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Mixed iota and kappa carrageenan gels in the presence of both calcium and potassium ions
Accepted Manuscript Title: Mixed Iota and Kappa Carrageenan Gels in the Presence of Both Calcium and Potassium Ions Authors: Viet T.N.T. Bui, Bach T. Nguyen, Taco Nicolai, Fr´ed´eric Renou PII: DOI: Article Number:
S0144-8617(19)30774-X https://doi.org/10.1016/j.carbpol.2019.115107 115107
Reference:
CARP 115107
To appear in: Received date: Revised date: Accepted date:
19 May 2019 17 July 2019 17 July 2019
Please cite this article as: Bui VTNT, Nguyen BT, Nicolai T, Renou F, Mixed Iota and Kappa Carrageenan Gels in the Presence of Both Calcium and Potassium Ions, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.115107 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mixed Iota and Kappa Carrageenan Gels in the Presence of Both Calcium and Potassium Ions
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Viet T.N.T. Bui1,2, Bach T. Nguyen2, Taco Nicolai1*, Frédéric Renou1
Le Mans Université, IMMM UMR-CNRS 6283, Polymères, Colloïdes et Interfaces, 72085 Le Mans,
cedex 9, France 2
Food Technology Faculty, Nha Trang University, Khanh Hoa, Vietnam
Email: [email protected]
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Graphical abstract
Highlights
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In the presence of both K+ and Ca2+, K+ initiates gelation of -car. In the presence of both K+ and Ca2+, Ca2+ initiates gelation of -car. Mixing K+ and Ca2+ has a synergistic effect on the gel stiffness Mixed -car /-car gels are more homogeneous than -car gels Structure and rheology of mixed -car /-car gels can be tuned by mixing K+ and Ca2+
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Abstract
The effect was studied of adding both KCl and CaCl2 on gelation of solutions of -
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carrageenan, -carrageenan and mixtures of both types. The gel temperature (Tg) of -car was found to be determined by the CaCl2 concentration and Tg of -car by the KCl concentration. At a given
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salt concentration, -car was stiffest with pure CaCl2, but -car gels and mixed carrageenan gels were stiffer when both KCl and CaCl2 were present. Gelation of -car increased the turbidity of mixed carrageenan gels in the presence of KCl or CaCl2, but when both salts were present it led to a
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drop of the turbidity. In mixed salt, K+ induces formation of a homogeneous -car network that causes the mixed network to become more homogeneous. Rheological and structural properties of carrageenan gels can be tuned for a given polymer and salt concentration by adding both KCl and CaCl2 to -car/-car mixtures. Keywords: -carrageenan; -carrageenan; gel; mixture; rheology; structure 2
1.
Introduction Carrageenan (Car) is a linear sulfated polysaccharide extracted from various species of edible
red algae and is widely used as thickener, stabilizer or gelling agent in food products, pharmaceutical applications and cosmetics. The molecular structure of Car is based on a
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disaccharide repeat of alternating units of D-galactose and 3,6-anhydro-galactose (3,6-AG) joined by α-1,4 and β-1,3-glycosidic linkage. It is classified into various types of Car, but kappa carrageenan (κ-car) and iota carrageenan (ι-car) are the most common types used in the industry due to their good gelling properties (Trius & Sebranek, 1996; Van De Velde et al., 2005; Van de Velde, Pereira & Rollema, 2004). Car forms a thermal-reversible gel in aqueous solution via the transition from a random coil to a helical conformation followed by aggregation of the helices to form a
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space-spanning network (Landry & Rochas, 1990; Morris, 1986). The coil-helix transition is induced by cooling in the presence of cations. The differences in structure of κ- and ι-car result in
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differences in their gelling properties. κ-Car can form a strong gel in the presence of specific
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monovalent cations, whereas the conformation transition of ι-car is particularly sensitive to divalent
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ions and forms a weak gel (Landry & Rochas, 1990; Robal et al., 2014; Rochas & Rinaudo, 1980). Mixtures of ι-car and κ-car were in first instance studied in order to measure the effect on
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gelation of κ-car samples containing a small fraction of ι-car and vice versa (Piculell, Nilsson & Muhrbeck, 1992) (Rochas & Rinaudo, 1980; Rochas, Rinaudo & Landry, 1989). Rochas et
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al.(1989) found that the elastic modulus of mixed gels decreased with increasing ι-car content. However, the yield stress of the mixed gels at a 50-50 ratio was higher than the sum of the
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individual Car gels. Later on, more studies were done on the behavior of Car in mixed systems by using other techniques such as DSC (Differential Scanning Calorimetry), CLSM, NMR, turbidity
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and rheological measurements (Brenner, Tuvikene, Parker, Matsukawa & Nishinari, 2014; Bui, Nguyen, Nicolai & Renou, 2019; Bui, Nguyen, Renou & Nicolai, 2019; Du, Brenner, Xie &
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Matsukawa, 2016; Hu, Du & Matsukawa, 2016; Lundin & Norton, 2000; Parker, Brigand, Miniou, Trespoey & Vallée, 1993; Ridout, Garza, Brownsey & Morris, 1996). These studies showed a twostep gelation process of the mixed gel during cooling with each step corresponding to gelation of the each type of Car with that of ι-car at higher temperatures followed by that of κ-car at lower temperatures. A strong synergy was observed between the two types of Car, which has in past been attributed to microphase separation between ι-car and κ-car when the latter gels (Brenner, Tuvikene, Parker, Matsukawa & Nishinari, 2014; Du, Brenner, Xie & Matsukawa, 2016; Hu, Du & 3
Matsukawa, 2016; Lundin & Norton, 2000; Piculell, Nilsson & Muhrbeck, 1992). Recently, we argued that it was more likely that the mixtures formed homogeneously interpenetrated or interconnected networks (Bui, Nguyen, Renou & Nicolai, 2019). Investigations of mixed ι-car and κ-car reported so far were done in the presence of either KCl or CaCl2. However, in applications often both types of salts are present. In addition, synergistic effects could potentially be obtained by
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adding both salts in a controlled manner.
The effect of adding mixtures of different types of salt on gelation of individual κ-car solutions has already been reported in the literature. Synergistic effects were found for the gelation of κ-car in the presence of a mixture of NaCl and KCl (Mangione, Giacomazza, Bulone, Martorana & San Biagio, 2003), or of a mixture of KCl or NaCl with CaCl2 (Hermansson, Eriksson & Jordansson, 1991; Nguyen, Nicolai, Benyahia & Chassenieux, 2014; Wang, Yuan, Cui & Liu, 2018;
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Williams, Phillips, Doyle, Giannouli, Philp & Morris, 2002). Stronger synergistic effects were
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found when CaCl2 was mixed with potassium than with sodium. Chen, Liao & Dunstan (2002)
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observed that the stiffness of gels formed by a commercial κ-car sample was much reduced after purification and concluded that this was caused by the synergy between Ca2+ and K+ in the
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unpurified sample. The effect of adding mixtures of salt on individual ι-car and mixed ι-car and κcar gels has not yet been investigated. Here we report on an investigation of the rheology and
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microstructure of individual and mixed ι-car and κ-car gels in the presence of mixtures of KCl and
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CaCl2. The principal objective is to establish if and to what extent the structure and stiffness of Car
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gels can be manipulated by using mixtures of ι-car and κ-car and mixtures of KCl and CaCl2.
Materials and methods
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2.1. Materials
Native ι-car and κ-car were extracted from two types of red seaweed: Kappaphycus alvarezii
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and Eucheuma denticulatum, respectively, by using the water extraction method as described by Bui et al. (Bui, Nguyen, Renou & Nicolai, 2018). The crude Car extracts were purified by dialysis first against 0.1M NaCl in order to exchange other cations by sodium and subsequently against Milli-Q water to remove excess salt. The purified Car was freeze-dried. The concentrations of Na+ and Ca2+ in the purified Car were 41.5 mg/g and 0.075 mg/g, respectively, for κ-car and 55.4 mg/g and 0.4 mg/g for ι-car, whereas no K+ was detected. The molar mass and hydrodynamic radius of κ-car 4
(1.0106 g/mol, 180 nm) and ι-car (1.1106 g/mol, 170 nm) were determined by light scattering techniques as described elsewhere (Bui, Nguyen, Renou & Nicolai, 2018). Fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC) and other chemicals were purchased from Sigma Aldrich (Germany). Sample preparation
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2.2.
The purified carrageenans were covalently labelled with FITC or RITC following the method described by Heilig et al. (Heilig, Göggerle & Hinrichs, 2009) with minor modifications. Briefly, 20 ml DMSO and 80 μl pyridine were mixed with 1 g Car and stirred at 70 °C for 30 min. After adding 0.1 g FITC or RBITC and 40 μl dibutyltin dilaurate, the mixture was incubated for 3 h at 70°C. The Car was then precipitated and washed many times with ethanol 95% until the waste solvent became
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colorless. The precipitate was dissolved and dialysed against Milli-Q water in order to remove any residual free fluorophore in dialysis bags with a 3.5 kDa cut-off. The fluorophore absorbance at 480
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nm of the bath water was checked and the dialysis process was considered complete when the
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absorbance was negligible. After purification the labelled Car was freeze-dried. Comparison of the
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absorbance of labelled Car with that of known concentrations of the fluorophore showed that about 1 in 100 repeat units was labelled. It was verified that the rheological properties of the labelled Car
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were the same as for unlabelled Car.
Stock solutions of labelled and non-labelled Car solutions were prepared by dissolving the
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freeze-dried Car at a concentration of 30 g/L in Milli-Q water with 200 ppm sodium azide added as a bacteriostastic agent by stirring for few hours at 70°C. The pH of the stock solution was 7.2.
