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Kastner, H., Einhorn-Stoll, U., & Senge, B. (2012). Structure formation in sugar containing pectin gels –
Influence of Ca2+ on the gelation of low-methoxylated pectin at acidic pH. Food Hydrocolloids, 27(1), 42–
49. https://doi.org/10.1016/j.foodhyd.2011.09.001
H. Kastner, U. Einhorn-Stoll, B. Senge
Structure formation in sugar containing
pectin gels – Influence of Ca2+ on the
gelation of low-methoxylated pectin at
acidic pH
Accepted manuscript (Postprint)Journal article |
1
Structure formation in sugar containing pectin gels - Influence of Ca2+ on the gelation of low-
methoxylated pectin at acidic pH
H. Kastner, U. Einhorn-Stoll, B. Senge
Technische Universitaet Berlin, Department of Food Science and Food Chemistry, Koenigin-Luise-
Strasse 22, D-14195 Berlin, Germany
Abstract
A new method for the examination of the pectin gelation process is presented as a complementation
of the most common determination of the gelling point (cross-over of G´ and G´´) from oscillation
measurements. It is based on the first derivation dG´/dt from oscillation measurements (named as
structuring velocity), and defines an initial as well as a critical structuring temperature. These allow
an exact determination of the start of structure formation and description of the structuring process
also in gels with pre-gelation that showed no clear GP. Moreover, phases and mechanisms of
gelation can be identified and structure developing rates can be calculated.
The application of this method on the gelation of low-methoxylated pectin at pH 3 and 30%
saccharose with different contents of Ca2+ was tested. The results show differences as well as
similarities between the GP and the newly defined structuring parameters that could be partly
explained by varying structuring mechanisms at different Ca-content. The initial structuring process
started probably with ionic interactions (egg-box junction zones and random crosslinks) via Ca-
bridges as well as hydrophobic interactions at temperatures ≥ 60 °C, it was nearly completed
around 40 °C. Hydrophilic interactions (below 50 °C) and inter-dimer aggregations (below 25 °C)
perhaps dominated the gelation during further cooling. In dependence on the Ca-content, two to
three phases could be identified during the structuring process. The properties of the gels after
cooling were tested by oscillation measurements as well as the USA-sag method. With increasing
calcium content, the elastic behaviour of the gels increased but they became also more and more
brittle.
2
1. Introduction
Pectins are typical gelling agents, traditionally applied in jam and jelly, but used also in other food
products such as soft drinks and milk products. The knowledge of the structuring properties and, in
particular, the gelling temperature of the pectins is of essential technological importance. The exact
determination of this sol – gel transition temperature, i.e. the “gel point” at which the material
properties change from more liquid – like to more solid – like, therefore, has been studied for
several decades.
Rheological methods, especially oscillation measurements, are often applied and frequently the
cross-over of G´ and G´´ with tan d = G´´/G´= 1 is defined as gel point (Arenaz & Lozano, 1998,
Gilsenan, Richardson & Morris, 2000, Löfgren, Walkenström & Hermansson, 2002, Lootens, Capel,
Durand, Nicolai, Boulenguer & Langendorff, 2003, Audebrand, Kolb & Axelos, 2006, Stang-Holst,
Kjöniksen, Bu, Sande & Nyström, 2006, Gigli, Garnier & Piazza, 2009, Slavov, Garnier, Crepeau,
Durand, Thibault & Bonnin, 2009). The method has been developed by Winter & Chambon (1986)
initially for chemical gelation. In food products, however, mostly physical gelation via junction zones
occurs. Moreover, the point of intersection partly was found to be a function of frequency (Winter &
Chambon, 1986, Rao, van Buren & Cooley, 1993, Lopes da Silva, 1994, Lopes da Silva, Goncalves
& Rao, 1995, Lopes da Silva & Rao, 2006). Strictly, the cross-over of G´ and G´´ might be defined
as gel point only when it is independent on frequency (Stang Holst et al., 2006). Sometimes it might
be close to but not identical with the real gel point and therefore is named also “apparent gel point”
(Lopes da Silva, 1994, Lopes da Silva, Goncalves & Rao, 1995).
