Food Hydrocolloids 39 (2014) 272e279

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

Rheology and structure of mixed kappa-carrageenan/iota-carrageenan

gels
Tom Brenner a, b, *, Rando Tuvikene c, d, Alan Parker e, Shingo Matsukawa b,
Katsuyoshi Nishinari a, f
a
Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585, Japan
b
Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
c
Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt 29, 10120 Tallinn, Estonia
d
National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia
e
Firmenich SA, Materials Science Department, Corporate Research Division, Meyrin 2, Geneva, Switzerland
f
Glyn O. Phillips Hydrocolloids Research Centre, School of Food and Pharmaceutical Engineering, Faculty of Light Industry, Hubei University of Technology,
Wuchang, Wuhan 430068, PR China

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

Article history: The rheology of mixed k-carrageenan/i-carrageenan gels was investigated in the presence of 75 or
Received 17 November 2013 150 mM Kþ. The addition of i-carrageenan to k-carrageenan led to an initial weak increase of the Young’s
Accepted 14 January 2014 modulus. A maximum occurred at concentrations ratios close to 1:1, followed by a weak decrease at
higher i-carrageenan concentrations. When k- and i-carrageenan were mixed at a constant total poly-
Keywords: saccharide concentration, a plateau of the Young’s modulus and a clear local minimum in the fracture
Carrageenan mixtures
strain in both compression and extension were identified at ratios between 2:1 and 4:3 (i:k). At all other
Mixed gels
ratios, the expected strong increase of the Young’s modulus and decrease of the fracture strain were
Rheological properties
Phase separation
observed with an increasing k-carrageenan fraction. A weak maximum of the fracture stress in
Bicontinuous structure compression and extension was observed at ratios between 1:1 and 1:2 (i:k). The structural and rheo-
logical findings support segregative phase-separation of mixed carrageenan gels in the presence of Kþ.
Phase separation could be promoted through curing of the gel network of i-carrageenan at temperatures
above the coil-to-helix transition of k-carrageenan.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
Red sea weed galactans, i.e., carrageenans and agars, form the
second largest group (weight-wise) of polysaccharides in the food
industry (Piculell, 2006). The main carrageenans, k and i, hence-
forth designated KC and IC, respectively, are linear polyelectrolytes
of different charge densities. Ideally, carrageenans have a disac-
charide repeating unit, comprising a 1,3 linked b-D-galactopyr-
anose (G) and a 1,4 linked 3,6-anhydro-a-D-galactopyranose (AG).
KC is sulphated at O-4 of the G residue, and IC carries one additional
sulphate group on O-2 of the AG residue. The different structures of
KC and IC lead to very different gelling behaviours. The helix for-
mation and subsequent helix aggregation of KC is strongly pro-
moted by the larger alkaline cations Kþ, Rbþ and Csþ, and especially
at low ionic strength, by divalent cations (Piculell, 2006; Piculell,
* Corresponding author. Department of Food Science and Technology, Tokyo
University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-
8477, Japan. Tel.: þ81 3 5463 0581.
E-mail address: physicalchemistrytom@gmail.com (T. Brenner).
Nilsson, & Muhrbeck, 1992). For IC, on the other hand, only weak
dependence of the coil-helix transition temperature on the identity
of the alkaline cation is observed, and no thermal hysteresis is
present between the DSC endothermic and exothermic peaks,
suggesting that the helices are not (or only very weakly) stabilized
by aggregation (Piculell, 2006; Piculell et al., 1992; Ridout, Garza,
Brownsey, & Morris, 1996).
In many applications, a mixture of carrageenans is present, and
in general, KC and IC powders (especially food-grade) can contain
non-negligible contaminations of the other (Parker, Brigand,
Miniou, Trespoey, & Vallee, 1993; Piculell et al., 1992; Rochas,
Domard, & Rinaudo, 1980; Rochas, Rinaudo, & Landry, 1989; van
de Velde, Peppelman, Rollema, & Tromp, 2001). These impurities
are present as entire chains of the contaminating carrageenan
(Rochas et al., 1989) or as blocks of the contaminating carrageenan
within a chain of the predominant carrageenan (van de Velde et al.,
2001), forming a KC/IC hybrid.
The more extensive aggregation of the helices of KC, compared
to that of IC helices, causes significant differences in the properties
of their gels. In general, KC gels are hard and brittle, whereas IC gels