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Aliquots of 1M KCl and/ or CaCl2 solutions were added in the required amounts to individual and mixed Car solutions at 90 °C while stirring and kept at 90 °C for 30 min. For observation with
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CLSM a small amount of labelled Car was added. 2.3.
Methods
2.3.1.
Rheological measurements
The rheological properties were determined using a rheometer (ARG2, TA Instruments) in
combination with a plate – plate geometry (diameter 40 mm, gap 1 mm). The storage (G′) and loss moduli (G″) were determined as a function of the temperature during heating and cooling ramps at 0.1 Hz at a rate of 2°C/min. The solutions were subsequently kept at 5°C during 1 h after which the frequency dependence of G′ and G″ was determined between 0.01 and 10 Hz. The temperature was 5
controlled by a Peltier system and the geometry was covered with paraffin oil to prevent water evaporation. Measurements were done at shear stresses within the linear response regime. 2.3.2.
Tubidity measurements
The turbidity of the pure carrageenan and mixtures was measured as a function of the
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temperature in 1 cm cells using a UV-visible spectrometer Varian Cary – 50 Bio. The temperature was varied from 85 °C to 5 °C at 2°C/ min at a wavelength = 600nm. 2.3.3.
Confocal Laser Scanning Microscopy (CLSM)
ι- and κ-car were visualized separately by using different fluorescent labelling. κ-Car was labelled with FITC, and ι-car was labelled with RITC. CLSM observations were made with a Zeiss LSM800 microscope (Jena, Germany) and with a water immersion objective 63x. The solutions
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were inserted between a concave slide and a coverslip and hermetically sealed. The incident light
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was emitted by a laser beam at 543 and/or 488 nm. The signal from FITC was collected at
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wavelengths below 520 nm where RITC does not emit light. The signal from RITC was detected at
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wavelengths above 550 nm where FITC emits relatively little light. Results Rheology
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Fig. 1a shows the evolution of the storage modulus (G’) during a cooling ramp at a rate of 2 C per minute for mixtures of ι-car and κ-car each at C = 5 g/L in the presence of different fractions
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of CaCl2 to KCl. The total salt concentration was kept fixed at 40 mM. A two step increase of G'
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was observed in all cases. As was mentioned in the Introduction earlier studies of mixtures of ι-car and κ-car in comparison with that of individual Car solutions have shown that the increase at higher
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temperatures corresponds to gelation of ι-car followed by that of κ-car at lower temperatures. Here the first step led to relatively weak gels formed by ι-car and the second step at lower temperatures
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led to stronger gels caused by the formation of a κ-car network. These results agree with reports on gelation of mixed Car in presence of individual salts KCl, NaCl or CaCl2 (Bui, Nguyen, Renou & Nicolai, 2019; Du, Brenner, Xie & Matsukawa, 2016; Lundin & Norton, 2000). With increasing fraction of CaCl2, Tg of κ-car decreases and Tg of ι-car increases.
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Fig.1 Storage modulus measured during cooling ramps at a rate of 2 °C/min for solutions of mixed
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ι- and κ-car each at 5 g/L at different compositions of CaCl2-KCl keeping the total salt concentration
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fixed at 40 mM (a) and the elastic modulus obtained after 1h at 5 °C as a function of the CaCl2 and
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KCl concentration (b).
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After the cooling ramp, the samples were kept at 5 °C for one hour and the frequency dependence of G' and G" was measured. G' of the gels was larger than G" and independent of the
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frequency at low frequencies (results not shown). The elastic modulus (Gel) taken as G' at low frequencies is plotted in Fig.1b as a function of the CaCl2 and KCl concentration keeping the total
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salt concentration fixed at 40 mM. Gel of the samples in the presence of mixed salts was higher than samples with just one type of salt at the same concentration. In particular, addition of even a small amount of KCl (5 mM) led to a strong increase of Gel, but replacement of some KCl by CaCl2 also
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increased the gel stiffness. Fig. 2a shows the storage modulus during a cooling ramp for mixtures of ι-car and κ-car
each at 5 g/L at different total salt concentrations with equal amounts of each salt. Two step gelation was again observed with an increase of Tg for both types of Car with increasing salt concentration. Tg was determined as the temperature where G' started to increased. For κ-car the increase was sharp and Tg could be determined without much ambiguity. For ι-car the increase of G' is more 7
progressive and we have taken Tg as the temperature where G' = 1 Pa. For mixtures we have taken Tg of κ-car as the temperature where G' shows a sharp inflexion. It is clear that the absolute values of Tg will depend to some extent on the manner they are determined and the rate of the temperature ramp. However, considering that the same manner is used for all systems the uncertainty in the relative changes as a function of the salt concentration is much smaller. Based on many
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measurements on different systems, not only those shown here, we estimate that the uncertainty in the value of Tg is 2 °C. Tg of ι-car and κ-car increased initially steeply with increasing salt concentration and more weakly at higher salt concentrations, see red symbols in Fig. 2b. 80
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Fig.2 Storage modulus measured during cooling ramps at a rate of 2 °C/min for mixtures of ι-car and κ-car containing 5 g/L of each in the presence of equal amounts of KCl and CaCl2 as indicated
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the figure (a) and the gel temperature of ι-car and κ-car in mixed systems as a function of the total salt concentration (b) with ι-car and κ-car (red symbols) or individual ι-car and κ-car gels formed in
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mixed salt (blue symbols) and individual ι-car and κ-car gels formed with only CaCl2 or KCl, respectively (green symbols). Notice that in the latter case the results are shown as a function of the
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same CaCl2 or KCl concentration as in the mixed salt case.
We have compared the results obtained for mixed Car solutions with those for individual ιcar and κ-car solutions at the same concentra tion as in the mixtures (5 g/L) both with mixed salt and with single salt, see in Fig. 2b. The values of Tg for individual ι-car solutions with mixed salt (blue symbols) were close to that with just CaCl2 at the same concentration as in the mixture (green 8
symbols). On the other hand, the values of Tg for individual κ-car solutions with mixed salt were close to that with just KCl. From this comparison we may conclude that ι-car and κ-car gelation in Car mixtures containing both salts is driven primarily by the cations for which they are most sensitive, i.e. CaCl2 for ι-car and KCl for κ-car. Fig. 3 shows the elastic moduli obtained after one hour at 5 °C as a function of the total salt
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concentration for individual ι-car (Fig. 3a) and κ-car (Fig. 3b) gels at 5 g/L as well as for mixed Car gels containing 5 g/L of each type (Fig. 3c). Based on many measurements on different systems, not only those shown here, we estimate that the uncertainty in the value of G' is about 20%. In all cases, Gel increased with increasing salt concentration until it reached a plateau at high salt concentrations. Gel of individual ι-car gels was higher in the presence of CaCl2 than in the presence of KCl, whereas the inverse was the case for κ-car gels. Gel of ι-car gels in the presence of both salts was close to that
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in the presence of just CaCl2, but κ-car gels were stiffer in the presence of both salts than with just
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Fig. 3. Elastic moduli of individual ι-car (a), κ-car (b) and mixed Car (c) gels obtained after cooling
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1 h at 5 °C as a function of the total salt concentration. The individual gels contained 5 g/L Car and the mixed gels contained 5 g/L of each type of Car. Results obtained with CaCl 2, KCl or equal
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quantities of both types of salt are indicated by different symbols as indicated in Fig. 3a.
We may also compare mixed Car gels with individual gels with the same ι-car or κ-car
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concentration as in the mixed Car gels. In the presence of CaCl2 the mixed Car gels are clearly
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stiffer than the sum of individual ι-car or κ-car gels demonstrating the synergy between the two types of Car. In the presence of KCl or both salts the elastic modulus of the mixed Car gels was
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quite close to that of individual κ-car gels. Though the values of G' were systematically slightly larger for the mixed Car gels the difference is too small to conclude that there is synergy between ι-
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car and κ-car in this case. It is clear that in the mixed Car gels the contribution of κ-car is in all cases much more important than that of ι-car.
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3.2 Turbidity The effect of gelation in the presence of both salts on the turbidity was measured during
cooling ramps at a rate of 2 °C per minute. Fig. 4 shows the effect of varying the total salt concentration at equal KCl and CaCl2 concentrations for individual κ-car (Fig. 4a) and ι-car (Fig. 4b) solutions at 10 g/L as well as for mixed Car solutions containing 5 g/L of each (Fig. 4c,d). As expected, the turbidity increased with increasing total salt concentration for all systems, which is 10
caused by an increase of the amplitude of the Car concentration fluctuations. However, there are remarkable differences in the temperature dependence. The turbidity of individual ι-car solutions depended only very weakly on the temperature, see Fig. 4a, whereas that of individual κ-car solutions increased sharply at Tg, see Fig. 4b. The increase of the turbidity of κ-car gels with
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decreasing temperature weakened first, but then showed again a faster increase below about 15°C.
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Fig. 4.Turbidity at 600 nm during cooling for ι-car (a) and κ-car (b) solutions at 10 g/L and for Car mixtures containing 5 g/L of each (c, d). The salt concentrations, CaCl2-KCl, in the systems are indicated in the figures.
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The turbidity of the mixed Car solutions with both salts increased progressively with decreasing temperature below Tg of κ-car, see Figs 4c and 4d. This increase has already been reported elsewhere for mixed Car gels in the presence of just CaCl2 (Bui, Nguyen, Renou & Nicolai, 2019). If only CaCl2 is present the progressive increase of the turbidity is followed by a sharp increase at Tg of κ-car, see Fig. 4d. However, when both salts are present the turbidity initially
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decreases sharply at Tg of κ-car followed by a weak increase at lower T. Fig. 4d shows that a relatively small fraction of KCl already has a strong influence on the turbidity.