Several attempts have been made to find another method for the gel point definition: researchers
from CP Kelco determined the gelling temperature via conductivity (Böttger, Christensen &
Stapelfeldt, 2008), and Dobies, Kozak & Jurga (2004) used NMR measurements. Oakenfull & Scott
(1984) and O´Brien, Philp & Morris (2009) used relatively simple visual tests. Dahme (1992) and
Neidhart, Hannak & Gierschner (1996, 2003) defined a strong decrease of tan d as an indicator for
the gel formation. Grosso & Rao (1998) and Fu & Rao (2001) studied the structuring kinetic of
pectin gels and defined the structure development rate SDR = dG´/dt in order to describe precisely
the moment, at which the formation of junction zones began. The problem of the gel point
determination is, however, not completely solved, yet.
Citrus and apple pectins, isolated from by-products of the fruit juice industry, are the most common
pectin types (Rolin, 2002). Their gelling properties vary in dependence on material and
environmental factors (Lopes da Silva et al., 2006). Among the material parameters, the number of
methoxylated carboxyl groups (degree of methoxylation DM) and their distribution in the
polygalacturonic acid backbone (degree of blockiness DB) are very important (Fraeye, Doungla,
Duvetter, Moldenaers, van Loey & Hendrickx, 2009, Fraeye, Colle, Vandevenne, Duvetter, van
3
Buggenhout, Moldenaers, van Loey & Hendrickx, 2010a). Materials with DM > 50% are named as
high-methoxylated pectins (HMP) and those with DM < 50% as low-methoxylated (LMP). The typical
DM for commercial use is about 60 to 77% for HMP and about 25 to 40% for LMP (Voragen, Pilnik,
Thibault, Axelos & Renard, 1995).
The pectin gelation processes are rather complex (Cardoso, Coimbra & Lopes da Silva, 2003). The
most important environmental factors are pH, Ca2+ and soluble solids (e.g. sugar). They have
different and partly opposite effects on the gelling process of different pectin types. High-
methoxylated pectins (HMP) form gels in the presence of > 55% soluble solids (mostly about 65%
sugar) and at pH < 3.5 (Rolin, 2002, Thakur, Singh & Handa, 1997, Lopes da Silva, 1995). Their
gelling mechanism is explained as a combination of hydrophobic interactions, favoured by higher
temperature, and hydrogen bonds between undissociated carboxyl groups, dominating at lower
temperature (Oakenfull & Fenwick 1977, Oakenfull & Scott 1984). Low-methoxylated pectins (LMP)
gel in the presence of Ca2+, forming intermolecular ionic junction zones between smooth regions of
neighboured chains (Morris, Powell, Gidley & Rees, 1982, Voragen et. al, 1995, Thakur et al., 1997,
Braccini & Perez, 2001). Several studies investigated the special influence of Ca2+ and / or pH on
the gelling process of LMP in a watery system with no or only small amounts of sugar (Thibault &
Rinaudo, 1986, Garnier, Axelos & Thibault, 1993, Gilsenan et al., 2000, Ralet, Dronnet, Buchholt &
Thibault, 2001, Braccini & Perez, 2001, Lootens et al., 2003, Cardoso et al., 2003, Dobies et al.,
2004, Capel, Nicolai, Durank, Boulenguer & Langendorff, 2005, 2006, Audebrand et al., 2006,
Ström, Ribelles, Lundin, Norton, Morris & Williams, 2007, Fang, Al-Assaf, Phillips, Nishinari, Funami
& Williams, 2008, Cardenas, Goycoolea & Rinaudo, 2008, Fraeye et al., 2009, Ngouemazong,
Tengweh, Fraeye, Duvetter, Cardinaels, van Loey, Moldenaers & Hendrickx, 2011a, Ngouemazong,
Kabuye, Fraeye, Cardinaels, van Loey, Moldenaers & Hendrickx, 2011b). Some authors also tested
the influence of higher sugar content (Grosso & Rao, 1998, Fu & Rao, 2001, Löfgren et al., 2002,
Löfgren, Guillotin & Hermansson, 2006). A current review (Fraeye, Duvetter, Doungla, van Loey &
Hendricks, 2010b) gives a good summery of the role of calcium ions in pectins. LMP gelation is
favoured by higher pH than that of HMP (above the pectin pKa 3.5) because the electrostatic
interactions via Ca-bridges require a certain number of dissociated carboxyl groups (Thakur et al.,
1997, Fraeye et al., 2009, 2010a, b).