0268-005X/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2014.01.024
 T. Brenner et al. / Food Hydrocolloids 39 (2014) 272e279
are much weaker and fracture at larger deformations. Because of
the huge difference in elastic properties of KC and IC in the presence
of Kþ, rheological measurements have been suggested as a cheap
alternative to more expensive 13C NMR determination of the
amount of KC contamination in IC samples (Parker, 1995). In mix-
tures containing Kþ, KC and IC show independent DSC endotherms
and exotherms (Ridout et al., 1996), and these correspond to in-
dependent gel networks as observed rheologically (Parker et al.,
1993). Both these results provide evidence that the two gel net-
works are uncoupled. However, probably owing to the huge dif-
ference in elastic moduli between pure KC and IC gels in the
presence of Kþ, determining whether the KC and IC elastic net-
works in mixed gels are phase-separated or interpenetrated has
proven rather difficult, with differing views expressed based on not
incompatible sets of data (Parker et al., 1993; Piculell et al., 1992;
Ridout et al., 1996).
Given the importance of KC/IC mixtures in applications, we
extensively map the small- and large-deformation rheological
properties of their mixed gels, using commercial food-grade sam-
ples. Our results are compared to the literature, where different
investigators have mooted either a phase-separation of the two
polymers (Parker et al., 1993; Piculell et al., 1992) or their inter-
penetration (Ridout et al., 1996) as structural possibilities. The
structure of the mixed gels affects not only their rheology, but also
the release of water or flavour compounds, and textural properties
that are difficult or impossible to relate to rheology (van den Berg,
van Vliet, van der Linden, van Boekel, & van de Velde, 2007a; Chen,
2009; Foegeding et al., 2011), like creaminess and spreadability. The
high permeability of bicontinuous structures improves flavour-
release that is mediated through the release of solvent (van den
Berg et al., 2007a; van den Berg, van Vliet, van der Linden, van
Boekel, & van de Velde, 2007b), and the emulsion-like (“water-in-
water emulsion”) structure of filled phase-separated gels contrib-
utes to their creaminess (e.g., Le Révérend, Norton, Cox, &
Spyropoulos, 2010). It is therefore clear that further evidence
regarding the structural transition in mixed KC/IC gels and the
respective structures at high and low i:k ratios holds potential in-
terest for the food industry.
2. Materials and methods
2.1. Ingredients
Food-grade powders of k-carrageenan (SAN SUPPORT Ò G-16)
and i-carrageenan (SAN SUPPORT Ò G-17) were provided by San Ei
Gen F.F.I. (Osaka, Japan) and used without further purification. KCl
was purchased from SigmaeAldrich.
2.2. Chemical analysis
1
H NMR spectroscopic analyses were performed on a Bruker
AVANCE III spectrometer operating at 800 MHz. The 1H NMR
spectra from 0.5% polysaccharide solutions in D2O (w/w) were
obtained at 65  C and 64 transients were collected with an inter-
pulse delay of 5 s. Sodium 4,4-dimethyl-4-silapentane-1-sulfonate
served as an internal reference (d ¼ 0 ppm). The molecular weight
of the polysaccharide samples was reduced prior to the NMR
measurement by sonicating in a water-bath at 25  C for 100 min
with an ultrasound homogenizer (Bandelin Sonopuls UW2070,
70 W, operating at 80%), followed by centrifugation and freeze-
drying. The molar ratio of KC and IC was estimated by integrating
the anomeric signals corresponding to AG residues from the
respective diads. Our NMR results indicated that, in molar terms,
the KC sample contains 80.5% KC and 19.5% IC, whereas the sample
of IC contains only 1.7% KC.
273
AG content was determined colorimetrically using a resorcinol-
acetal method (Yaphe & Arsenault, 1965) with fructose as a stan-
dard sugar. Galactose content was obtained by reductive hydrolysis
of the polysaccharides followed by the acetylation process (Jol,
Neiss, Penninkhof, Rudolph, & De Ruiter, 1999); the obtained aldi-
tol acetate derivatives were analysed by gas chromatography
coupled with flame ionization detection (GC-FID) using inositol as
an internal standard (Stevenson & Furneaux, 1991). The ratio of G to
AG units was 1.03 and 1.15 in the KC and IC samples. The lower AG
content in the IC powder is a general feature of IC preparations, as
they are known to contain non-trivial amounts of substituted gal-
actopyranosyl units, carrying mainly methyl and pyruvate groups
(see, e.g., Tuvikene et al., 2009; Usov, 2011), which we did not
quantify. We note that the presence of only trace amounts of G-
2S,6S, i.e., the n-form carrageenan, could be inferred from the 1H
NMR spectra. The total polysaccharide content (without counter-
ions), recalculated assuming idealized structure with the ratio of i
and k diads estimated from NMR, was 64.8% and 71.9% in the KC and
IC samples, respectively. The moisture content of these powders
was respectively 8.7% and 10.3%.
Inductively coupled plasma atomic absorption elemental anal-
ysis was carried out on an SPS7800 Plasma Spectrometer (SII
Group). About 0.1 g of powder was microwave-digested in 5 mL
nitric acid and 5 mL hydrogen peroxide, then diluted x10 with
water and x5 with a mixture of 1/9 nitric acid/water. Contents of
Naþ, Kþ, Ca2þ, Mg2þ and S are given in Table 1.
Size exclusion chromatography (SEC) of polysaccharides was
performed on a chromatograph equipped with a PerkinElmer Se-
ries 200 pump, a Knauer Smartline 2300 refractive index detector, a
Knauer Smartline column thermostat, and two Shodex OHpak SB-
806MHQ columns in series. Elution was carried out using a 0.1 M
NaNO3 solution as the mobile phase at a flow rate of 0.8 mL/min.
The temperature of the columns was maintained at 60.0  C. A
calibration curve was constructed using 12 pullulan standards
(peak molecular weight Mp values 2400, 1560, 710, 380, 200, 106,
45.9, 22.0, 11.2, 5.6, 1.08 and 0.342 kDa). The elution volume was
corrected to the internal marker of ethylene glycol (0.01% in sam-
ple) at 23.04 mL. The polymer concentration used was 0.07%, and
the sampling volume 100 mL. Powders were dissolved in the SEC
eluent solvent by vigorously stirring for 10 min at the boiling point.
The hot (60  C) sol was filtered through a 0.45 mm RC membrane,
allowed to cool down, and then injected into the HPLC system.
Fig. 1 shows the SEC elution profiles of the samples. The peak
molecular weight values (Mp), relative to those of the pullulan
standards, were 1860 and 1435 kDa for KC and IC, respectively.
Another finding of note regarding the KC sample is the large (15%)
peak for Cl (at 21.37 mL), which, based on the data of Table 1, is in
excess of the theoretical limit even if no other anions are present.
This peak, however, decreases as a function of keeping time at high
temperature, indicating that some heat sensitive oligosaccharides
are eluted at the same retention volume as Cl. Our mono-
saccharide analysis also detected trace amounts of glucose in the KC
powder, supporting the conclusion that some oligosaccharide has
been added to the sample. An important finding of the IC powder is
the presence of excess SO2 4 (2.2%; retention volume 20.33 mL), so
Table 1
Elemental analysis results of KC and IC powders.
Element IC (%) KC (%)
Na 0.84 0.32
K 3.3 9.1
Ca 3.3 0.88
Mg 0.21 0.18
S 13.1 7.7
274 T. Brenner et al. / Food Hydrocolloids 39 (2014) 272e279

