affected by
several parameters related to the pectin structure, including the
molar mass, charge distribution along the backbone, presence of
side chains, DA and DE. DE is a particularly important parameter;
low-methoxyl (LM pectins: DE < 50%) and high-methoxyl (HM
* Corresponding author. Tel.: 55 41 3361 1661; fax: 55 41 3266 2042.
E-mail address: clop@ufpr.br (C.L.O. Petkowicz).
pectins: DE > 50%) pectins have distinct gel formation mechanisms
(Axelos & Thibault, 1991; ONeill, Darvill, & Albersheim, 2001; Rolin,
1993).
After heating, the HM pectins gel at a low pH (frequently
pH < 3.5) in the presence of high co-solute concentrations (typi-
cally 60e65% sucrose), whereas the gelation of LM pectins occurs in
the presence of calcium ions (ONeill et al., 2001; Oakenfull, 1991;
Rolin, 1993; Thakur, Singh, & Handa, 1997). In the rst case, the
gelling mechanism primarily involves hydrogen bonding combined
with hydrophobic interactions (Oakenfull, 1991), while ionic in-
teractions play an important role in the gelation of LM pectins
(Axelos & Thibault, 1991).
HM pectins are very important in the food industry because
many processed fruit products are acidic and contain a high sucrose
content (e.g., jams and jellies) (ONeill et al., 2001). Therefore,
several studies have investigated the gelling properties of low-pH,
high-sugar HM pectin gels by varying the sucrose concentration
(El-Nawawi & Heikel, 1997; Evageliou, Richardson, & Morris, 2000;
Sharma, Liptay, & Le Maguer, 1998; Vriesmann, Silveira, &
Petkowicz, 2010), pH (El-Nawawi & Heikel, 1997; Evageliou et al.,
2000; Sharma et al., 1998), pectin concentration (Ptitchkina,
Danilova, Doxastakis, Kasapis, & Morris, 1994; Sharma et al., 1998;
Vriesmann et al., 2010) and the type of co-solute employed
(Evageliou et al., 2000; Tsoga, Richardson, & Morris, 2004).
However, few studies have investigated the gelling properties of
HM pectins with high acetyl content (high DA), which is known to