In principal, three possible types of junction zones can be formed by LMP: hydrophobic interactions
between methoxyl ester groups, hydrophilic interactions between undissociated carboxyl groups
and / or hydroxyl groups via hydrogen bonds as well as ionic interactions between dissociated
carboxyl groups via Ca-bridges (Figure 1). The latter require a minimum number of 6 to 14
consecutive dissociated carboxyl groups in order to form the typical egg-box structure (Fraye et al.,
2010b). In case the total number of such groups is rather low (below the pKa of pectin), these typical
junction zones can be limited. Instead, Ca2+ could interact with single dissociated carboxyl groups in
4
an undissociated neighbourhood, forming monocomplexes by charge reversal on a single chain or
random (unspecific) crosslinking between separate chains (Siew & Willams, 2005, Fang et al.,
2008). Though these complexes are no typical Ca2+ -junction zones, they additionally reduce the
electrostatic repulsion, possibly create even electrostatic attraction and, in any case, promote a
closer contact of the pectin molecules and support gel structure formation. Similar effects are
described also by Cardoso et al. (2003), Fraeye et al. (2009) and Ngouemazong et al. (2011a).
Figure 1 Structure formation mechanisms in pectin gelation. A hydrogen bonds between undissociated carboxyl groups,
b hydrophobic interactions, c random ionic interactions (crosslinks) between dissociated carboxyl groups.
Ca-bridges at subsequent free dissociated carboxyl groups can form egg-box junction zones as known from
many references (e.g. Braccini et al., 2001).
Electrostatic repulsion between pectin molecules is low at pH < pKa because of a high share of
undissociated carboxyl groups. This favours association and aggregation of pectin chains by
intermolecular hydrogen bonds and additionally stabilises these systems in the absence of calcium
(Gilsenan et al., 2000, Cardoso et al., 2003). Moreover, Cardoso et al. (2001) and Cardenas et al.
(2008) assume that, after initial dimer formation, also inter-dimer interactions and cross-linking of
pectin molecules occur by associations of threefold helices with contributions of hydrophobic
interactions and hydrogen bonds.
In contrast to HMP, high sugar content is not essential for LMP gelation but could support it by
binding water and promoting close contact of neighboured molecules.
5
Altogether, the typical calcium-mediated LMP gelation can be seen as a two-step process: After
initial dimerisation by strong electrostatic interchain associations via calcium ions with contributions
of hydrophobic interactions at high temperature and hydrogen bonds at lower temperature, follows a
subsequent aggregation of dimers that additionally increases gel strength (Lopes da Silva & Rao,
2006, Cardenas et al., 2008).
The aim of the presented paper is (I) to present a new method for the characterisation of the pectin
gel structuring process by using the first derivation of the storage modulus dG´/dt from oscillation
measurements and (II) to apply this method for the investigation of calcium influence on gelation of
LMP at pH 3.0 and in the presence of 30% saccharose.
6
2. Materials and methods
The gel composition and preparation is based on a method that is applied in the pectin industry for
testing the gelling properties by the Ridgelimeter (USA-sag method, IFT Committee, 1959)
according to Cox & Higby (1944). The quantities were slightly modified in order to get the necessary
amount of pectin solution. All experiments were made four times, the control tests without calcium in
duplicate.
2.1. Materials
The pectin was a commercial low-methoxylated non-standardized citrus pectin with 81.5%
galacturonic acid content, DM 30.2% and intrinsic viscosity = 336 cm3/g. Citric acid, tri-sodium
citrate dehydrate and calcium chloride dehydrate were of analytical grade (Sigma-Aldrich),
saccharose was of food quality from a local supermarket.
2.2. Methods
Gel preparation
637.5 g demineralised water, 7.5 ml 54.3% w/v citric acid solution and 15 ml 6% w/v sodium citrate
solution were mixed in a steel pot and 6 g dry pectin powder, mixed with about 40 g saccharose,
was added while stirring. The suspension was heated quickly until boiling, 224 g saccharose was
added in 3 portions and the solution was boiled again. Afterwards, the required amount of 2.205%
w/v calcium chloride dehydrate solution (25 / 31 / 37.5 / 44 / 50 ml, respectively) was added and
while further boiling and stirring the total mass was reduced to 900 g. The whole process should
take no more than about 5 min.