different parameters were chosen for technical reasons, and cy-

21.37 lindrical gel samples (height ¼ 9.0 mm, diameter ¼ 11 mm) were
23.04 compressed (10 mm/s, strain rate 1.1 s1) to fracture using a square
PMMA plunger. Ring extension was as described elsewhere
(Brenner, Wang, Achayuthakan, Nakajima, & Nishinari, 2013). En-
gineering fracture strain and stress were calculated from the
compression test (εfc and sfc), while for the ring extension test the
engineering fracture strain and stress (εfe and sfe) were approxi-
A
mated from the elongation of the inner side of ring as described
elsewhere (Brenner et al., 2013).
22.33

20.33 2.6. Rotational rheometry

B
The shear storage (G0 ) and loss (G00 ) moduli were measured on a
10 15 20 25 Haake Mars II rheometer (Thermo Scientific) using a chemically
roughened parallel plate geometry (diameter 35 mm). Contact with
Retention volume, mL
the sample was ensured through use of the Autotension option.
Fig. 1. Size exclusion chromatograms of KC (A) and IC (B) samples obtained at 60  C.
2.7. Turbidity measurements
we can conclude the presence of 13.1%e0.7% ¼ 12.4% S in the IC
polysaccharide. The turbidity of gels (300e1000 nm) was determined using a V-
630 Biospectrophotomer (Jasco, Tokyo, Japan) using 10 mm quartz
2.3. Preparation of the gels cells. Aqueous 0.15 M KCl solution was used as a blank.