In order to investigate further the drop of the turbidity of the mixed Car solutions when κ-car gels, we measured the turbidity as a function of time during and after decreasing the temperature as fast as possible to different values close to Tg of κ-car, see Fig. 5. It took between 8 and 10 min to
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reach the set temperature to within less than 0.5 °C. The turbidity in the presence of both KCl and CaCl2 at 20 mM shown in Fig. 5a may be compared with those solutions containing only 20 mM
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CaCl2 shown in Fig. 5b. When only CaCl2 was present the turbidity increased progressively with
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decreasing temperature and continued to increase, albeit more slowly, once the set temperature had been reached implying that the gel structure continued to evolve. However, when in addition 20 mM
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KCl was present, the turbidity passed through a minimum at T < 30 °C, i.e. when κ-car gelled. It appears that gelation of κ-car initially caused a decrease of the amplitude of the Car concentration
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fluctuations in the system. With time the κ-car network becomes more densely crosslinked and more
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heterogeneous leading eventually to a larger turbidity than in the absence of KCl.
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Fig. 5.Turbidity at 600 nm as a function of time of mixed ι-car and κ-car (5-5 g/L) in the presence of 20 mM KCl and 20 mM CaCl2 (a) or only 20 mM CaCl2 (b) at different temperatures indicated in Fig. 5a. 3.3 Microstructure
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The turbidity is related to the amplitude of the overall Car concentration fluctuations, but does not inform about the microstructure of the ι-car and κ-car networks within the mixed gel. The microstructure of each network can be probed using CLSM microscopy utilizing different fluorescent labels for each type of Car. We checked that the brightness in the images is proportional to the concentration of the labelled Car. CLSM images were obtained at 20 °C after cooling from 90 °C solutions containing 5 g/L of ι-car and κ-car. Fig. 6 shows that the heterogeneity of the labelled
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κ-car network increased with increasing concentration of CaCl2 in approximately the same manner whether the total salt concentration was fixed or the concentration of KCl was kept the same as that
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of CaCl2. Notice, however, that in all cases the contrast in brightness is very weak, because the
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amplitude of the concentration fluctuations of labelled Car is very small. Elsewhere, we showed that
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in the presence of only CaCl2 the amplitude of the concentration fluctuations of the κ-car network in mixed Car gels was smaller than for individual the κ-car gels (Bui, Nguyen, Renou & Nicolai,
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2019). We also showed that the characteristic length scale of the concentration fluctuations of κ-car obtained from the pair correlation function of the fluorescence intensity fluctuations was
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approximately one µm in the presence of CaCl2. Here we find that this length scale was not significantly different in mixed salts. The labelled ι-car network in the mixed Car gel was in all
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cases homogenous on length scales accessible to CLSM (images not shown).
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10 µm
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Fig.6. CLSM images at 20 °C of the labelled κ-car network in mixed ι- and κ-car gels containing 5
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g/L of each Car in the presence of different amounts of CaCl2-KCl keeping the total salt concentration fixed at 40 mM (top) or keeping the KCl equal to that of CaCl2 (bottom). The
Discussion
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4.
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brightness is proportional to the κ-car concentration.
Here we have shown the effect of adding both CaCl2 and KCl on the gel temperature, the gel
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stiffness and the microstructure of individual and mixed ι-car and κ-car gels. Synergistic effects were observed both between the two types of salt and between the two types of Car. In order to
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appreciate the effect of adding both salts in mixtures of Car we will first briefly discuss individual ιcar and κ-car gelation as well as that of Car mixtures in the presence of either CaCl2 of KCl. Then
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we discuss the effect of adding both CaCl2 and KCl on individual Car gelation and on mixed Car gelation..
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Individual ι-car and κ-car gelation in the presence of either CaCl2 or KCl. Gelation of ι-car
and κ-car occurs during cooling at a temperature that increases with increasing CaCl2 or KCl concentration. Tg of ι-car is higher in the presence of CaCl2 than in the presence of KCl at the same salt concentration, whereas for κ-car the inverse is the case. Here we found that for both types of Car, Gel increased initially with increasing salt concentration and remained approximately constant at higher salt concentration. We note, however, a difference between the dependence of G el on the 14
CaCl2 concentration between the native κ-car used here and the commercial κ-car used in other studies reported in the literature. For commercial κ-car, Gel was found to decrease at higher CaCl2 concentrations (MacArtain, Jacquier & Dawson, 2003; Nguyen, Nicolai, Benyahia & Chassenieux, 2014; Thrimawithana, Young, Dunstan & Alany, 2010), whereas here we observed that Gel remained approximately the same in this concentration range. Individual κ-car gels are more turbid
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and have a more heterogeneous microstructure in the presence of CaCl2 than in the presence of KCl. Interestingly, the effect of CaCl2 on the turbidity was found to be stronger for commercial κ-car (MacArtain, Jacquier & Dawson, 2003; Nguyen, Nicolai, Benyahia & Chassenieux, 2014) than for the native κ-car used here (Bui, Nguyen, Renou & Nicolai, 2019). Apparently, native κ-car forms less heterogeneous gels with Ca2+ than commercial κ-car, which is perhaps related to the difference in the dependence of Gel on the CaCl2 concentration. ι-car gels are more homogeneous than κ-car
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gels both in the presence of KCl and CaCl2.
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Mixed ι-car and κ-car gelation in the presence of either CaCl2 or KCl. Gelation of ι-car and κ-
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car in mixtures occurs at the same temperatures as for individual solutions (Brenner, Tuvikene, Parker, Matsukawa & Nishinari, 2014; Bui, Nguyen, Nicolai & Renou, 2019; Bui, Nguyen, Renou
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& Nicolai, 2019; Du, Brenner, Xie & Matsukawa, 2016; Lundin & Norton, 2000; Parker, Brigand, Miniou, Trespoey & Vallée, 1993; Piculell, Nilsson & Muhrbeck, 1992). Tg is higher for ι-car than
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for κ-car and the elastic modulus of the mixed Car gel is close to that of individual ι-car gels as long
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as κ-car is not crosslinked. However, Gel increases sharply below Tg of κ-car and becomes larger than the sum of the elastic moduli of the individual Car networks at the same concentration as in the
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mixtures. As was mentioned in the introduction, this synergy has been attributed either to microphase separation between ι-car and κ-car when the latter gels or to modification of the interpenetrated or interconnected networks. In the presence of CaCl2 the turbidity of the mixed Car
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systems increases with decreasing temperature between Tg of ι-car and κ-car, see for example Fig. 4d. This means the presence of κ-car within the ι-car network increases the amplitude of the
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concentration fluctuation. At Tg of κ-car the turbidity starts to increase more sharply, but remarkably the increase caused by κ-car gelation in the mixed Car systems is less than for individual κ-car solutions (Bui, Nguyen, Renou & Nicolai, 2019; Lundin & Norton, 2000). This observation was confirmed by CLSM images that showed that the κ-car network in mixed Car gels was less heterogeneous than individual κ-car networks (Bui, Nguyen, Renou & Nicolai, 2019). The ι-car
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network was found to be homogeneous on length scales accessible to CLSM for both individual and mixed gels. Individual ι-car and κ-car gelation in the presence of both CaCl2 and KCl. At most conditions studied here gelation of ι-car was induced by the CaCl2 in the system and that of κ-car was induced by the KCl. The gel temperatures with mixed salts were close to that obtained with just
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CaCl2 and KCl at the same concentrations as in the mixtures for ι-car and κ-car, respectively, see Fig. 1b. The elastic modulus of ι-car gels in the presence of mixed salts was the same as in the absence of KCl. However, Gel of κ-car gels was higher when both types of salt were present, which means that CaCl2 reinforced the κ-car network. Synergy between CaCl2 and KCl with respect to the stiffness of κ-car gels had already been reported in the literature (Hermansson, Eriksson & Jordansson, 1991; Nguyen, Nicolai, Benyahia & Chassenieux, 2014; Robal et al., 2014; Wang,
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Yuan, Cui & Liu, 2018). The turbidity of ι-car gels increased weakly with increasing salt
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concentration, but remained low in the presence of either or both types of salt. Nguyen et al.
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(Nguyen, Nicolai, Benyahia & Chassenieux, 2014) observed that the turbidity of commercial κ-car gels in the presence of CaCl2 was much reduced if KCl was present as well. The turbidity of native
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κ-car gels was also found to be lower in mixed salt than with just CaCl2 at the same concentration as in the mixed salt system. However, the turbidity of κ-car gels in mixed salt is still larger than in the
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presence of just KCl indicating that CaCl2 influences the structure of the κ-car network even if its
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formation is induced by K+. It appears that K+ does not have a significant influence on the ι-car network formation induced by the presence of Ca2+, but that Ca2+ renders the κ-car network induced
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by K+ stiffer and more heterogeneous. Mixed ι-car and κ-car gelation in the presence of both CaCl2 and KCl. In mixed Car solutions
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that contain both types of salt, Tg of ι-car is determined by the CaCl2 concentration and Tg of κ-car is determined by the KCl concentration, see Fig. 2b. The gelation temperature of each Car in the mixtures is not influenced by the presence of the other type of Car nor by the presence of the other
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type of salt. However, adding both salts led to an increase of Gel of mixed Car gels similar to that of individual κ-car gels. Between Tg of ι-car and κ-car, the turbidity of mixed Car solutions in mixed salt increased with decreasing temperature similar to mixed Car systems containing just CaCl2 and was determined by the amount of CaCl2 in the system. Remarkably, gelation of κ-car induced a sharp decrease of the turbidity when KCl was present together with CaCl2. Gelation of κ-car in the mixed salt is induced by KCl, which causes formation of a homogeneous network. It appears that 16
gelation of κ-car induced by KCl causes a decrease of the amplitude of the concentration fluctuations in the Car mixture. It was reported in the literature that gelation of κ-car induced by KCl can inverse microphase separation in mixtures with whey protein aggregates, which was attributed to the elasticity of the κ-car network (Ako, Durand & Nicolai, 2011; Baussay, Durand & Nicolai, 2006). The reduction of the amplitude of the concentration fluctuations in mixed Car
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systems containing both salts may have the same origin. The synergistic effect between ι-car and κcar on the gel stiffness was maintained when both salt were present, but the sharp drop of the turbidity when κ-car gelled renders microphase separation between the two types of Car an unlikely explanation of this synergy. As the crosslink density of the κ-car network increased with decreased temperature, the turbidity was observed to increased again, which we attribute to the effect of CaCl2 on the κ-car network structure. Measurements at different constant temperatures illustrate the
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consecutive effects of κ-car network formation induced by KCl and the coarsening of the network
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induced by CaCl2, that cause first a decrease of the turbidity followed by an increase, see Fig. 5a.