0268-005X/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2013.02.010
 L.C. Vriesmann, C.L.O. Petkowicz / Food Hydrocolloids 33 (2013) 58e65
modify pectin solubility and gelation properties (Voragen, Pilnik,
Thibault, Axelos, & Renard, 1995; Willats, McCartney, Mackie, &
Knox, 2001). A content value of 4% acetyl was considered the crit-
ical limit for gel formation for previously studied systems (Iglesias
& Lozano, 2004; Pilnik & Voragen, 1992).
Therefore, a high degree of acetylation appears to be important
for the gelling capacity of pectin. This factor has been a limitation in
the use of sugar beet pectin as a gelling additive (Mc Cready, 1966;
Pippen, McCready, & Owens, 1950; Rolin, 1993). Many studies have
investigated the chemical or enzymatic removal of acetyl groups
from sugar beet pectins and various extraction procedures to
improve their gelling properties (Faulds & Williamson, 1990;
Matthew, Howson, Keenan, & Belton, 1990; Oosterveld, Beldman,
Searle-van Leeuwen, & Voragen, 2000; Pippen et al., 1950;
Turquois, Rinaudo, Taravel, & Heyraud, 1999, 2000; Williamson
et al., 1990).
Because the characteristics of pectin can determine its applica-
tions, e.g., as a gelling agent, the analysis of its physicochemical
properties is essential for its optimal use. Therefore, we report the
rheological properties of the highly acetylated pectin (OP) previ-
ously obtained from cacao pod husk under optimised conditions.
2. Materials and methods
2.1. Pectin sample
OP is a highly acetylated HM pectin (DE 56.6  1.4%; DA
17.1  0.5%). The OP used in this study was obtained by the opti-
mised nitric-acid-mediated extraction from cacao pod husk and
previously characterised (Vriesmann, Telo et al., 2011).
2.2. Preparation of pectin for rheological analysis
First, OP was solubilised in deionised water at 5% (w/w) with
agitation over 16 h at 25
C. The solution was allowed to rest for 4 h
prior to the rheological experiments.
The OP samples were then processed under the appropriate
conditions to obtain HM pectin gels. The OP solutions were pre-
pared at 1.32% (as galacturonic acid equivalents in the fraction, w/
w) with 60% sucrose (w/w) at various acidic pH values (2.5e3.3) to
determine the appropriate pH for future experiments. Weighed
amounts of the sample were hydrated in aqueous citric acid with
agitation over 16 h at 25
C. After solubilisation, the mixtures were
heated and sucrose was added in the appropriate proportion. The
mixtures were heated in a boiling bath with continuous stirring for
15 min and cooled to room temperature. Next, their pH was
measured, and the samples were stored under refrigeration for 16 h
and analysed at 25
C. The OP solutions were also prepared with
different GalA quantities (0.33e1.98%) at pH 2.7 and processed as
described above.
2.3. Rheological analyses
The rheological measurements were performed using a Rheo-
Stress1 rheometer (Haake GmbH, Germany) equipped with a Haake
DC30 circulating bath at 25
C using a C60/2
spindle (cone and
plate geometry) with a 1 mm gap between the cone and the plate.
The mechanical responses of the materials were determined by
subjecting them to a frequency sweep from 0.01 to 10 Hz at 25
C
under strain in the viscoelastic-linear region (g < 5%; obtained by
strain sweep tests at 1 Hz) where the network structure was pre-
served. The ow behaviour of the samples was evaluated using
continuous shear rate curves in the CR mode (controlled shear
rate). The sensor was programmed to increase the shear rate from
0 to 300 s1 over 360 s at 25
C. The data from the rheological
59
measurements were evaluated using the HAAKE RheoWin 4 soft-
ware. All of the experiments were repeated at least twice. The re-
ported results are the averages of these multiple trials and
exhibited excellent reproducibility.
3. Results and discussion
Rheological analysis was employed to characterise the OP in
solution and as a gel and will provide important information for
future studies on their properties and applications.
3.1. OP solution
The viscosity curve of an aqueous solution containing 5% OP (w/
w) is shown in Fig. 1. OP exhibited a low initial viscosity (<1 Pa s),
which is similar to the 5% pectin solutions chemically extracted
from apple pomace (Min et al., 2011). A non-Newtonian shear-
thinning behaviour was observed for OP, with decreases in the
viscosity with increases in shear rate, as described for other pectin
solutions (Evageliou, Ptitchkina, & Morris, 2005; Hwang & Kokini,
1992; Min et al., 2011; Sengkhamparn et al., 2010; Singthong,
Ningsanond, Cui, & Goff, 2005).
The viscoelastic properties of the 5% OP were evaluated using
frequency sweeps (Fig. 2). G0 refers to the elastic (solid) behaviour,
and G00 refers to the viscous (liquid) behaviour. The 5% OP solution