Potassium chloride and carrageenan powders were added in 2.8. Differential scanning calorimetry
this order under stirring at room temperature. The mixtures were
heated with stirring for 120 min at 40  C and 30 min at 90  C. The Differential scanning calorimetry (DSC) was performed on a
stirring was stopped for the last 15 min of the heating procedure to Micro-DSC VII (Setaram, Caluire, France). Samples (0.8 mL) were
allow any trapped air to escape. Following heating, the hot solu- loaded into Hastelloy cells, heated to 85  C to erase the thermal
tions were poured into moulds of different dimensions and then history, and then cooled to 5  C at 0.5  C/min to identify the sol-to-
kept in a refrigerator (5  C) for 16e20 h, except for experiments on gel transition temperatures of the two polysaccharides.
the effect of the curing method. In these experiments, the mould
with hot solution was immediately placed in a water bath kept at 3. Results and discussion
52  C for a fixed time, and then immersed in ice water for 60 min
before being placed in a refrigerator (5  C) for 16e20 h. Gels were 3.1. Small and large deformation rheology
equilibrated to room temperature (20  2  C) (about 60e120 min)
before measurement. MQ-grade water was used, and the total Fig. 2 shows the Young’s modulus, E, for KC:IC mixtures as a
concentration of Kþ was fixed to 75 or 150 mM. function of the IC weight fraction, cIC ¼ CIC/(CIC þ CKC), in the
presence of 150 mM Kþ and at a fixed total polysaccharide content
2.4. Linear extensional rheology e Young’s modulus CIC þ CKC ¼ 1.6 wt%. A near-plateau of E is observed at IC:KC ratios of
2:1 to 4:3, i.e., cIC z 0.55e0.65. Fig. 3A and B shows the fracture
The complex Young’s modulus of the gels was measured as strain and stress of these mixed gels in compression and extension,
described by Nishinari et al. (1980). Cylindrical gels
(height ¼ 31 mm, diameter ¼ 20 mm) were glued to the upper and
bottom faces of a Rheolograph Gel (Toyo Seiki Seisakusho, Tokyo,
Japan) with alpha cyano acrylate glue (Aron Alpha; Toa Gosei Ltd.,
Japan). The Rheolograph measures the storage (E0 ) and loss (E00 )
Young’s moduli at frequency of 3 Hz and an amplitude of 0.1 mm,
which for these gel specimens corresponds to a strain of 0.32%. For
all gels it was found that E0 z E, while E00 was not reliably deter-
mined for most gels, i.e., it was lower than or comparable to the
lower limit of the Rheolograph, 0.1 kPa. Therefore, we will only
discuss E.

2.5. Mechanical tests

Compression and extension tests were carried out on an XT.T2

Texture Analyser (Stable Micro Systems, Surrey, UK), or on a RCT-
2002D-D Fudoh Rheometer (Rheotech, Tokyo, Japan). Lubrication
in compression was not necessary because the gels exude water. For
compression tests using the Texture Analyser, cylindrical gel sam-
ples (height ¼ 15 mm, diameter ¼ 25 mm) were compressed
(10 mm/s, strain rate 0.67 s1) to fracture using a square aluminium
plunger. For compression tests using the Fudoh Rheometer,
Fig. 2. Young’s modulus E (circles) as a function of cIC ¼ CIC/(CIC þ CKC) for a fixed total
carrageenan content of 1.6% and [Kþ] ¼ 150 mM. Dotted: upper (isostrain, x in Eq.
(1) ¼ 1) bound, solid: lower (isostress, x ¼ 1) bound, dot-dashed: additivity, short
dashed: bicontinuous (x ¼ 0.2), long dashed: fit to Eq. (1) with x ¼ 0.46. Error-bars
(from two preparations) are too small to be seen.
 T. Brenner et al. / Food Hydrocolloids 39 (2014) 272e279
Fig. 3. Engineering fracture strain (open symbols) and stress (filled symbols). A, compression, B, extension. Error-bars are calculated from at least 4 samples from each of 2
preparations.
respectively, and demonstrate that at the same ratio range, a clear
local minimum of the fracture strain is observed, and as a result,
this is where the strongest decrease of the fracture stress with
increasing IC content is observed. When a composite gel contains
two gelling components, the structure formed may comprise a
single coupled network, two interpenetrating networks or two
phase-separated networks (e.g., Morris, 1995). This simplified tax-
onomy does not take into account the distribution of each
component in different phases, phase continuity/connectivity,
different interactions (other than direct binding) or swelling/
deswelling. There are different models for estimating the elastic
modulus of the composite gel G0 from the individual moduli Gi and
volume fractions Øi. For phase-separated networks, the elastic
contributions of each phase may be added arithmetically (isostrain)
or harmonically (isostress) to give theoretical upper and lower
bound estimates (e.g., Ross-Murphy, 1995):