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Conclusion
ι- and κ-car solutions form gels when cooled in the presence of either CaCl 2 or KCl, but they
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have different sensitivities for CaCl2 and KCl. As a consequence in the presence of both types of salt κ-car gelation is induced by KCl and gelation of ι-car by CaCl2. This is the case both for
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individual Car solutions and for mixtures of both types of Car. In mixed salt, ι-car gels at higher temperatures than κ-car, but forms much weaker gels. The elastic modulus of the ι-car network is
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determined by the CaCl2 concentration and is not influenced significantly by the presence of KCl nor by the presence of κ-car in the coil conformation. The elastic modulus of the κ-car network in
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mixed salt is larger than in pure KCl or pure CaCl2 at the same total salt concentration. The same synergy is observed for individual κ-car gels and for mixed Car gels. The heterogeneity of κ-car gels
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formed in the presence of CaCl2 can be reduced by adding ι-car or adding KCl. Gelation of κ-car induced by K+ in mixed Car systems containing both types of salt cause the mixed gel to be more homogeneous, which renders it unlikely that microphase separation between ι-car and κ-car is the origin of the synergy. Mixing different types of Car and different types of salt can be used to tune the mechanical and structural properties of Car gels for given total Car and salt concentrations.
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References
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Ako, K., Durand, D., & Nicolai, T. (2011). Phase separation driven by aggregation can be reversed by elasticity in gelling mixtures of polysaccharides and proteins. Soft Matter, 7(6), 2507-2516. Baussay, K., Durand, D., & Nicolai, T. (2006). Coupling between polysaccharide gelation and micro-phase separation of globular protein clusters. Journal of colloid and interface science, 304(2), 335-341. Brenner, T., Tuvikene, R., Parker, A., Matsukawa, S., & Nishinari, K. (2014). Rheology and structure of mixed kappa-carrageenan/iota-carrageenan gels. Food Hydrocolloids, 39, 272-279. Bui, V. T. N. T., Nguyen, B. T., Nicolai, T., & Renou, F. (2019). Mobility of carrageenan chains in iota- and kappa carrageenan gels. Colloids and Surfaces A, 562(October 2018), 113-118. Bui, V. T. N. T., Nguyen, B. T., Renou, F., & Nicolai, T. (2018). Structure and rheological properties of carrageenans extracted from different red algae species cultivated in Cam Ranh Bay, Vietnam. Journal of Applied Phycology. Bui, V. T. N. T., Nguyen, B. T., Renou, F., & Nicolai, T. (2019). Rheology and microstructure of mixtures of iota and kappa-carrageenan. Food Hydrocolloids, 89, 180-187. Chen, Y., Liao, M.-L., & Dunstan, D. E. (2002). The rheology of K+ -carrageenan as a weak gel. Carbohydrate Polymers, 50(2), 109-116. Du, L., Brenner, T., Xie, J., & Matsukawa, S. (2016). A study on phase separation behavior in kappa/iota carrageenan mixtures by micro DSC, rheological measurements and simulating water and cations migration between phases. Food Hydrocolloids, 55, 81-88. Heilig, A., Göggerle, A., & Hinrichs, J. (2009). Multiphase visualisation of fat containing β-lactoglobulin-κ-carrageenan gels by confocal scanning laser microscopy, using a novel dye, V03-01136, for fat staining. LWT - Food Science and Technology, 42(2), 646-653. Hermansson, A., Eriksson, E., & Jordansson, E. (1991). Effects of Potassium , Sodium and Calcium on the Microstructure and Rheologicai Behaviour of KappaCarrageenan Gels. Carbohydrate Polymers, 16, 297-320. Hu, B., Du, L., & Matsukawa, S. (2016). NMR study on the network structure of a mixed gel of kappa and iota carrageenans. Carbohydrate Polymers, 150, 57-64. Landry, M. R. S., & Rochas, C. (1990). Role of the Molecular Weight on the Mechanical Cels Properties of Kappa Carrageenan previously. 2, 255-266. Lundin, L. O. K. F. T. J., & Norton, I. T. (2000). Phase separation in mixed carrageenan systems. Supermolecular and colloidal structures in Biomaterials and Biosubstrates pp. 436-449): ICP. 18
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MacArtain, P., Jacquier, J. C., & Dawson, K. A. (2003). Physical characteristics of calcium induced κ-carrageenan networks. Carbohydrate Polymers, 53(4), 395-400. Mangione, M. R., Giacomazza, D., Bulone, D., Martorana, V., & San Biagio, P. L. (2003). Thermoreversible gelation of κ-Carrageenan: Relation between conformational transition and aggregation. Biophysical Chemistry, 104(1), 95-105. Morris, E. R. (1986). Molecular Interactions in Polysaccharide Gelation. British Polymer Journal, 18(1), 14-21. Nguyen, B. T., Nicolai, T., Benyahia, L., & Chassenieux, C. (2014). Synergistic effects of mixed salt on the gelation of -carrageenan. Carbohydrate Polymers, 112, 10-15. Parker, A., Brigand, G., Miniou, C., Trespoey, A., & Vallée, P. (1993). Rheology and fracture of mixed - and i-carrageenan gels: Two-step gelation. Carbohydrate Polymers, 20(4), 253-262. Piculell, L., Nilsson, S., & Muhrbeck, P. (1992). Effects of small amounts of kappacarrageenan on the rheology of aqueous iota-carrageenan. Carbohydrate Polymers, 18(3), 199-208. Ridout, M. J., Garza, S., Brownsey, G. J., & Morris, V. J. (1996). Mixed iota-kappa carrageenan gels. International Journal of Biological Macromolecules, 18(1-2), 5-8. Robal, M., Brenner, T., Matsukawa, S., Ogawa, H., Truus, K., Rudolph, B., & Tuvikene, R. (2014). Monocationic salts of carrageenans: Preparation and physicochemical properties. Food Hydrocolloids, 63, 656-667. Rochas, C., & Rinaudo, M. (1980). Activity Coefficients of Counterions and Conformation in Kappa-Carrageenna System. Biopolymers, 19, 1675-1687. Rochas, C., Rinaudo, M., & Landry, S. (1989). Relation between the molecular structure and mechanical properties of carrageenan gels. Carbohydrate Polymers, 10(2), 115-127. Thrimawithana, T. R., Young, S., Dunstan, D. E., & Alany, R. G. (2010). Texture and rheological characterization of kappa and iota carrageenan in the presence of counter ions. Carbohydrate Polymers, 82(1), 69-77. Trius, A., & Sebranek, J. G. (1996). Carrageenans and Their Use in Meat Products. Critical Reviews in Food Science and Nutrition, 36(1-2), 69-85. Van De Velde, F., Antipova, A. S., Rollema, H. S., Burova, T. V., Grinberg, N. V., Pereira, L., Gilsenan, P. M., Tromp, R. H., Rudolph, B., & Grinberg, V. Y. (2005). The structure of /-hybrid carrageenans II. Coil-helix transition as a function of chain composition. Carbohydrate Research, 340, 1113-1129. Van de Velde, F., Pereira, L., & Rollema, H. S. (2004). The revised NMR chemical shift data of carrageenans. Carbohydrate Research, 339, 2309-2313. Wang, Y., Yuan, C., Cui, B., & Liu, Y. (2018). Influence of cations on texture, compressive elastic modulus, sol-gel transition and freeze-thaw properties of kappacarrageenan gel. Carbohydrate Polymers, 202, 530-535. 19
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Williams, P. A., Phillips, G. O., Doyle, J., Giannouli, P., Philp, K., & Morris, E. R. (2002). Effect of K+ and Ca2+ cations on gelation of -carrageenan. Gums and Stabilisers for the Food Industry 11 pp. 158-164.