The stochiometric ratio between calcium and carboxyl content R = 2Ca2+/COOH was 0.46 / 0.58 /
0.70 / 0.82 and 0.94, respectively. This means 0.42 / 0.52 / 0.62 / 0.73 and 0.83 mM CaCl2 per 100
g gel. The regular amount of CaCl2 in the standard procedure is 37.5 ml (0.62 mM/100 g gel, R
0.70); variations were made in both directions.
Rheological measurements
The applied rheometer was a Physica MCR 301 (Anton Paar, Germany). Oscillation measurements
(temperature sweep) of storage modulus G´ and loss modulus G´´ were made using a double gap
rotational cylinder CC27/P1 with Peltier cylinder temperature system TEZ 150P. Samples were
transferred onto the pre-heated rheometer (100 °C) and cooled to 10 °C with a cooling rate of 1
K/min. The sample was coated with silicone oil and the cylinder was closed with a special lid in
order to avoid evaporation. Dynamic rheological parameter (G´ and G´´) were recorded during
cooling at a frequency of 1 Hz and a deformation amplitude of g 0.001.
Ridgelimeter (USA-sag method)
7
This method is rather empirical but frequently used for routine tests in the pectin industry, yet. The
hot pectin solution was filled into three special glasses which were stored at 25 °C for 24 h before
measurement. The single gels were removed carefully from the glasses and transferred on a plate.
The percentage of sagging of the gel cone under its own weight within 2 min is measured. From this
value the °SAG can be calculated.
pH
The pH was determined in the gel after Ridgelimeter measurement using a Lab850 pH-meter
(Schott Instruments) and a special penetration electrode.
Examination of structure formation
From the G´ data, the first derivation was calculated and smoothed using Origin 8.1 software. Two
characteristic temperatures were determined from this first derivation as shown in Figure 2. The
initial structuring temperature IST is the temperature at which the value dG´/dt was different from 0
for the first time and the critical structuring temperature CST is the extrapolated temperature of the
first strong increase of dG´/dt.
The average structure developing rates SDRa was calculated from differences of storage moduli
during cooling time for the total gelling process:
G´IST and tIST are parameters at the initial structuring temperature IST and G´end and tend are the final
values at 10 °C.
ISTend
ISTend
att
GG
SDR
-
-
=´´
8
3. Results and discussion
3.1. Structure formation parameters
Rheological tests of the gelling behaviour of pectins sometimes give no clear (apparent) “gel point”
(GP) as a cross-over of G´ and G´´. Either G´ may be higher than G´´ already from the start of the
rheological measurements, or the curves are more or less parallel during a longer cooling period
without a clear intercept. The difficulties of studying pectin gelation in general are discussed by
Lopes da Silva & Rao (2006) and those of LMP by Fraeye et al. (2010a, b). Therefore, an additional
method might be helpful in order to describe the structure formation process.
Figure 2 Evaluation of the first derivation dG´/dt in a gel with 0.62 mM CaCl2. Full curve = dG´/dt, dotted curve = G´;
vertical lines give IST and CST (---) and the start of structuring phases (
٠٠٠٠
); IST = initial structuring
temperature and CST = critical structuring temperature; end = end level at 10 °C. The GP is marked as X on
the G´ curve.
The first derivation dG´/dt was used already for the description of the gelling kinetic of pectins and
calculation of the structure developing rate SDR (Grosso & Rao, 1998, Fu & Rao, 2001, Cardoso et
al., 2003). dG´/dt can be seen as structuring velocity and changes of this velocity are indicators for
the start of structuring process as well as for further alterations (phases) during cooling. After
smoothing the first derivation of the G´ curve the new parameters initial structuring temperature IST
and the critical structuring temperature CRT (Figure 2) were determined. The IST is an indicator for
the start of structure formation and the CRT for a first acceleration in structure formation. These two
temperatures could be found for any pectin we have ever studied, not only in the experiments
described in this paper. Therefore, this method seems to be a good alternative or complement for
the classical “gel point”, defined as cross-over of G´ and G´´.
015 30 45 60 75 90
0
1
2
3
4
dG'/dt (Pa/min)
Time (min)
CST
IST
end
III
III
GP
0
25
50
75
100
Storage modulus (Pa)
100 90 80 70 60 50 40 30 20 10
X
Temperature (°C)
9
3.2. Application of the structure formation parameters temperature on the gelation of LMP
The characteristic temperatures defined above shall be applied for the discussion of the gelation of
LMP at pH about 3 and with 30% saccharose in the presence of different amounts of Ca2+.