1=x
G0 ¼ B1 Gx1 þ B2 Gx2
The exponent x determines the behaviour of the composite: the
values x ¼ 1 and x ¼ 1 correspond to the upper (isostrain) and
lower (isostress) bounds, respectively, which were suggested for
filled gels where only one phase is continuous (the other one being
a filler). Isostrain behaviour is expected when the continuous phase
has the higher elastic modulus, as the deformation will be deter-
mined by the network. On the other hand, when the filler is more
rigid than the network, its deformation is lower than that of the
network, and the limit of equal stress in both phases is more
appropriate (Ross-Murphy, 1995). When both networks are
continuous, i.e., for the bicontinuous case, Davies (1971) suggested
the exponent x ¼ 1/5.
When two independent networks span the entire volume and
do not phase-separate on a length scale larger than the scale of
heterogeneities in each network (the interpenetrating case; see
discussion below), the starting point for calculating G0 is assuming
additivity, i.e.:
G0 ðC1 þ C2 Þ ¼ G1 ðC1 Þ þ G2 ðC2 Þ
where C1 and C2 are concentrations of the individual gelling
(polymer) components (Zhang & Rochas, 1990).
Piculell et al. (1992) and Parker et al. (1993) substituted KC for
up to 10% of the IC content in the presence of 0.1 M KCl and 0.2 M
NaCl, respectively, and concluded that the gels were phase-
separated, because the small KC fraction affected the rheology
much more than expected if it were not excluded from the large
volume occupied by the dominant iota fraction. Ridout et al. (1996)
275
replaced 25%, 50% and 75% of the IC content with KC, in the pres-
ence of 0.08 M KCl. As in the other two studies, the data of Ridout
et al. (1996) lay between the upper and lower bounds predicted by
Eq. (1). However, because no obvious phase-inversion was indi-
cated by their data, Ridout et al. (1996) concluded that an inter-
penetrating KC/IC network was formed. At this point we should
clarify the distinction between the bicontinuous and inter-
penetrating cases for the networks of mixed carrageenans. Obvi-
ously, each of the elastic networks is formed by phase-separating
from both the solvent and the other polysaccharide, as double-
helices are formed strictly from two chains of the same poly-
saccharide, and junction-zones of double-helices include only one
type of polysaccharide. The question to be answered is what the
scale of phase-separation, or inhomogeneity, between the two
polysaccharide networks is. In the limit of interpenetrating net-
works, it is assumed that the density of each network is the same as
that in absence of the other network, i.e., no densification takes
place, and a junction-zone of one network will be on average at the
(1)
same distance from a junction-zone of the other polysaccharide
network as from another junction-zone of its own network when
the total density of junction-zones of both networks is equal. When
one network causes densification of the other but both networks
keep their continuity, we can speak of phase-separation. In this
case, at equal total junction-zone density of both networks, a
junction-zone of one network will be on average closer to a
junction-zone of its own network than to a junction-zone of the
other networks, thus leading to well-defined areas (phases) where
one network is concentrated and the other is depleted, on a length
scale larger than that of inhomogeneities of either network. Of
course, the two cases of interpenetrating and phase-separated
networks are approximate limiting cases, while the reality of dis-
tribution of the two networks can be much more complicated (see,
e.g., discussion in Ross-Murphy, 1995).
Our data do not contradict the results reported by Parker et al.
(1993), Piculell et al. (1992) and Ridout et al. (1996), and
resemble rheological data by Rochas et al. (1989), who, like us,
prepared mixed gels using a nearly pure i-form and a substantially
(2) impure (11% i-impurity) k-form. As in our Fig. 2, they reported a
monotonous decrease of the elastic modulus with increasing i-
fraction, however, did not identify a near-plateau as in our case.
Furthermore, Rochas et al. (1989) also found, as in our Fig. 3A, a
maximum of the fracture stress in compression at about equal
mixing ratios of the two polysaccharides.
Our results indicate that the elastic modulus is indeed close to
that predicted from simple additivity at low i:k ratios, but the effect
of KC is far greater than predicted by additivity at low kappa con-
tents. Without over-emphasizing the theoretical values for the
276 T. Brenner et al. / Food Hydrocolloids 39 (2014) 272e279
interpenetrating networks and bicontinuous cases as presented in
Fig. 2, we can say that both models predict the data better than
either the lower or upper bounds. However, while the inter-
penetrating networks and bicontinuous cases are both close to the
data at low i:k ratios, only the bicontinuous model seems plausible
at high IC content. Indeed, the interpenetrating limit leads to a
minimum of the elastic modulus at high IC content, but we, like
Rochas et al. (1989), found that the elastic modulus monotonously
decreased with increasing IC content, with a weak plateau at
around ratios of 2:1 to 4:3 (i:k). As shown in Fig. 2, the monotonous
form Eq. (1) provides a satisfactory fit to the data, except for around
the plateau, where the fit overestimates the data. The monotonous
change in the elastic modulus points to the bicontinuous phase,
even if the exponent of the fit in Eq. (1) is different to that predicted
by (Davies, 1971). Furthermore, the transition seen in our data at