exhibited G00 > G0 in the analysed frequency range, which indicated
the predominance of the liquid behaviour. The value of both moduli
increased proportionally with the frequency.
The predominance of the liquid behaviour was also observed for
the 5% solutions of the pectins from apple pomace (58 and 69% DE;
Min et al., 2011). However, the apple pomace samples exhibited
higher elastic properties at higher frequency oscillations.
3.2. OP gels
The OP is composed of highly acetylated HM pectins and is ex-
pected to form gels at low pH in the presence of a high content of
low-molecular-weight co-solutes (i.e., sucrose). Acidic pH (<3.5)
leads to the protonation of the carboxylate groups of the unes-
teried galacturonate units, reducing the negative charge along the
pectin chain and minimising the electrostatic chain repulsion. In
addition, a high sucrose concentration decreases the water activity,
Fig. 1. Inuence of shear rate on the apparent viscosity of the 5% OP aqueous solution
at 25
C.
60 L.C. Vriesmann, C.L.O. Petkowicz / Food Hydrocolloids 33 (2013) 58e65
Fig. 2. Frequency sweep of the 5% OP aqueous solution at 25
C.
competing with the pectin for hydrogen bonding to water and
allowing more interactions between the distinct pectin chains
compared to the interactions of the chains with water. Therefore,
the formation of junction zones is facilitated through the stabilising
hydrophobic attractions between the methoxyl groups and the
hydrogen bonding between undissociated carboxyl and secondary
alcohol groups (Thakur et al., 1997; Voragen et al., 1995;
Walkinshaw & Arnott, 1981).
In this work, the rheological properties of the OP prepared with
a conventional procedure for preparing the gels of HM pectins (i.e.,
low pH and high sucrose content) were evaluated. Initially, the
mixtures were prepared with a xed sucrose (60%, w/w) and pectin
(1.32% GalA, w/w) content at a pH ranging from 2.5 to 3.3.
3.2.1. Effect of pH on OP rheological behaviour
The viscosity curves of the OP samples prepared under low-pH,
high-sucrose conditions are shown in Fig. 3. The OP samples
exhibited a non-Newtonian shear-thinning behaviour in which the
Fig. 3. Inuence of the shear rate on the apparent viscosity of the OP at 1.32% GalA,
60% sucrose (w/w) at 25
C and various pH values.
viscosities decreased as the shear rate increased. The apparent
viscosity of all of the samples was more than 1000-fold higher than
that of the OP solution at 5%. The highest apparent viscosity was
observed for the sample prepared at pH 2.7 (Fig. 4).
According to Rolin and de Vries (1990), pH 3.1 is typical for HM
pectin gelation. A very low pH is critical for HM pectin gelation, and
a gel will not form above pH 3.5 (Rolin & de Vries, 1990). Evageliou
et al. (2000) observed that the gel strength increased with
decreasing pH. Sharma et al. (1998), who studied the gelation of
HM pectins obtained from tomato pulp with different sucrose
contents (50e70%) and pH values (2.0e5.0), observed that gels
were formed with 60% sucrose at pH < 3.0.
Fig. 5A shows the elastic moduli (G0 ) of the OP samples prepared
at different pH values, and Fig. 5B shows the respective viscous
moduli (G00 ). In contrast to the 5% OP solution, all of the mixtures
exhibited an elastic modulus that was higher than the viscous
modulus (G0 > G00 ) for the studied oscillation frequencies. In addi-
tion, G0 was found to be less dependent on the frequency than G00 ,
which is characteristic of gel-like behaviour. At lower frequencies,
G0 is more than three times higher than G00 .
A high concentration of acetyl groups has been reported to
disturb pectin gelation (Mc Cready, 1966; Pilnik & Voragen, 1992;
Voragen et al., 1995; Willats et al., 2001). Williamson et al. (1990)
proposed that the presence of acetyl groups inhibits the gel for-
mation of both HM and LM pectins by steric hindrance of the chain
association. According to Sengkhamparn et al. (2010), acetylation
plays a signicant role in increasing the viscosity of okra LM and
HM pectin solutions by promoting hydrophobic associations.
However, the gelation of these pectins was not evaluated. In this
work, the OP displayed gel-like behaviour despite having a high
acetyl content.
When the gels prepared at different pH values are compared,
the mixture at pH 2.7 formed the gel with the highest G0 values and
the greatest difference between the moduli, while the mixture
prepared at pH 3.3 displayed the opposite behaviour. The mixtures
with a pH of 2.5e2.6 and 2.8-3.0 exhibited similar behaviour. Fig. 6
Fig. 4. pH dependence of the apparent viscosity at 0.003 s1 of the OP at 1.32% GalA
and 60% sucrose (25
C).
 L.C. Vriesmann, C.L.O. Petkowicz / Food Hydrocolloids 33 (2013) 58e65 61