Typically, gelation of LMP is dominated initially at high temperature by formation of egg-box junction
zones via calcium-bridges and hydrophobic interactions. During further cooling, the influence of
hydrogen bonds should increase, supported by interchain inter-dimer associations. Random
electrostatic interactions of Ca2+ with single dissociated carboxyl groups of pectin chains (calcium
crosslinking) could promote the structuring process.
In case of the applied pectin system, some divergences from this typical behaviour were expected:
the number of methoxyl groups was rather low at 30% DM but should not be ignored; the high
starting temperature of the measurements (100 °C) is favourable for hydrophobic interactions. The
free carboxyl groups were assumed to be randomly distributed as it is typical for most commercial
pectins after chemical demethoxylation (Fraeye et al., 2010b, Ngouemazong et al., 2011a). The
number of dissociated free carboxyl groups should be relatively low at pH about 3 (below the
pKa 3.5). Therefore, it was expected that the formation of typical egg-box junction zones would be
limited and more interactions between undissociated carboxyl groups via hydrogen bonds would be
formed instead. The number of random (unspecific) calcium crosslinking via single dissociated
carboxyl groups should increase with rising Ca2+. The high sugar content in the gels could
additionally promote the interchain interactions as it is known from HMP gelation.
Figure 3 Comparison of the structuring temperatures in dependence on calcium content. GP = gel point = intersection of
G´ and G´´ (―); IST = initial structuring temperature (---); CST = critical structuring temperature (- • -).
20.0
40.0
60.0
80.0
100.0
0.4 0.5 0.6 0.7 0.8 0.9
mM CaCl2 / 100 g gel
structuring temperature (°C)
GP IST CST
10
G´ and G´´
dG´/dt
gel properties
CaCl2
As can be seen from Figure 3, the gel point temperature as well as the initial and the critical
structuring temperatures increased at higher calcium content. This confirmed the crucial role of Ca2+
for the gelling of LMP (e.g. Grosso & Rao, 1998, Lootens et al., 2003, Cardoso et al., 2003,
Cardenas et al. 2008, Fraeye et al., 2009, 2010a, b). IST and CST developed in a nearly parallel
way; IST was always about 10 K higher than CST. The gel point temperature GP behaved different:
at low calcium concentration (0.42 mM, R=0.46) it was found about 25 K below the IST and also
CST, and at high calcium content (>0.73 mM, R>0.82) GP was above IST. It should be considered,
however, that at high calcium content the GP could not be determined for all samples (Table 1) and
the values were therefore rather vague. This will be discussed in detail below.
Table 1 Data of structure formation. CaCl2 is given as mM content in the final gel mass of 900 g as well as R =
stoichiometric ratio Ca2+ / COO-; GP = gel point = intersection of G´ and G´´; tan
d
end = loss factor at 10 °C; IST
= initial structuring temperature; CST = critical structuring temperature; M = mean value
11
The detailed discussion of the gelling process with respect to possible structuring mechanisms at
different Ca-contents is illustrated by Figure 2, 4 and 5 a-d. The Figures 2 and 5 are single
measurement curves, the data of IST and CST in the text are medium values of four repeated
measurements. For reproducibility see Figure 4 and Table 1. Some of the structuring velocity curves
allowed the hypothesis of a two- or three-phase gelling process (Figure 4). Two phases of gelation
are also described by Fu & Rao (2001), the according temperature ranges and activation energies
for the first (70 to 50 °C) and second phase (50 and 20 °C) varied in different pectins.
Figure 4 Structure velocity dG´/ dt in dependence on calcium content in 100 g gel: 1 0.42 mM, 2 0.52 mM, 3 0.62 mM, 4
0.73 mM, 5 0.83 mM.
i. Mixtures without any calcium did not gel at all but started a kind of “structure formation” below
20 °C (Table 1). A possible explanation gives Ngouemazong et al. (2011a) who suggest a
gel-like characteristic in concentrated LMP systems in the absence of calcium and without
junction zone formation. Even a high share of sugar, that should allow a gelation of LMP also
under these conditions as described by Gilsenan et al. (2000) and Cardoso (2003), had no
real gel-promoting effect.
ii. A low number of Ca2+ (0.42 mM / 100 g, R 0.46) were already sufficient to initiate a certain gel
formation as can be seen in comparison to (i). A continuous structuring process started at IST
57 °C (Figure 5a) probably with a small number of ionic egg-box and / or crosslinking via Ca2+
as well as by hydrophobic interactions. They were, however, not strong enough for a real
gelation. With further cooling increasing formation of hydrogen bonds started (Gilsenan et al.,
2000, Cardoso et al., 2003) and the structuring process was accelerated despite of the
decreasing influence of hydrophobic interactions. This is indicated by the CST 47 °C.