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S0144-8617(19)30774-X https://doi.org/10.1016/j.carbpol.2019.115107 115107
Reference:
CARP 115107
To appear in: Received date: Revised date: Accepted date:
19 May 2019 17 July 2019 17 July 2019
Please cite this article as: Bui VTNT, Nguyen BT, Nicolai T, Renou F, Mixed Iota and Kappa Carrageenan Gels in the Presence of Both Calcium and Potassium Ions, Carbohydrate Polymers (2019), https://doi.org/10.1016/j.carbpol.2019.115107 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mixed Iota and Kappa Carrageenan Gels in the Presence of Both Calcium and Potassium Ions
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Viet T.N.T. Bui1,2, Bach T. Nguyen2, Taco Nicolai1*, Frédéric Renou1
Le Mans Université, IMMM UMR-CNRS 6283, Polymères, Colloïdes et Interfaces, 72085 Le Mans,
cedex 9, France 2
Food Technology Faculty, Nha Trang University, Khanh Hoa, Vietnam
Email: [email protected]
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Tel: 33-(0)43833119
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Graphical abstract
Highlights
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In the presence of both K+ and Ca2+, K+ initiates gelation of -car. In the presence of both K+ and Ca2+, Ca2+ initiates gelation of -car. Mixing K+ and Ca2+ has a synergistic effect on the gel stiffness Mixed -car /-car gels are more homogeneous than -car gels Structure and rheology of mixed -car /-car gels can be tuned by mixing K+ and Ca2+
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Abstract
The effect was studied of adding both KCl and CaCl2 on gelation of solutions of -
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carrageenan, -carrageenan and mixtures of both types. The gel temperature (Tg) of -car was found to be determined by the CaCl2 concentration and Tg of -car by the KCl concentration. At a given
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salt concentration, -car was stiffest with pure CaCl2, but -car gels and mixed carrageenan gels were stiffer when both KCl and CaCl2 were present. Gelation of -car increased the turbidity of mixed carrageenan gels in the presence of KCl or CaCl2, but when both salts were present it led to a
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drop of the turbidity. In mixed salt, K+ induces formation of a homogeneous -car network that causes the mixed network to become more homogeneous. Rheological and structural properties of carrageenan gels can be tuned for a given polymer and salt concentration by adding both KCl and CaCl2 to -car/-car mixtures. Keywords: -carrageenan; -carrageenan; gel; mixture; rheology; structure 2
1.
Introduction Carrageenan (Car) is a linear sulfated polysaccharide extracted from various species of edible
red algae and is widely used as thickener, stabilizer or gelling agent in food products, pharmaceutical applications and cosmetics. The molecular structure of Car is based on a
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disaccharide repeat of alternating units of D-galactose and 3,6-anhydro-galactose (3,6-AG) joined by α-1,4 and β-1,3-glycosidic linkage. It is classified into various types of Car, but kappa carrageenan (κ-car) and iota carrageenan (ι-car) are the most common types used in the industry due to their good gelling properties (Trius & Sebranek, 1996; Van De Velde et al., 2005; Van de Velde, Pereira & Rollema, 2004). Car forms a thermal-reversible gel in aqueous solution via the transition from a random coil to a helical conformation followed by aggregation of the helices to form a
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space-spanning network (Landry & Rochas, 1990; Morris, 1986). The coil-helix transition is induced by cooling in the presence of cations. The differences in structure of κ- and ι-car result in
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differences in their gelling properties. κ-Car can form a strong gel in the presence of specific
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monovalent cations, whereas the conformation transition of ι-car is particularly sensitive to divalent
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ions and forms a weak gel (Landry & Rochas, 1990; Robal et al., 2014; Rochas & Rinaudo, 1980). Mixtures of ι-car and κ-car were in first instance studied in order to measure the effect on
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gelation of κ-car samples containing a small fraction of ι-car and vice versa (Piculell, Nilsson & Muhrbeck, 1992) (Rochas & Rinaudo, 1980; Rochas, Rinaudo & Landry, 1989). Rochas et
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al.(1989) found that the elastic modulus of mixed gels decreased with increasing ι-car content. However, the yield stress of the mixed gels at a 50-50 ratio was higher than the sum of the
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individual Car gels. Later on, more studies were done on the behavior of Car in mixed systems by using other techniques such as DSC (Differential Scanning Calorimetry), CLSM, NMR, turbidity
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and rheological measurements (Brenner, Tuvikene, Parker, Matsukawa & Nishinari, 2014; Bui, Nguyen, Nicolai & Renou, 2019; Bui, Nguyen, Renou & Nicolai, 2019; Du, Brenner, Xie &
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Matsukawa, 2016; Hu, Du & Matsukawa, 2016; Lundin & Norton, 2000; Parker, Brigand, Miniou, Trespoey & Vallée, 1993; Ridout, Garza, Brownsey & Morris, 1996). These studies showed a twostep gelation process of the mixed gel during cooling with each step corresponding to gelation of the each type of Car with that of ι-car at higher temperatures followed by that of κ-car at lower temperatures. A strong synergy was observed between the two types of Car, which has in past been attributed to microphase separation between ι-car and κ-car when the latter gels (Brenner, Tuvikene, Parker, Matsukawa & Nishinari, 2014; Du, Brenner, Xie & Matsukawa, 2016; Hu, Du & 3
Matsukawa, 2016; Lundin & Norton, 2000; Piculell, Nilsson & Muhrbeck, 1992). Recently, we argued that it was more likely that the mixtures formed homogeneously interpenetrated or interconnected networks (Bui, Nguyen, Renou & Nicolai, 2019). Investigations of mixed ι-car and κ-car reported so far were done in the presence of either KCl or CaCl2. However, in applications often both types of salts are present. In addition, synergistic effects could potentially be obtained by
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adding both salts in a controlled manner.
The effect of adding mixtures of different types of salt on gelation of individual κ-car solutions has already been reported in the literature. Synergistic effects were found for the gelation of κ-car in the presence of a mixture of NaCl and KCl (Mangione, Giacomazza, Bulone, Martorana & San Biagio, 2003), or of a mixture of KCl or NaCl with CaCl2 (Hermansson, Eriksson & Jordansson, 1991; Nguyen, Nicolai, Benyahia & Chassenieux, 2014; Wang, Yuan, Cui & Liu, 2018;
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Williams, Phillips, Doyle, Giannouli, Philp & Morris, 2002). Stronger synergistic effects were
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found when CaCl2 was mixed with potassium than with sodium. Chen, Liao & Dunstan (2002)
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observed that the stiffness of gels formed by a commercial κ-car sample was much reduced after purification and concluded that this was caused by the synergy between Ca2+ and K+ in the
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unpurified sample. The effect of adding mixtures of salt on individual ι-car and mixed ι-car and κcar gels has not yet been investigated. Here we report on an investigation of the rheology and
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microstructure of individual and mixed ι-car and κ-car gels in the presence of mixtures of KCl and
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CaCl2. The principal objective is to establish if and to what extent the structure and stiffness of Car
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gels can be manipulated by using mixtures of ι-car and κ-car and mixtures of KCl and CaCl2.
Materials and methods
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2.1. Materials
Native ι-car and κ-car were extracted from two types of red seaweed: Kappaphycus alvarezii
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and Eucheuma denticulatum, respectively, by using the water extraction method as described by Bui et al. (Bui, Nguyen, Renou & Nicolai, 2018). The crude Car extracts were purified by dialysis first against 0.1M NaCl in order to exchange other cations by sodium and subsequently against Milli-Q water to remove excess salt. The purified Car was freeze-dried. The concentrations of Na+ and Ca2+ in the purified Car were 41.5 mg/g and 0.075 mg/g, respectively, for κ-car and 55.4 mg/g and 0.4 mg/g for ι-car, whereas no K+ was detected. The molar mass and hydrodynamic radius of κ-car 4
(1.0106 g/mol, 180 nm) and ι-car (1.1106 g/mol, 170 nm) were determined by light scattering techniques as described elsewhere (Bui, Nguyen, Renou & Nicolai, 2018). Fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RITC) and other chemicals were purchased from Sigma Aldrich (Germany). Sample preparation
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2.2.
The purified carrageenans were covalently labelled with FITC or RITC following the method described by Heilig et al. (Heilig, Göggerle & Hinrichs, 2009) with minor modifications. Briefly, 20 ml DMSO and 80 μl pyridine were mixed with 1 g Car and stirred at 70 °C for 30 min. After adding 0.1 g FITC or RBITC and 40 μl dibutyltin dilaurate, the mixture was incubated for 3 h at 70°C. The Car was then precipitated and washed many times with ethanol 95% until the waste solvent became
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colorless. The precipitate was dissolved and dialysed against Milli-Q water in order to remove any residual free fluorophore in dialysis bags with a 3.5 kDa cut-off. The fluorophore absorbance at 480
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nm of the bath water was checked and the dialysis process was considered complete when the
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absorbance was negligible. After purification the labelled Car was freeze-dried. Comparison of the
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absorbance of labelled Car with that of known concentrations of the fluorophore showed that about 1 in 100 repeat units was labelled. It was verified that the rheological properties of the labelled Car
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were the same as for unlabelled Car.
Stock solutions of labelled and non-labelled Car solutions were prepared by dissolving the
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freeze-dried Car at a concentration of 30 g/L in Milli-Q water with 200 ppm sodium azide added as a bacteriostastic agent by stirring for few hours at 70°C. The pH of the stock solution was 7.2.
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Aliquots of 1M KCl and/ or CaCl2 solutions were added in the required amounts to individual and mixed Car solutions at 90 °C while stirring and kept at 90 °C for 30 min. For observation with
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CLSM a small amount of labelled Car was added. 2.3.
Methods
2.3.1.
Rheological measurements
The rheological properties were determined using a rheometer (ARG2, TA Instruments) in
combination with a plate – plate geometry (diameter 40 mm, gap 1 mm). The storage (G′) and loss moduli (G″) were determined as a function of the temperature during heating and cooling ramps at 0.1 Hz at a rate of 2°C/min. The solutions were subsequently kept at 5°C during 1 h after which the frequency dependence of G′ and G″ was determined between 0.01 and 10 Hz. The temperature was 5
controlled by a Peltier system and the geometry was covered with paraffin oil to prevent water evaporation. Measurements were done at shear stresses within the linear response regime. 2.3.2.
Tubidity measurements
The turbidity of the pure carrageenan and mixtures was measured as a function of the
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temperature in 1 cm cells using a UV-visible spectrometer Varian Cary – 50 Bio. The temperature was varied from 85 °C to 5 °C at 2°C/ min at a wavelength = 600nm. 2.3.3.