Fig. 6. pH dependence of G0 and G00 at a frequency of 0.1 Hz (25
C) for the OP prepared
with 1.32% GalA and 60% sucrose (w/w).

reports cited above, as the pectin in OP fraction has a DE of

56.6  1.4% and the gel with highest elastic behaviour was obtained
at pH 2.7, followed by that obtained at pH 2.6 (Fig. 6).
After ANOVA and t-test analysis (p < 0.05), a pH of 2.7 was
selected to prepare the OP gels with different pectin concentrations.

3.2.2. Effect of pectin concentration on the OP rheological behaviour

The ow behaviour of OP samples at different concentrations
(0.33e0.98% GalA, w/w) at pH 2.7 was evaluated at 25
C (Fig. 7). In
the low-shear-rate region, the samples exhibited a Newtonian
plateau followed by a decrease in viscosity with an increase in shear
rate. This result is characteristic of a shear-thinning ow behaviour
under the examined conditions, in which the apparent viscosities of
the samples increased as the concentration increased. The largest
difference in the apparent viscosity was observed at lower
concentrations.

Fig. 5. Frequency sweeps at 25
C of the OP with 1.32% GalA (w/w) and 60% sucrose
(w/w) at different pH values: (A) Elastic modulus, G0 , and (B) Viscous modulus, G00 .

shows the predominance of G0 over G00 for OP mixtures at all of the

studied pH values (0.1 Hz). A maximum G0 value is reached at pH
2.7. For G00 , only a slight difference is observed for the different pH
values. The OP exhibited the worst gelling ability at pH 3.3.
Evageliou et al. (2000) evaluated the rheological properties of an
HM pectin (70.3% DE, 65% sucrose) at pH values ranging from 3.0 to
4.7. They observed that both G0 and G00 decreased sharply when the
pH was increased above w3.4, which is the pKa of the galacturonate
units in the polymer.
Pectins with 65.9e73.7% DE have been reported to exhibit little
difference in gelation at pH w3.0 and a loss of the ability to form
gels at higher pH values (Smit & Bryant, 1968). According to the
literature, at lower DEs, stronger gels were obtained when the pH
value decreased (El-Nawawi & Heikel, 1997; Smit & Bryant, 1968).
For a DE of 43.7%, Smit and Bryant (1968) found the best gel
forming ability at pH 2.2. For pectins with DE of 51% the jelly grade
was higher at pH 2.5e2.3 (El-Nawawi & Heikel, 1997). Pectins with
61% DE exhibited a higher jelly grade at pH values of 2.7 and 2.3,
and a slightly lower jelly grade was observed at pH 2.5 (El-Nawawi Fig. 7. Inuence of the shear rate on the apparent viscosity of the OP prepared at
& Heikel, 1997). Our results appear to be in agreement with the different GalA concentrations at pH 2.7 and 60% sucrose (w/w) (25
C).
62 L.C. Vriesmann, C.L.O. Petkowicz / Food Hydrocolloids 33 (2013) 58e65

At low shear rates, the sample with the highest concentration

(1.98% GalA) exhibited an apparent viscosity value over one hun-
dred times higher than that with the lowest concentration (0.33%
GalA) under the same experimental conditions. Fig. 8 shows the
variation in the apparent viscosity (Pa s) with pectin concentration
at 0.003 s1. The magnitude of the viscosity increased as the GalA
concentration increased. For this shear rate, the 1.98% GalA sample
exhibited an apparent viscosity of 6228 Pa s, while the 0.33% GalA
sample exhibited a value of 42 Pa s. Therefore, a 6-fold difference in
concentration generated a 148-fold difference in the apparent
viscosity for this shear rate, which indicates the presence of more
entanglements at a higher concentration.
Fig. 9A and B show the variation in elastic (G0 ) and viscous
moduli (G00 ) as a function of frequency at 25
C for OP with different
concentrations prepared at pH 2.7 with 60% (w/w) sucrose.
Although acetyl groups are believed to hamper gelation, the fre-
quency sweeps of the OP (degree of acetylation of 17.1  0.5%)
demonstrated gel behaviour at all of the concentrations analysed
(Fig. 9). G0 was higher than G00 over the entire frequency range and
less dependent on the frequency than G00 , which indicates the
predominance of a solid response similar to that present in elastic
networks (Endress, Dschl-Volle, & Dengler, 1996; Morris, 2008).
Polymer gels and networks can be divided into three main classes:
covalently cross-linked materials, entanglement networks and
physical gels. For entanglement networks at low frequency, G0 N f2;
in contrast, G0 is frequency insensitive for cross-linked networks,
with G0 N f0 (Kavanagh & Ross-Murphy, 1998). In the present work,
G0 N fp where p ranges from 0.1448 to 0.1917 (R2 > 0.98, data not
shown). According to Kavanagh and Ross-Murphy (1998), pectin
gels are classied as physical gels.
Pippen et al. (1950) prepared partially acetylated pectins from
commercial citrus pectin. They observed that the presence of acetyl
inhibited gel formation with acid/sugar in samples containing 5%
acetyl, while samples with 3.5e4% acetyl formed weak gels upon
resting for from one to four weeks. They also veried that the
percentage of acetyl required to prevent gelation was not
signicantly inuenced by the DE of the pectins. In addition, the
removal of the acetyl groups restored the gelling ability when the
percentage of acetyl dropped to 2.4 or less. The same authors
studied the inuence of removing the acetyl groups from the
sugar beet pectin and found that gelling pectin was produced