030 60 90
0
5
10
15
dG'/dt (Pa/min)
Time (min)
1
2
3
4
5
12
Additionally, dimer associations could become more important with decreasing temperature. A
low GP of 32 °C below CST confirmed the transition from the liquid-like to a dominating solid-
like system.
a c
b d
Figure 5 Diagrams of gels with different calcium content in 100 g gel: a 0.42 mM, b 0.52 mM, c 0.73 mM, d 0.83 mM; full
curve = dG´/dt, dotted curve = G´; vertical lines give IST and CST (---) and the start of structuring phases
(
٠٠٠٠
); X on the G´ curves marks the GP. For 0.62 mM/100 g gel see Figure 2.
iii. At higher Ca2+ concentration (0.52 mM / 100g, R 0.58) IST and CST were comparable to (ii)
but this time the GP at 53 °C was found already shortly after IST 57 °C (Figure 5b). Two clear
phases could be defined: phase 1 started from IST, probably with formation of egg-box and
other ionic interactions as explained by Cardoso et al. (2003), Siew & Williams (2005) and
Fraye et al. (2009) as well as hydrophobic junction zone. It was strongly supported by
hydrogen bonds (especially below CST 42 °C) as well as by high sugar content and
accelerated with increasing contact between pectin chains during cooling. The second phase
started rather late at about 20 °C and could be possibly ascribed mainly to increasing dimer
associations and inter-dimer aggregations.
iv. The structuring process of gels containing 0.62 mM CaCl2 / 100 g (R 0.7) began earlier than in
(iii) at IST 67 °C (Figure 3), the higher calcium content obviously accelerated the structure
formation considerably by formation of more ionic interactions. The GP 58 °C was found again
between IST and CST (55 °C), but on a higher level than in (iii). This time even three phases
015 30 45 60 75 90
0,0
0,1
0,2
0,3
0,4
0,5
dG'/dt (Pa/min)
Time (min)
0
1
2
3
4
5
6
7
8
Storage modulus (Pa)
100 90 80 70 60 50 40 30 20 10
X
Temperature (°C)
015 30 45 60 75 90
0
2
4
6
8
dG'/dt (Pa/min)
Time (min)
0
100
200
300
Storage modulus (Pa)
100 90 80 70 60 50 40 30 20 10
X
Temperature (°C)
III III
III III
015 30 45 60 75 90
-0,5
0,0
0,5
1,0
1,5
X
dG'/dt (Pa/min)
Time (min)
0
10
20
30
40
50
Storage modulus (Pa)
100 90 80 70 60 50 40 30 20 10
Temperature (°C)
III
015 30 45 60 75 90
0
4
8
12
16
dG'/dt (Pa/min)
Time (min)
0
100
200
300
400
500
Storage modulus (Pa)
100 90 80 70 60 50 40 30 20 10
X
Temperature (°C)
III III
13
could be identified in the structuring process. It was assumed that ionic junction zones via Ca-
bridges together with hydrophobic interactions dominated the first phase, and that hydrogen
bonds became a supporting force in the second. This second phase seemed to be partly
comparable to the first one of (iii). The transition from phase 1 to 2 was near 40 °C. Dimer
interactions and inter-dimer aggregations could be ascribed to a third phase below 20 °C,
comparable to phase 2 of the gels in (iii).