Confocal Laser Scanning Microscopy (CLSM)
ι- and κ-car were visualized separately by using different fluorescent labelling. κ-Car was labelled with FITC, and ι-car was labelled with RITC. CLSM observations were made with a Zeiss LSM800 microscope (Jena, Germany) and with a water immersion objective 63x. The solutions
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were inserted between a concave slide and a coverslip and hermetically sealed. The incident light
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was emitted by a laser beam at 543 and/or 488 nm. The signal from FITC was collected at
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wavelengths below 520 nm where RITC does not emit light. The signal from RITC was detected at
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wavelengths above 550 nm where FITC emits relatively little light. Results Rheology
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Fig. 1a shows the evolution of the storage modulus (G’) during a cooling ramp at a rate of 2 C per minute for mixtures of ι-car and κ-car each at C = 5 g/L in the presence of different fractions
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of CaCl2 to KCl. The total salt concentration was kept fixed at 40 mM. A two step increase of G'
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was observed in all cases. As was mentioned in the Introduction earlier studies of mixtures of ι-car and κ-car in comparison with that of individual Car solutions have shown that the increase at higher
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temperatures corresponds to gelation of ι-car followed by that of κ-car at lower temperatures. Here the first step led to relatively weak gels formed by ι-car and the second step at lower temperatures
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led to stronger gels caused by the formation of a κ-car network. These results agree with reports on gelation of mixed Car in presence of individual salts KCl, NaCl or CaCl2 (Bui, Nguyen, Renou & Nicolai, 2019; Du, Brenner, Xie & Matsukawa, 2016; Lundin & Norton, 2000). With increasing fraction of CaCl2, Tg of κ-car decreases and Tg of ι-car increases.
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40 - 0 mM 30 - 10 20 - 20 10 - 30 0 - 40
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Fig.1 Storage modulus measured during cooling ramps at a rate of 2 °C/min for solutions of mixed
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ι- and κ-car each at 5 g/L at different compositions of CaCl2-KCl keeping the total salt concentration
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fixed at 40 mM (a) and the elastic modulus obtained after 1h at 5 °C as a function of the CaCl2 and
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KCl concentration (b).
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After the cooling ramp, the samples were kept at 5 °C for one hour and the frequency dependence of G' and G" was measured. G' of the gels was larger than G" and independent of the
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frequency at low frequencies (results not shown). The elastic modulus (Gel) taken as G' at low frequencies is plotted in Fig.1b as a function of the CaCl2 and KCl concentration keeping the total
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salt concentration fixed at 40 mM. Gel of the samples in the presence of mixed salts was higher than samples with just one type of salt at the same concentration. In particular, addition of even a small amount of KCl (5 mM) led to a strong increase of Gel, but replacement of some KCl by CaCl2 also
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increased the gel stiffness. Fig. 2a shows the storage modulus during a cooling ramp for mixtures of ι-car and κ-car
each at 5 g/L at different total salt concentrations with equal amounts of each salt. Two step gelation was again observed with an increase of Tg for both types of Car with increasing salt concentration. Tg was determined as the temperature where G' started to increased. For κ-car the increase was sharp and Tg could be determined without much ambiguity. For ι-car the increase of G' is more 7
progressive and we have taken Tg as the temperature where G' = 1 Pa. For mixtures we have taken Tg of κ-car as the temperature where G' shows a sharp inflexion. It is clear that the absolute values of Tg will depend to some extent on the manner they are determined and the rate of the temperature ramp. However, considering that the same manner is used for all systems the uncertainty in the relative changes as a function of the salt concentration is much smaller. Based on many
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measurements on different systems, not only those shown here, we estimate that the uncertainty in the value of Tg is 2 °C. Tg of ι-car and κ-car increased initially steeply with increasing salt concentration and more weakly at higher salt concentrations, see red symbols in Fig. 2b. 80
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Fig.2 Storage modulus measured during cooling ramps at a rate of 2 °C/min for mixtures of ι-car and κ-car containing 5 g/L of each in the presence of equal amounts of KCl and CaCl2 as indicated
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the figure (a) and the gel temperature of ι-car and κ-car in mixed systems as a function of the total salt concentration (b) with ι-car and κ-car (red symbols) or individual ι-car and κ-car gels formed in
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mixed salt (blue symbols) and individual ι-car and κ-car gels formed with only CaCl2 or KCl, respectively (green symbols). Notice that in the latter case the results are shown as a function of the
A
same CaCl2 or KCl concentration as in the mixed salt case.
We have compared the results obtained for mixed Car solutions with those for individual ιcar and κ-car solutions at the same concentra tion as in the mixtures (5 g/L) both with mixed salt and with single salt, see in Fig. 2b. The values of Tg for individual ι-car solutions with mixed salt (blue symbols) were close to that with just CaCl2 at the same concentration as in the mixture (green 8
symbols). On the other hand, the values of Tg for individual κ-car solutions with mixed salt were close to that with just KCl. From this comparison we may conclude that ι-car and κ-car gelation in Car mixtures containing both salts is driven primarily by the cations for which they are most sensitive, i.e. CaCl2 for ι-car and KCl for κ-car. Fig. 3 shows the elastic moduli obtained after one hour at 5 °C as a function of the total salt
SC RI PT
concentration for individual ι-car (Fig. 3a) and κ-car (Fig. 3b) gels at 5 g/L as well as for mixed Car gels containing 5 g/L of each type (Fig. 3c). Based on many measurements on different systems, not only those shown here, we estimate that the uncertainty in the value of G' is about 20%. In all cases, Gel increased with increasing salt concentration until it reached a plateau at high salt concentrations. Gel of individual ι-car gels was higher in the presence of CaCl2 than in the presence of KCl, whereas the inverse was the case for κ-car gels. Gel of ι-car gels in the presence of both salts was close to that
U
in the presence of just CaCl2, but κ-car gels were stiffer in the presence of both salts than with just
a
CaCl2
M
103
b
103
D
102
100 0
EP
101
20
40
Gel (Pa)
KCl mixed salt
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Gel (Pa)
A
N
KCl. Gel of mixed Car gels was also highest in the presence of both salts.
102
101
100 60
80
0
total salt concentration (mM)
20
40
60
A
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total salt concentration (mM)
9
80
c
102
SC RI PT
Gel (Pa)
103
101
100 0
20
40
60
80
total salt concentration (mM)
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Fig. 3. Elastic moduli of individual ι-car (a), κ-car (b) and mixed Car (c) gels obtained after cooling
N
1 h at 5 °C as a function of the total salt concentration. The individual gels contained 5 g/L Car and the mixed gels contained 5 g/L of each type of Car. Results obtained with CaCl 2, KCl or equal
M
A
quantities of both types of salt are indicated by different symbols as indicated in Fig. 3a.
We may also compare mixed Car gels with individual gels with the same ι-car or κ-car
D
concentration as in the mixed Car gels. In the presence of CaCl2 the mixed Car gels are clearly
TE
stiffer than the sum of individual ι-car or κ-car gels demonstrating the synergy between the two types of Car. In the presence of KCl or both salts the elastic modulus of the mixed Car gels was
EP
quite close to that of individual κ-car gels. Though the values of G' were systematically slightly larger for the mixed Car gels the difference is too small to conclude that there is synergy between ι-
CC
car and κ-car in this case. It is clear that in the mixed Car gels the contribution of κ-car is in all cases much more important than that of ι-car.
A
3.2 Turbidity The effect of gelation in the presence of both salts on the turbidity was measured during
cooling ramps at a rate of 2 °C per minute. Fig. 4 shows the effect of varying the total salt concentration at equal KCl and CaCl2 concentrations for individual κ-car (Fig. 4a) and ι-car (Fig. 4b) solutions at 10 g/L as well as for mixed Car solutions containing 5 g/L of each (Fig. 4c,d). As expected, the turbidity increased with increasing total salt concentration for all systems, which is 10
caused by an increase of the amplitude of the Car concentration fluctuations. However, there are remarkable differences in the temperature dependence. The turbidity of individual ι-car solutions depended only very weakly on the temperature, see Fig. 4a, whereas that of individual κ-car solutions increased sharply at Tg, see Fig. 4b. The increase of the turbidity of κ-car gels with
0.40
0.30
a
b
5-5 mM 10-10 20-20 30-30
0.35 0.30
5-5 mM 10-10 20-20 30-30 40-40
0.25 turbidity (cm-1)
0.25 0.20
0.20
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turbidity (cm-1)
SC RI PT
decreasing temperature weakened first, but then showed again a faster increase below about 15°C.
0.15
0.10
0.10
20
40
60
80
A
0
M
T (°C) 0.30 c
TE
20
40
60
80
T (°C)
d
40 - 0 mM 30 - 10 20 - 20 10 - 30 0 - 40
0.35
EP
0.20
0
0.40
turbidity (cm-1)
D
5- 5 mM 10-10 20-20 30-30 40-40
0.25 turbidity (cm-1)
N
0.15
0.30 0.25 0.20
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0.15
0.15
0.10
0
0.10 20
40
60
80
0
T (°C)
20
40
60
80
T (°C)
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Fig. 4.Turbidity at 600 nm during cooling for ι-car (a) and κ-car (b) solutions at 10 g/L and for Car mixtures containing 5 g/L of each (c, d). The salt concentrations, CaCl2-KCl, in the systems are indicated in the figures.
11
The turbidity of the mixed Car solutions with both salts increased progressively with decreasing temperature below Tg of κ-car, see Figs 4c and 4d. This increase has already been reported elsewhere for mixed Car gels in the presence of just CaCl2 (Bui, Nguyen, Renou & Nicolai, 2019). If only CaCl2 is present the progressive increase of the turbidity is followed by a sharp increase at Tg of κ-car, see Fig. 4d. However, when both salts are present the turbidity initially
SC RI PT
decreases sharply at Tg of κ-car followed by a weak increase at lower T. Fig. 4d shows that a relatively small fraction of KCl already has a strong influence on the turbidity.