Fig. 9. Frequency sweeps at 25
C of the OP at 0.33e1.98% GalA (w/w) with 60% su-
crose (w/w): (A) Elastic modulus, G0 , and (B) Viscous modulus, G00 .

when the percentage of acetyl decreased below a value of 1

Fig. 8. Dependence of the apparent viscosity at 0.003 s1 on the concentration of the
OP samples.
(Pippen et al., 1950). However, the conditions used to prepare the
gels were not described by the authors.
In contrast to the results reported by Pippen et al. (1950), the OP
fraction containing a DA of 17.1  0.5% exhibited gelling ability even
at the lowest concentration analysed (0.33% GalA w/w). It should be
noted that the total inhibition of pectin gelation occurs at
DA  12.5% (Rolin, 1993). However, gel formation was observed for
the OP fraction pectins, which have a higher DA, suggesting that
other molecular factors must assist in the gelation or that the high
acetyl content only modies the gel characteristics of the sample.
Not only the DA but also the distribution of the groups can play
an important role in the gelling properties of the pectins. Depending
on the plant source, acetyl groups may be present in HG or RG-I
regions, attached to galacturonic acid units at O-2 and/or O-3
(Bonnin et al., 2008; Willats et al., 2001). However, in okra pectins,
 L.C. Vriesmann, C.L.O. Petkowicz / Food Hydrocolloids 33 (2013) 58e65
the acetyl groups were not only attached to galacturonic acid units
in HG and RG-I, but also to rhamnose and/or galactose units in side
chains of RG-I (Sengkhamparn, Verhoef, Schols, Sajjaanantakul, &
Voragen, 2009). It is possible that in OP, acetylated units of gal-
acturonic acid might be arranged in blocks. Also, the acetyl groups
could be mainly attached to neutral sugar units of RG-I. In this way,
long runs of unacetylated material could exist along the HG back-
bone chains and the gel formation could take place by the typical
mechanism for HM pectins (low pH in the presence of a high content
of sucrose).
Furthermore, the results in Fig. 9 indicate that a higher pectin
concentration in the OP gels results in a G0 value with a larger
magnitude (Fig. 9A), which indicates the presence of stronger gels
at higher concentrations, as observed for other pectins (Lfgren,
Walkenstrm, & Hermansson, 2002; Ptitchkina et al., 1994;
Sharma et al., 1998; Vriesmann et al., 2010). A doubling of the
concentration of the OP gels resulted in an approximately 5-fold
increase in G0 and an approximately 4-fold increase in G00 . When
the concentration of the OP samples was increased 6-fold (from
0.33% GalA to 1.98% GalA), G0 increased by at least 70-fold.
Lfgren et al. (2002) reported that HM pectin gels are greatly
inuenced by changes in concentration. In their study, doubling the
concentration from 0.75% to 1.5% increased the G0 by 3-fold and the
G00 by 4-fold. These authors observed the presence of a denser gel
network at increased concentrations. However, they reported that
the nature of the network structure did not change.
Fig. 10 shows the variation in the rigidity (G0 ) of the OP samples
as a function of the pectin concentration measured at 0.1 Hz. The
magnitude of G0 increased as the GalA concentration increased,
indicating an increase in the quantity of elastic junction zones. In
addition, G0 was found to be power-law dependent on the GalA
concentration, G0 N C2.4.
Although the nature of the intermolecular junctions in a
network depends signicantly on the primary structure of the
polymer, this type of plot exhibits a slope of 2 at high concentra-
tions, which is a common pattern for most gelling polysaccharides
and indicates a (concentration)2 dependence of the gel strength
(Morris, 2008). According to Morris (2008), the value of the slope
increases when the polymer concentration decreases towards the