v. With further increasing calcium content of 0.73 mM / 100g (R 0.82), the determination of the
gel point was possible only for one of four samples and the resulting value was, therefore,
rather vague. In contrast, IST and CST could be determined without any problems but this
time below GP (Figure 5c). It seems that partly pre-gelation (formation of micro-gel particles)
happened already during the preparation process (no GP found by temperature sweep tests)
or immediately after preparation in the starting phase of the measurements (GP > IST),
though the pectin mixtures seemed to be homogenous, yet. A similar effect was described by
Morris (2009). He defined a “weak gel structure”, formed by some egg-box junction zones
rapidly after gel preparation and a “true gel structure”, developing on cooling by other
mechanisms. The micro-gels of the “weak gel structures” seemed to be irregular solid
particles that caused an “apparent gel point” but a real network was formed later during further
cooling. The structuring process of the tested pectin system could be divided into three
phases again, the first below IST 70 °C with acceleration at CST 61 °C, the second beginning
at around 40 °C and the third below 22 °C. The structuring processes in these phases were
assumed to be comparable to those explained in (iv).
vi. At the highest calcium content (0.83 mM / 100 g gel, R 0.94) less clear structuring phases
could be defined and the GP, found already at 86 °C, was as vague as in (v). The “true”
gelling process (phase 1) started at IST 77 °C and was accelerated at about 65 °C (CST). The
high number of Ca2+ probably was able to increase the structuring velocity by forming more
ionic interactions that were dominating the whole gelling process. Some small peaks or
shoulders below 45 °C could be perhaps ascribed to the supporting effect of hydrogen bonds
but formed no single second phase. The clear peak below 20 °C, resulting from increasing
dimer interactions, corresponds again to phase 3.
Altogether, during gelation of LMP with different Ca-content two to three phases were found with
differing starting and final temperatures. The first phase could be ascribed to ionic egg-box and
random crosslinks together with hydrophobic interactions; it was clearly detected in gels with more
than 0.52 mM CaCl2 / 100 g and shifted to higher temperature with increasing Ca2+. The second
phase (in gels with less Ca2+ also found as first one) perhaps indicated the contribution of hydrogen
bonds and the third phase (in 2-phase processes the second one) might be dominated by inter-
dimer interactions. At the highest tested Ca-content, the ionic interactions seem to dominate the
14
whole gelling process with no clear difference between phase 1 and 2. Figure 6 shows a possible
model of the structuring process.
Figure 6 Model scheme of the structuring process of the tested low-methoxylated pectin system during cooling for 3
phases or 2 phases process.
3.3. Gel structure after cooling
The question is, whether the calcium content influenced not only the gelation process but also the
final gel structure. This will be discussed considering different gel parameters.
The final values of G´ and G´´ after cooling at 10 °C as well as the Ridgelimeter tests gave
information about different gel properties. All three parameters strongly correlated with the
increasing Ca-content (Table 1 and Figures 7, 8).
The °SAG value is characteristic for the ability of a gel to keep its shape under its own weight (gel
form stability). The samples with no Ca2+ did not really gel and could not be measured. Those with
low content (0.42 mM) were rather weak and deformed quickly within the 2 min measuring time, it
was impossible to get results by this method. The gels became stiffer and less sagging at higher
Ca-content and, moreover, more and more brittle. The increase of °SAG was not linear and,
altogether, moderate (the highest value was about six times higher than the smallest (Figure 7).
15
Figure 7 Influence of the calcium content on the gel form stability (°SAG) in the Ridgelimeter method.
The storage modulus at the end of cooling (G´end) characterised the elastic material properties of the
samples. These increased with higher content of Ca2+ (maximum increase factor > 50, Figure 8),
which made the gels more stable and elastic. That was found also by Ngouemazong et al. (2011a)
for gels without sugar.
Figure 8 Influence of the calcium content on the storage modulus G´ (full line) and the loss factor tan
d
end (dotted line) at
the end of the cooling phase at 10 °C.
The influence of the loss modulus G´´ can be found in the loss factor tan d = G´´ / G´ (Figure 8).
Though G´´ also increased with higher Ca2+ (factor about 15), the differences were not as high as in
case of G´ and thus G´ dominated. After complete cooling, tan dend was the highest in mixtures
without any calcium (about 10, Table 1), confirming their dominating liquid-like viscous material
properties. With increasing Ca2+, tan dend decreased and above 0.62 mM it was nearly constant.