In order to investigate further the drop of the turbidity of the mixed Car solutions when κ-car gels, we measured the turbidity as a function of time during and after decreasing the temperature as fast as possible to different values close to Tg of κ-car, see Fig. 5. It took between 8 and 10 min to
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reach the set temperature to within less than 0.5 °C. The turbidity in the presence of both KCl and CaCl2 at 20 mM shown in Fig. 5a may be compared with those solutions containing only 20 mM
N
CaCl2 shown in Fig. 5b. When only CaCl2 was present the turbidity increased progressively with
A
decreasing temperature and continued to increase, albeit more slowly, once the set temperature had been reached implying that the gel structure continued to evolve. However, when in addition 20 mM
M
KCl was present, the turbidity passed through a minimum at T < 30 °C, i.e. when κ-car gelled. It appears that gelation of κ-car initially caused a decrease of the amplitude of the Car concentration
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fluctuations in the system. With time the κ-car network becomes more densely crosslinked and more
0.18
0.18
b
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a
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heterogeneous leading eventually to a larger turbidity than in the absence of KCl.
A
Turbidity (cm-1)
0.17
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Turbidity (cm-1)
0.17
0.16
30 oC 27 oC
0.15
25 oC 23 oC
0.14
0.16
0.15
0.14
20 oC 0.13
0.13 1
10
100
1
Time (min)
10 Time (min)
12
100
Fig. 5.Turbidity at 600 nm as a function of time of mixed ι-car and κ-car (5-5 g/L) in the presence of 20 mM KCl and 20 mM CaCl2 (a) or only 20 mM CaCl2 (b) at different temperatures indicated in Fig. 5a. 3.3 Microstructure
SC RI PT
The turbidity is related to the amplitude of the overall Car concentration fluctuations, but does not inform about the microstructure of the ι-car and κ-car networks within the mixed gel. The microstructure of each network can be probed using CLSM microscopy utilizing different fluorescent labels for each type of Car. We checked that the brightness in the images is proportional to the concentration of the labelled Car. CLSM images were obtained at 20 °C after cooling from 90 °C solutions containing 5 g/L of ι-car and κ-car. Fig. 6 shows that the heterogeneity of the labelled
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κ-car network increased with increasing concentration of CaCl2 in approximately the same manner whether the total salt concentration was fixed or the concentration of KCl was kept the same as that
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of CaCl2. Notice, however, that in all cases the contrast in brightness is very weak, because the
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amplitude of the concentration fluctuations of labelled Car is very small. Elsewhere, we showed that
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in the presence of only CaCl2 the amplitude of the concentration fluctuations of the κ-car network in mixed Car gels was smaller than for individual the κ-car gels (Bui, Nguyen, Renou & Nicolai,
D
2019). We also showed that the characteristic length scale of the concentration fluctuations of κ-car obtained from the pair correlation function of the fluorescence intensity fluctuations was
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approximately one µm in the presence of CaCl2. Here we find that this length scale was not significantly different in mixed salts. The labelled ι-car network in the mixed Car gel was in all
A
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cases homogenous on length scales accessible to CLSM (images not shown).
13
40 – 0 mM
30 – 10
20 -20
10 - 30
0 - 40
0 mM
10 - 10
20-20
SC RI PT
10 µm
30-30
40-40
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Fig.6. CLSM images at 20 °C of the labelled κ-car network in mixed ι- and κ-car gels containing 5
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g/L of each Car in the presence of different amounts of CaCl2-KCl keeping the total salt concentration fixed at 40 mM (top) or keeping the KCl equal to that of CaCl2 (bottom). The
Discussion
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4.
M
A
brightness is proportional to the κ-car concentration.
Here we have shown the effect of adding both CaCl2 and KCl on the gel temperature, the gel
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stiffness and the microstructure of individual and mixed ι-car and κ-car gels. Synergistic effects were observed both between the two types of salt and between the two types of Car. In order to
EP
appreciate the effect of adding both salts in mixtures of Car we will first briefly discuss individual ιcar and κ-car gelation as well as that of Car mixtures in the presence of either CaCl2 of KCl. Then
CC
we discuss the effect of adding both CaCl2 and KCl on individual Car gelation and on mixed Car gelation..
A
Individual ι-car and κ-car gelation in the presence of either CaCl2 or KCl. Gelation of ι-car
and κ-car occurs during cooling at a temperature that increases with increasing CaCl2 or KCl concentration. Tg of ι-car is higher in the presence of CaCl2 than in the presence of KCl at the same salt concentration, whereas for κ-car the inverse is the case. Here we found that for both types of Car, Gel increased initially with increasing salt concentration and remained approximately constant at higher salt concentration. We note, however, a difference between the dependence of G el on the 14
CaCl2 concentration between the native κ-car used here and the commercial κ-car used in other studies reported in the literature. For commercial κ-car, Gel was found to decrease at higher CaCl2 concentrations (MacArtain, Jacquier & Dawson, 2003; Nguyen, Nicolai, Benyahia & Chassenieux, 2014; Thrimawithana, Young, Dunstan & Alany, 2010), whereas here we observed that Gel remained approximately the same in this concentration range. Individual κ-car gels are more turbid
SC RI PT
and have a more heterogeneous microstructure in the presence of CaCl2 than in the presence of KCl. Interestingly, the effect of CaCl2 on the turbidity was found to be stronger for commercial κ-car (MacArtain, Jacquier & Dawson, 2003; Nguyen, Nicolai, Benyahia & Chassenieux, 2014) than for the native κ-car used here (Bui, Nguyen, Renou & Nicolai, 2019). Apparently, native κ-car forms less heterogeneous gels with Ca2+ than commercial κ-car, which is perhaps related to the difference in the dependence of Gel on the CaCl2 concentration. ι-car gels are more homogeneous than κ-car
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gels both in the presence of KCl and CaCl2.
N
Mixed ι-car and κ-car gelation in the presence of either CaCl2 or KCl. Gelation of ι-car and κ-
A
car in mixtures occurs at the same temperatures as for individual solutions (Brenner, Tuvikene, Parker, Matsukawa & Nishinari, 2014; Bui, Nguyen, Nicolai & Renou, 2019; Bui, Nguyen, Renou
M
& Nicolai, 2019; Du, Brenner, Xie & Matsukawa, 2016; Lundin & Norton, 2000; Parker, Brigand, Miniou, Trespoey & Vallée, 1993; Piculell, Nilsson & Muhrbeck, 1992). Tg is higher for ι-car than
D
for κ-car and the elastic modulus of the mixed Car gel is close to that of individual ι-car gels as long
TE
as κ-car is not crosslinked. However, Gel increases sharply below Tg of κ-car and becomes larger than the sum of the elastic moduli of the individual Car networks at the same concentration as in the
EP
mixtures. As was mentioned in the introduction, this synergy has been attributed either to microphase separation between ι-car and κ-car when the latter gels or to modification of the interpenetrated or interconnected networks. In the presence of CaCl2 the turbidity of the mixed Car
CC
systems increases with decreasing temperature between Tg of ι-car and κ-car, see for example Fig. 4d. This means the presence of κ-car within the ι-car network increases the amplitude of the
A
concentration fluctuation. At Tg of κ-car the turbidity starts to increase more sharply, but remarkably the increase caused by κ-car gelation in the mixed Car systems is less than for individual κ-car solutions (Bui, Nguyen, Renou & Nicolai, 2019; Lundin & Norton, 2000). This observation was confirmed by CLSM images that showed that the κ-car network in mixed Car gels was less heterogeneous than individual κ-car networks (Bui, Nguyen, Renou & Nicolai, 2019). The ι-car
15
network was found to be homogeneous on length scales accessible to CLSM for both individual and mixed gels. Individual ι-car and κ-car gelation in the presence of both CaCl2 and KCl. At most conditions studied here gelation of ι-car was induced by the CaCl2 in the system and that of κ-car was induced by the KCl. The gel temperatures with mixed salts were close to that obtained with just
SC RI PT
CaCl2 and KCl at the same concentrations as in the mixtures for ι-car and κ-car, respectively, see Fig. 1b. The elastic modulus of ι-car gels in the presence of mixed salts was the same as in the absence of KCl. However, Gel of κ-car gels was higher when both types of salt were present, which means that CaCl2 reinforced the κ-car network. Synergy between CaCl2 and KCl with respect to the stiffness of κ-car gels had already been reported in the literature (Hermansson, Eriksson & Jordansson, 1991; Nguyen, Nicolai, Benyahia & Chassenieux, 2014; Robal et al., 2014; Wang,
U
Yuan, Cui & Liu, 2018). The turbidity of ι-car gels increased weakly with increasing salt
N
concentration, but remained low in the presence of either or both types of salt. Nguyen et al.