ratios of 2:1 to 4:3 (i:k) is indicated not only from measurements of
the elastic modulus (Fig. 2), but also from fracture measurements in
compression and extension (Fig. 3). While KC forms relatively
brittle and strong gels in the presence of KCl, IC forms weaker gels
with higher fracture strains. The increase in E and decrease in
fracture strain with a decreasing IC:KC ratio are therefore expected.
However, it is very difficult to explain the local minimum in the
fracture strain in compression and the near-plateaus in E and
fracture strain in extension around ratios of 2:1 to 4:3 (i:k) without
invoking some structural transition. The presence of a transition
could be an indicator of phase-separation. The degree of phase-
separation is such that it affects the rheological properties in a
manner that negates the assumption that neither network is
affected by presence of the other, as in the interpenetrating model.
We are therefore led to conclude the presence of sufficient densi-
fication of either polysaccharide network by the other, so that we
speak of “phase separation”.
Regarding fracture of the mixed gels in compression, Rochas
et al. (1989) claimed (without showing the data) that the fracture
strain was identical for all mixing ratios, which is in clear
disagreement with our data (Fig. 3A). In our case, the maximum of
the fracture stress can be understood to arise from the two
opposing trends of increasing fracture strain and decreasing elastic
modulus with increasing cIC. From the report by Rochas et al.
(1989), however, it must have been concluded that the strain
hardening of the gels in compression increases with increasing
content of i-carrageenan, because otherwise the fracture stress
could not increase with increasing i-carrageenan content at a
constant fracture strain with decreasing elastic modulus. All our
gels showed similar strain hardening, although at cIC ¼ 0.5, strain
hardening was indeed slightly stronger, see Fig. 4. While Rochas
et al. (1989) concluded from the maximum of the fracture stress
that interaction must be present between k- and i-chains, we
Fig. 4. Engineering stress s, normalized with the Young’s modulus E and the engi-
neering strain ε, as a function of the strain in compression. cIC values are indicated in
the figure.
attribute this maximum to both the opposing trends of the elastic
modulus and the fracture strain and to the slightly stronger strain
hardening. We do not consider the data of Fig. 4 to contradict phase
separation of the two polysaccharides in the gel phase, as to our
knowledge, no universally accepted model has been put forth that
explains the large deformation rheology of mixtures.
The Young’s modulus E of mixed gels as a function of KC and IC
concentrations is plotted in Fig. 5A. This figure clearly demonstrates
the higher moduli of KC gels in comparison with IC gels, but also
shows that when IC is added to KC at a constant KC concentration,
an initial increase is observed, with a subsequent peak and an
eventual weak decrease. This leads to a certain plateau in the 3D
plot when following a straight line connecting points of the same
value on the (positive) CKC and CIC axes, i.e., straight lines for con-
stant total polysaccharide concentrations and different IC:KC ratios.
This plateau appears at the same ratio range seen in Fig. 2, namely,
about 4:3 to 2:1 (i:k). The findings of Fig. 5 are also indicative of a
certain structural transition between low and high i:k ratios. The
fracture parameters from both compression and extension also
support this conclusion, but because their three-dimensional rep-
resentation tends to be undescriptive, we chose to show only the
fracture stress in compression, see Fig. 5B. At constant KC concen-
trations, the fracture stress goes through a maximum at around
equal additions of IC, emphasizing the general nature of this
finding, which was first noted from Fig. 3 at a fixed total carra-
geenan content.
Eq. (1) can be applied to binary mixtures of arbitrary concen-
trations of the two components, but this requires knowledge of the
elastic modulus of the pure components over the entire range of
concentrations. In this study, we found the elastic modulus of KC to
scale as a power law of the KC concentration:
2:55
EKC fCKC (3)
Because for all relevant concentrations, the elastic modulus of
KC is much higher than that of IC, the elastic modulus will be
dependent only on the volume fraction and elastic modulus of the
KC phase, especially if Eq. (1) is valid with a sufficiently high value
of the exponent x. As we saw from the data obtained at a constant
total polysaccharide content (Fig. 2), the best fit to Eq. (1) is ob-
tained with x ¼ 0.46. If the elastic modulus is determined by the
volume fraction and elastic modulus of KC, then it is clear that x
should be larger than 1/2.55 z 0.4 to allow for an increase in the
elastic modulus with increasing IC concentration at a fixed KC
concentration:
  x 1=x
E ¼ ð1BKC ÞA ð1BKC Þ2:55 þBIC EIC
x
  x 1=x
z ð1BKC ÞA ð1BKC Þ2:55
¼ A1=x ð1BKC Þð12:55xÞ=x (4)
where A is a constant. Thus, Eq. (1) with x ¼ 0.46, which is slightly
higher than 1/2.55, can explain the slight increase in E with
increasing CIC at a constant CKC, although it cannot explain the
maximum at around equal concentrations of KC and IC or the
subsequent decrease in E with increasing CIC.
For mixtures of agarose and KC, Zhang and Rochas (1990)
concluded the presence of an interpenetrating network, but their
investigation was mainly structural rather than rheological. As
expressed by Parker et al. (1993), it is interesting if the KC network
is indeed phase separated from that of its closest relative, IC, but is