0
20
40
60
80
100
120
140
160
0.4 0.5 0.6 0.7 0.8 0.9
mM CaCl2 / 100 g gel
°SAG
0
50
100
150
200
250
300
350
400
00.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
mM CaCl2 / 100 g gel
G´end (Pa)
0.1
1
10
lg tan d end
16
These gels were more solid-like and elastic but also increasingly brittle as found already during the
Ridgelimeter tests. A comparable effect of the calcium concentration on gel properties was found
also by Cardenas et al. (2008), Fraye et al. (2010a) and Ngouemazong et al. (2011a).
The varying properties of the final gels confirmed the assumption of varying gel structures, resulting
from different structuring mechanisms in dependence on the calcium content. Systems with low Ca-
content, gelled mainly by non-ionic interactions, were very weak and deformable. Gels with a higher
(optimum) amount of Ca2, formed by a combination of ionic and other interactions were form-stable
and rather elastic. Gels with the highest concentration of Ca2+, dominated by ionic structuring
mechanisms and showing pre-gelation, were brittle and susceptible to mechanical destruction.
3.4. Structure developing rate
The average structure developing rates SDRa was calculated for the total gelation process
(Figure 9). It increased strongly and non-linear with the calcium contents of the gels and confirmed
the well-known role of Ca2+ for the gelation of low methoxylated pectins.
Figure 9 Influence of the calcium content on the structure developing rate during the whole structure formation (SDRa).
0.00
2.00
4.00
6.00
0.4 0.5 0.6 0.7 0.8 0.9
mM CaCl2 / 100 g gel
SDR [Pa/min]
17
4. Conclusions
i. The application of the first derivation dG´/dt, the structuring velocity curve, supports the
understanding of the gelation process. The suggested new parameters initial structuring
temperature IST and critical structuring temperature CST do not describe the same event in
pectin gelation like the widely used cross-over of G´ and G´´ (gel point GP). The IST was
higher than the GP as long as the latter could be determined regularly. In these cases, IST
can be seen as the beginning of the structuring process in a system with dominating liquid-
like character. The CST of the tested gels was found mainly below the GP. It seems that the
structuring process was strongly accelerated after a certain critical number of junction zones
had been formed in an increasingly solid-like material. The classical GP, however, seemed
to be not always an indicator of real structure formation. It might also result from pre-gelation
and the formation of irregular solid particles in micro-gels.
The two new parameters have proved to be good indicators for the start and the further
development of structure formation in low methoxylated pectin gels at varying calcium
concentrations. All samples could be evaluated, also those with no clear GP. IST and CST
are not seen as complete substitutes of the GP, but they give valuable complementary
information and, in case of no clear GP, they are an alternative method for the examination
of structure formation. Moreover, the application of the dG´/dt curve allowed the identification
of single phases of the gelation, detected by increasing and decreasing structuring
velocities. The calculation of structure developing rates, which was made only for the total
gelation process, could be applied possibly also to single phases.
ii. The tested LMP gels at pH 3 and with 30% saccharose required at least a small amount of
calcium in ionic junction zones for successful gelation; hydrophobic interactions, hydrogen
bonds and other mechanisms alone have been proved to be not sufficient, even at the
promoting high sugar content. The first phase of the structuring process started at
temperatures of ≥ 60 °C by formation of egg-box junction zones and random crosslinks via
Ca-bridges. It was supported by hydrophobic interactions and seemed to be nearly
completed at about 40 °C. The second and third phase of the structuring process until the
end at 10 °C were probably dominated by hydrophilic interactions (assumed below 50 °C)
and dimer aggregations (below 25 °C), respectively. The transition of the GP to higher
temperature with increasing Ca-content clearly confirmed the importance of the ions for the
gelation process. All types of Ca-bridges obviously increased the gelation velocity during
cooling considerably and supported the formation of stable elastic gels. Above a certain
calcium content, pre-gelation could take place during or immediately after gel preparation
that changed the gel structure. These gels were very elastic but became more and more
18
brittle. The properties of the final gels confirmed the varying gel structures, resulting from
different structuring mechanisms in dependence on the calcium content.
iii. The presented interpretations of the rheological parameters and their relation to structuring
mechanisms are partly assumptions, yet, and have to be confirmed by further experiments
using additional methods and pectin types. The application of the first derivation dG´/dt and
the calculated new initial and critical structuring temperatures on the gelation of low
methoxylated pectins is, however, a first step into the examination of their general
importance for the pectin gelation process.
Acknowledgements
Karla Kern is acknowledged for her skilful technical assistance during the pectin gel preparations.
19
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