A
(Nguyen, Nicolai, Benyahia & Chassenieux, 2014) observed that the turbidity of commercial κ-car gels in the presence of CaCl2 was much reduced if KCl was present as well. The turbidity of native
M
κ-car gels was also found to be lower in mixed salt than with just CaCl2 at the same concentration as in the mixed salt system. However, the turbidity of κ-car gels in mixed salt is still larger than in the
D
presence of just KCl indicating that CaCl2 influences the structure of the κ-car network even if its
TE
formation is induced by K+. It appears that K+ does not have a significant influence on the ι-car network formation induced by the presence of Ca2+, but that Ca2+ renders the κ-car network induced
EP
by K+ stiffer and more heterogeneous. Mixed ι-car and κ-car gelation in the presence of both CaCl2 and KCl. In mixed Car solutions
CC
that contain both types of salt, Tg of ι-car is determined by the CaCl2 concentration and Tg of κ-car is determined by the KCl concentration, see Fig. 2b. The gelation temperature of each Car in the mixtures is not influenced by the presence of the other type of Car nor by the presence of the other
A
type of salt. However, adding both salts led to an increase of Gel of mixed Car gels similar to that of individual κ-car gels. Between Tg of ι-car and κ-car, the turbidity of mixed Car solutions in mixed salt increased with decreasing temperature similar to mixed Car systems containing just CaCl2 and was determined by the amount of CaCl2 in the system. Remarkably, gelation of κ-car induced a sharp decrease of the turbidity when KCl was present together with CaCl2. Gelation of κ-car in the mixed salt is induced by KCl, which causes formation of a homogeneous network. It appears that 16
gelation of κ-car induced by KCl causes a decrease of the amplitude of the concentration fluctuations in the Car mixture. It was reported in the literature that gelation of κ-car induced by KCl can inverse microphase separation in mixtures with whey protein aggregates, which was attributed to the elasticity of the κ-car network (Ako, Durand & Nicolai, 2011; Baussay, Durand & Nicolai, 2006). The reduction of the amplitude of the concentration fluctuations in mixed Car
SC RI PT
systems containing both salts may have the same origin. The synergistic effect between ι-car and κcar on the gel stiffness was maintained when both salt were present, but the sharp drop of the turbidity when κ-car gelled renders microphase separation between the two types of Car an unlikely explanation of this synergy. As the crosslink density of the κ-car network increased with decreased temperature, the turbidity was observed to increased again, which we attribute to the effect of CaCl2 on the κ-car network structure. Measurements at different constant temperatures illustrate the
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consecutive effects of κ-car network formation induced by KCl and the coarsening of the network
A
N
induced by CaCl2, that cause first a decrease of the turbidity followed by an increase, see Fig. 5a.
M
Conclusion
ι- and κ-car solutions form gels when cooled in the presence of either CaCl 2 or KCl, but they
D
have different sensitivities for CaCl2 and KCl. As a consequence in the presence of both types of salt κ-car gelation is induced by KCl and gelation of ι-car by CaCl2. This is the case both for
TE
individual Car solutions and for mixtures of both types of Car. In mixed salt, ι-car gels at higher temperatures than κ-car, but forms much weaker gels. The elastic modulus of the ι-car network is
EP
determined by the CaCl2 concentration and is not influenced significantly by the presence of KCl nor by the presence of κ-car in the coil conformation. The elastic modulus of the κ-car network in
CC
mixed salt is larger than in pure KCl or pure CaCl2 at the same total salt concentration. The same synergy is observed for individual κ-car gels and for mixed Car gels. The heterogeneity of κ-car gels
A
formed in the presence of CaCl2 can be reduced by adding ι-car or adding KCl. Gelation of κ-car induced by K+ in mixed Car systems containing both types of salt cause the mixed gel to be more homogeneous, which renders it unlikely that microphase separation between ι-car and κ-car is the origin of the synergy. Mixing different types of Car and different types of salt can be used to tune the mechanical and structural properties of Car gels for given total Car and salt concentrations.
17
References
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Ako, K., Durand, D., & Nicolai, T. (2011). Phase separation driven by aggregation can be reversed by elasticity in gelling mixtures of polysaccharides and proteins. Soft Matter, 7(6), 2507-2516. Baussay, K., Durand, D., & Nicolai, T. (2006). Coupling between polysaccharide gelation and micro-phase separation of globular protein clusters. Journal of colloid and interface science, 304(2), 335-341. Brenner, T., Tuvikene, R., Parker, A., Matsukawa, S., & Nishinari, K. (2014). Rheology and structure of mixed kappa-carrageenan/iota-carrageenan gels. Food Hydrocolloids, 39, 272-279. Bui, V. T. N. T., Nguyen, B. T., Nicolai, T., & Renou, F. (2019). Mobility of carrageenan chains in iota- and kappa carrageenan gels. Colloids and Surfaces A, 562(October 2018), 113-118. Bui, V. T. N. T., Nguyen, B. T., Renou, F., & Nicolai, T. (2018). Structure and rheological properties of carrageenans extracted from different red algae species cultivated in Cam Ranh Bay, Vietnam. Journal of Applied Phycology. Bui, V. T. N. T., Nguyen, B. T., Renou, F., & Nicolai, T. (2019). Rheology and microstructure of mixtures of iota and kappa-carrageenan. Food Hydrocolloids, 89, 180-187. Chen, Y., Liao, M.-L., & Dunstan, D. E. (2002). The rheology of K+ -carrageenan as a weak gel. Carbohydrate Polymers, 50(2), 109-116. Du, L., Brenner, T., Xie, J., & Matsukawa, S. (2016). A study on phase separation behavior in kappa/iota carrageenan mixtures by micro DSC, rheological measurements and simulating water and cations migration between phases. Food Hydrocolloids, 55, 81-88. Heilig, A., Göggerle, A., & Hinrichs, J. (2009). Multiphase visualisation of fat containing β-lactoglobulin-κ-carrageenan gels by confocal scanning laser microscopy, using a novel dye, V03-01136, for fat staining. LWT - Food Science and Technology, 42(2), 646-653. Hermansson, A., Eriksson, E., & Jordansson, E. (1991). Effects of Potassium , Sodium and Calcium on the Microstructure and Rheologicai Behaviour of KappaCarrageenan Gels. Carbohydrate Polymers, 16, 297-320. Hu, B., Du, L., & Matsukawa, S. (2016). NMR study on the network structure of a mixed gel of kappa and iota carrageenans. Carbohydrate Polymers, 150, 57-64. Landry, M. R. S., & Rochas, C. (1990). Role of the Molecular Weight on the Mechanical Cels Properties of Kappa Carrageenan previously. 2, 255-266. Lundin, L. O. K. F. T. J., & Norton, I. T. (2000). Phase separation in mixed carrageenan systems. Supermolecular and colloidal structures in Biomaterials and Biosubstrates pp. 436-449): ICP. 18
A
CC
EP
TE
D
M
A
N
U
SC RI PT
MacArtain, P., Jacquier, J. C., & Dawson, K. A. (2003). Physical characteristics of calcium induced κ-carrageenan networks. Carbohydrate Polymers, 53(4), 395-400. Mangione, M. R., Giacomazza, D., Bulone, D., Martorana, V., & San Biagio, P. L. (2003). Thermoreversible gelation of κ-Carrageenan: Relation between conformational transition and aggregation. Biophysical Chemistry, 104(1), 95-105. Morris, E. R. (1986). Molecular Interactions in Polysaccharide Gelation. British Polymer Journal, 18(1), 14-21. Nguyen, B. T., Nicolai, T., Benyahia, L., & Chassenieux, C. (2014). Synergistic effects of mixed salt on the gelation of -carrageenan. Carbohydrate Polymers, 112, 10-15. Parker, A., Brigand, G., Miniou, C., Trespoey, A., & Vallée, P. (1993). Rheology and fracture of mixed - and i-carrageenan gels: Two-step gelation. Carbohydrate Polymers, 20(4), 253-262. Piculell, L., Nilsson, S., & Muhrbeck, P. (1992). Effects of small amounts of kappacarrageenan on the rheology of aqueous iota-carrageenan. Carbohydrate Polymers, 18(3), 199-208. Ridout, M. J., Garza, S., Brownsey, G. J., & Morris, V. J. (1996). Mixed iota-kappa carrageenan gels. International Journal of Biological Macromolecules, 18(1-2), 5-8. Robal, M., Brenner, T., Matsukawa, S., Ogawa, H., Truus, K., Rudolph, B., & Tuvikene, R. (2014). Monocationic salts of carrageenans: Preparation and physicochemical properties. Food Hydrocolloids, 63, 656-667. Rochas, C., & Rinaudo, M. (1980). Activity Coefficients of Counterions and Conformation in Kappa-Carrageenna System. Biopolymers, 19, 1675-1687. Rochas, C., Rinaudo, M., & Landry, S. (1989). Relation between the molecular structure and mechanical properties of carrageenan gels. Carbohydrate Polymers, 10(2), 115-127. Thrimawithana, T. R., Young, S., Dunstan, D. E., & Alany, R. G. (2010). Texture and rheological characterization of kappa and iota carrageenan in the presence of counter ions. Carbohydrate Polymers, 82(1), 69-77. Trius, A., & Sebranek, J. G. (1996). Carrageenans and Their Use in Meat Products. Critical Reviews in Food Science and Nutrition, 36(1-2), 69-85. Van De Velde, F., Antipova, A. S., Rollema, H. S., Burova, T. V., Grinberg, N. V., Pereira, L., Gilsenan, P. M., Tromp, R. H., Rudolph, B., & Grinberg, V. Y. (2005). The structure of /-hybrid carrageenans II. Coil-helix transition as a function of chain composition. Carbohydrate Research, 340, 1113-1129. Van de Velde, F., Pereira, L., & Rollema, H. S. (2004). The revised NMR chemical shift data of carrageenans. Carbohydrate Research, 339, 2309-2313. Wang, Y., Yuan, C., Cui, B., & Liu, Y. (2018). Influence of cations on texture, compressive elastic modulus, sol-gel transition and freeze-thaw properties of kappacarrageenan gel. Carbohydrate Polymers, 202, 530-535. 19
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Williams, P. A., Phillips, G. O., Doyle, J., Giannouli, P., Philp, K., & Morris, E. R. (2002). Effect of K+ and Ca2+ cations on gelation of -carrageenan. Gums and Stabilisers for the Food Industry 11 pp. 158-164.
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