miscible with the network of its more distant relative agarose. On
the other hand, Piculell et al. (1992) noted that the separation of the
KC and IC networks is reasonable as they are both anionic
 T. Brenner et al. / Food Hydrocolloids 39 (2014) 272e279
Fig. 5. Young’s modulus E (A) and fracture strain in compression sfc (B) as a function of KC and IC concentrations in the presence of 0.15 M Kþ.
polyelectrolytes, and that KC should indeed be closer to complete
miscibility with the neutral agarose. We also note that mainly
based on structural investigations, KC was also concluded to form a
phase separated gel with gellan, another anionic polysaccharide
(Nishinari, Watase, Rinaudo, & Milas, 1996), and parenthetically
note that the rheological additivity was not indicated correctly in
Fig. 6 of that publication. Our results indicate that a phase-
separated bicontinuous structure is formed in the KC/IC mixtures
at high i:k ratios. While strictly, the equation suggested by Davies
(1971) for the elastic modulus of the bicontinuous structure un-
derestimates the values we find experimentally at low i:k ratios, we
recognize that conceptually, as an intermediate case between the
upper and lower bounds of the phase separated system, it explains
the values of the elastic modulus at all ratios of the two carra-
geenans. The slight underestimate of Davies’s (1971) model can be
eliminated by using a higher exponent x in Eq. (1), as was shown in
Fig. 2. As explained above, this higher exponent (x ¼ 0.46) can also
explain the slight increase in the elastic modulus with increasing IC
content at fixed CKC, but it cannot explain the peak and eventual
decrease in E with further increase of CIC.
The presence of a bicontinuous structure at all i:k ratios is
consistent with the phase-separation best indicated by the strong
effect of low concentrations of KC on the elastic modulus of IC gels,
and is also consistent with a structural transition between high and
low ratios i:k, reflected in both small and large deformation tests.
We note that as our sample of KC contains 80.5 mol% KC and
Fig. 6. Fractional ratios of turbidity R; open symbols, R400/800, closed symbols, R300/
900. Error-bars indicate one standard deviation.
277
19.5 mol% IC, the transition suggested from Figs. 2e4 around
cIC z 0.6 suggests a molar ratio close to 2:1 i:k.
3.2. Structural investigations
To substantiate the presence of a structural transition, we
measured the turbidity of gels in the UV-VIS range. While the
turbidity is a measure of structural homogeneity, it is not often
appreciated that many parameters determine the turbidity, among
which are the concentration of scattering units, the contrast be-
tween the scattering units and the medium (in this case, the sol-
vent), the length scale of concentration fluctuations and the
compressibility of the system (see, e.g., De Kruif, 1993; Pouzot,
Durand, & Nicolai, 2004), and in our case, also direct absorption
of light, and all of these factors have different wavelength de-
pendencies. For this reason, instead of looking at the turbidity at a
single wavelength, we decided to search for clues of structural
changes by calculating the ratio of the turbidity at two different
wavelengths. Because we are not using any models to quantify the
turbidity, using such ratios of the turbidity offers no advantage over
the more convenient fractional ratio R, where we fix the ratio for
one pure component to zero and for the other pure component to
unity:
si s
i ðICÞ
sj  s ðICÞ
Ri=j ¼ s ðKCÞ j s ðICÞ (5)
sj ðKCÞ  sj ðICÞ
i i
where i and j refer to two wavelengths, si is the turbidity at
wavelength i, and s(KC) and s(IC) are the turbidities of the pure KC
and IC samples, respectively. The advantage of this fractional ratio
over the turbidity ratios themselves is that curves for different
combinations of wavelengths can be compared directly.
Fig. 6 shows the fractional ratios of the turbidity for
400&800 nm (open symbols) and 300&900 nm (closed symbols).
Our turbidity measurements at these two different combinations of
wavelengths show no evidence for the structural transition close to
cIC z 0.6 implied by our rheological measurements. We hope that
more detailed spectroscopic measurements we are making, on
which we will report separately, will resolve this enigma.
3.3. Controlling the structure through the curing method
As should be clear from the previous sections, our suggestion of
a bicontinuous structure of k- and i-carrageenan networks is based
on rheological findings, as we have failed to produce convincing
structural results to support this conclusion. However, as noted by a
278 T. Brenner et al. / Food Hydrocolloids 39 (2014) 272e279
Fig. 7. Storage shear modulus G0 (circles, g ¼ 0.02, u ¼ 1 rad s1) and DSC heat flow
(solid line) during cooling. The arrow indicates exothermic heat flow direction.
CKC ¼ 0.4%, CIC ¼ 1.2%, [Kþ] ¼ 75 mM. The cooling rate was 0.5  C/min in both
experiments.
Fig. 8. Stress relaxation curves of a mixed gel (CKC ¼ 1.2%, CIC ¼ 0.8%, [Kþ] ¼ 75 mM) at
15% compression. Samples were cured at 52  C (curing time in min indicated in figure)
before being quenched in ice water; C indicates the control sample cured at 5  C.
reviewer of this paper, as i-carrageenan gels at a higher tempera-
ture than k-carrageenan (unless the concentration of cations that
promote k-carrageenan aggregation is very high), it should be
possible to allow different degrees of formation of the i-carra-
geenan elastic network by keeping the mixtures at a temperature
between the gelation temperatures of the two polysaccharides