minimum critical gelling concentration (c*), which is the lowest
concentration at which gel formation is possible.
Fig. 10. G0 as a function of the GalA concentration of the OP gels at different fre-
quencies (25
C).
63
The highly acetylated pectins in OP fraction demonstrated
weaker gel properties than other HM pectin samples prepared with
an acidic pH and a high sucrose content (Lfgren et al., 2002;
Ptitchkina et al., 1994; Vriesmann et al., 2010).
A high acetyl content is known to negatively affect pectin
gelation (Matthew et al., 1990; Mc Cready, 1966; Oosterveld et al.,
2000; Pilnik & Voragen, 1992; Pippen et al., 1950; Rombouts &
Thibault, 1986; Voragen et al., 1995; Willats et al., 2001) and can
inuence the gel characteristics of OP samples.
Other features related to the pectin structure can affect the
gelling properties, such as the presence of side chains, the DE, the
charge distribution along the backbone and the degree of poly-
merisation (Axelos & Thibault, 1991; Rolin, 1993).
The gel strength of the pectins is positively related to the molar
mass of the molecule. If this molar mass is very low, the pectin will
not form a gel under any conditions, whereas increasing the pectin
molar mass promotes the formation of strong gels (ODonoghue &
Somereld, 2008; Pagn & Ibarz, 1999). Low molar mass
(<5.104 g/mol) has been used to explain the poor gelling properties
of sugar beet pectins (Michel, Thibault, Mercier, Heitz, & Pouillaude,
1985; Phatak, Chang, & Brown, 1988; Pilnik & Voragen, 1992;
Thibault, 1986). However, this explanation does not hold for OP,
which has a high molar mass (3.498  105 g/mol; Vriesmann,
Telo et al., 2011).
The presence of a-1,2 rhamnopyranose units (which possess
neutral sugar side chains at O-4) interspersing the a-1,4 poly-
galacturonate backbone was suggested introducing kinks that limit
the junction zone size or interrupt it in pectin gels (Rees & Wight,
1971; Walkinshaw & Arnott, 1981). Rhamnose is localized in re-
gions of GalAeRha disaccharide repeating units (rhamnogalactur-
onan segments) along the HG regions of pectins. Single rhamnose
units interspersing HG regions are scarce (Nakamura, Furuta,
Maeda, Nagamatsu, & Yoshimoto, 2000; Voragen, Coenen,
Verhoef, & Schols, 2009; Yapo, 2011; Yapo, Lerouge, Thibault, &
Ralet, 2007; Zhan, Janssen, & Mort, 1998). The presence of 10%
Rha in the OP (Vriesmann, Telo et al., 2011) could also inuence
gel formation.
The presence, length and distribution of side branches in pectins
can interfere with their gelation because junction zone formation is
only possible via smooth regions in the molecule (Hwang, Pyun, &
Kokini, 1993; Keenan, Belton, Matthew, & Howson, 1985; Matthew
et al., 1990; Schmelter, Wientjes, Vreeker, & Klaffke, 2002; Thibault,
Guillon, & Rombouts, 1991; Thibault & Ralet, 2008). Schmelter et al.
(2002) observed that pectins with shorter side chains exhibited
better gelation properties than those with longer side branches.
However, the dimension or length of the side branches must be
sufcient to provide additional interchain entanglements in the
polymer network compared to those of linear polymers with
similar molecular weight because the backbone of these chains is
shorter than that of linear polymers (Schmelter et al., 2002). It has
been reported that neutral sugar side chains could be directly
involved in the gelation mechanism of HM pumpkin pectin
(Ptitchkina et al., 1994). However, the long backbone in citric-acid-
extracted squash pectin may have counteracted the interference of
the side chains in its gelation in low-pH, high-sugar solutions
(ODonoghue & Somereld, 2008). Neutral sugar side chains are
present in OP (Vriesmann, Telo et al., 2011), but their length and
distribution are not yet known.
On the other hand, the presence of rhamnose and neutral sugar-