for varying lengths of time before inducing gelation of the
Fig. 9. Schematic representations of the bicontinuous structure suggested for KC/IC mixed gels. Left panel, structure obtained following fast curing; right panel, enhanced phase
separation following curing of IC at temperatures above the coil-to-helix transition temperature of KC.
k-carrageenan network. In theory, promoting the network forma-
tion of i-carrageenan before the k-carrageenan is allowed to gel
should lead to phase-separation on a larger length scale. In practice,
testing this idea becomes more feasible because the curing of IC is
very slow, as noted by Nono, Durand, and Nicolai (2012). We per-
formed DSC and rotational rheometry tests on a mixture
(CKC ¼ 0.4%, CIC ¼ 1.2%) containing 75 mM Kþ. The reason for the
lower potassium concentration chosen was to allow a larger sepa-
ration between the gelation temperatures of the i and k forms, as
the gelation temperature of the latter strongly increases with
increasing [Kþ] while the gelation temperature of the former is
hardly affected (see, e.g., Ridout et al., 1996). Our data clearly show
the distinct gelations of the two polysaccharides, with two separate
exotherms and two increases of G0 . The data are of course not novel,
as the distinct gelation of the i and k forms was demonstrated by
other researchers using rotational rheometry (Parker et al., 1993)
and DSC (Ridout et al., 1996). The data enabled us, however, to
choose an optimal temperature for the curing experiments to
promote IC gelation while KC chains are still in the coil conforma-
tion. Based on the data shown in Fig. 7, we chose to cure samples at
52  C for different periods of time, and, to maximize the effect of
the curing, we then quenched the samples in ice-water at 0  C, to
allow as fast a curing as possible of the k-carrageenan. After 60 min
at 0  C, gels were transferred to a refrigerator (5  C) for curing over
16e20 h. We found, however, that to obtain reliable stress-
relaxation data with the equipment available to us we needed to
prepare gels at higher KC concentrations than that used for
obtaining the data of Fig. 7. We therefore opted for a concentration
of 1.2% KC with 0.8% IC while keeping [Kþ] ¼ 75 mM. The stress
relaxation curves of this gel are shown in Fig. 8. We found that to
obtain clean signals a compression strain of 15% was needed. Ac-
cording to our preliminary tests, this strain is outside the linear
regime of the gels investigated. The same results could be obtained
using lower strains, but the signals tended to be noisier. From Fig. 8,
we clearly see that increasing the curing time at 52  C had a weak
but consistent effect to increase the effective instantaneous
modulus (i.e., the stress/strain at t ¼ 0) of the mixed gel. This
finding serves, therefore, as another evidence of phase separation;
enhancing the phase separation by allowing selective curing of one
network before the other affects the rheology. This effect is rather
weak, as could be expected from the data of Fig. 5A and the prior
discussion of the Eq. (1) exponent x ¼ 0.46 (see Fig. 2), which was
noted to be only slightly higher than 1/2.55 z 0.4. We will
comment parenthetically on the effect of the cooling rate on the
rheology of the gel. Logically, the lateral aggregation of KC is
 T. Brenner et al. / Food Hydrocolloids 39 (2014) 272e279
arrested by gelation. Therefore, slower curing of the gel should
allow for more extensive KC aggregation that in turn will lead to a
higher elastic modulus. As seen from Fig. 8, the control sample that
was cured in a refrigerator at 5  C, showed a higher effective
modulus at t ¼ 0 than all the gels that were quenched in ice water,
which is what we expect for a gel cured using a lower cooling rate.
Our last conclusion, inferred from the findings of Fig. 8, can be
summarized schematically as in the drawings of Fig. 9. In the left
panel, we drew the bicontinuous structure we consider to be
formed in the KC/IC mixed gels. In the right panel, the extent of the
phase separation is enhanced by an initial curing of IC at temper-
atures above the coil-to-helix transition temperature of KC, and this
was represented by drawing each network on a larger scale.
4. Summary
IC addition to KC led to an initial increase in the elastic modulus
that passed through a peak, followed by a decrease to values close
to those of the pure KC gel. At small KC additions to IC, addition of
the elastic moduli strongly underestimated the elastic modulus.
These results support the presence of a bicontinuous structure at
high i:k ratios. A clear transition was seen at i:k ratios of 2:1 to 4:3
in measurements at both small and large deformation. We
conclude that phase separation takes place at the concentration
range tested, and it leads to formation of a bicontinuous structure.
The phase separation could be enhanced by allowing for curing of
IC at temperatures above the coil-to-helix transition temperature of
KC, and this coarsening of the structure led to a slight increase in
the elastic modulus. Further investigations are required to provide
spectroscopic evidence of the phase separated bicontinuous
structure and the structural transitions, and also to reproduce our
rheological findings using pure forms of KC and IC.
Acknowledgements
The present work was financially supported by Research and
Development Projects for Application in Promoting New Policy of
Agriculture, Forestry and Fisheries (No. 22026). R.T. acknowledges
receipt of financial support from the Estonian Ministry of Educa-
tion, targeted financing No. SF0690034s09.
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