branched regions along with the HG regions in pectins from OP
fraction may also prevent undesirable phenomena in the gels.
Thibault and Ralet (2008) proposed that these components are
junction-zone-terminating structural elements that prevent
turbidity, precipitation or syneresis in acidic gels with 55e65%
sugar contents.
64 L.C. Vriesmann, C.L.O. Petkowicz / Food Hydrocolloids 33 (2013) 58e65
Furthermore, the DE and the distribution of methyl-esteried
GalA units along the pectin chain (block-wise or random) are pri-
marily responsible for the gelling behaviour of pectins, which
changes their aggregation and network properties (Powell, Morris,
Gidley, & Rees, 1982; Strm et al., 2007; Thibault & Rinaudo, 1986;
Willats et al., 2001).
Neidhart, Hannak, and Gierschner (2003) veried that distinct
intermolecular arrangements of ester groups strongly affected the
gelation properties of pectins. In particular, there were clear dif-
ferences in molecules with similar uronic acid and esterication
content (ODonoghue & Somereld, 2008). For LM pectins, a more
block-wise distribution led to gel-like behaviour at higher DE and
lower calcium or pectin concentration compared to pectins with a
random (Fraeye et al., 2009). However, investigations of the
methyl-esteried GalA arrangement on the rheological properties
of low-pH, high-sugar HM pectin gels are scarce.
The formation of junction zones in HM pectin gels occurs in the
smooth regions and is assisted through hydrophobic interactions
between the methoxyl groups and hydrogen bonds between un-
dissociated carboxyl and secondary alcohol groups at acidic pH and
high sucrose content (Thakur et al., 1997; Voragen et al., 1995;
Walkinshaw & Arnott, 1981).
Apart to the presence of acetyl groups, the occurrence of
methyl-esteried GalA units in short blocks along the HG backbone
of OP could result in weaker gels than those described for other HM
pectins (Lfgren et al., 2002; Ptitchkina et al., 1994; Vriesmann
et al., 2010).
Prior to this study, native pectins rich in acetyl and neutral
sugars and/or having low molecular mass, such as those from
chicory roots (Robert, Devillers, Wathelet, Van Herck, & Paquot,
2006) and sugar beets (Michel et al., 1985; Pippen et al., 1950;
Yapo, Robert, Etienne, Wathelet, & Paquot, 2007), had been found to
exhibit decient gelling ability (Yapo, 2009). For the OP fraction,
obtained from cacao pod husks, the gel formation was not impeded
under the conditions employed in the present work despite the
high DA of their pectins.
4. Conclusions
Although the OP from cacao pod husks contains a high acetyl
content (DA 17.1  0.5%), which is known to alter the solubility and
gelation properties of the pectins, the obtained results indicated
that this pectin was able to form gel networks in low-pH, high-
sugar solutions.
For a concentration of 1.32% GalA, the best gel properties were
observed using 60% sucrose and a pH of 2.7. When the pectin
concentration was varied (0.33e1.98% GalA), the samples exhibi-
ted gel properties with a solid-like response (G0 > G00 ), even at
lower concentrations. The viscosities increased as the concentra-
tion increased, and the samples exhibited shear-thinning
behaviour.
The obtained results show that acetylation did not impede the
gelation of the pectins from OP fraction but might interfere with the
nal gel properties. Although a more detailed study is necessary,
we can suggest the use of this pectin, which was obtained from
cacao pod husks, as a gelling, stabilising or thickening additive,
which can contribute to an improved use of the primary compo-
nent of cocoa industry waste.
Acknowledgements
The authors wish to thank CEPLAC/CEPEC for supplying the
cacao pod husks used to obtain the OP fraction and CNPq for
nancial support.
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