Impact of xanthan gum, sucrose and fructose on the viscoelastic properties of agarose hydrogels

Food Hydrocolloids 29 (2012) 298e307

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Impact of xanthan gum, sucrose and fructose on the viscoelastic properties of agarose hydrogels Sania Maurer*, Ann Junghans 1, Thomas A. Vilgis 2 Max-Planck-Institute of Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 August 2011 Accepted 12 March 2012

Mixed carbohydrate systems are of special interest for the food and non-food industry as they offer a versatile range of unique and novel functional properties. However, intense research is required to understand the complex processes occurring in such systems on a molecular level and to be able to modify them aim-oriented. In food, characteristic properties are based on the physicochemical functions of the biopolymers added. Thus, small deformation tests and moisture analysis have been applied to study the impact of xanthan gum and two types of sugar on the viscoelastic properties, the solegel transition and the water holding capacity of 1% agarose hydrogels. Agarose gels are very elastic, turbid and prone to synaeresis, which impinges on their mouth feeling. Additions of xanthan gum revealed less elastic gels with an unaffected water holding capacity. Progressive addition of two different types of up to 40% of sugar yield an increase of the elasticity of agarose gels, whereby sugar concentrations of 60% partially result in a structural breakdown and thus a significant lower network structure but better water holding. In ternary systems, the effect of the sugar concentration and sugar type used is diminished by xanthan gum. The gelation mechanism of agarose gels with a distinct amount of co-solutes is presumably mainly affected by the water shortage evolved from the competition for it of all solutes present. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Agarose Xanthan gum Fructose Sucrose Rheology Gelation

1. Introduction The demand for carbohydrates with unique physicochemical properties is high and propels the research of such additives to understand the basic principles of their properties and function as precise knowledge about the rheological behaviour of thickeners and gelling agents can be helpful for process design and product development. Textural characteristics, mouth feeling and the general appearance of a product, as well as fluid flows and flow conditions during processing are thus more predictable and controllable (Marcotte, Hoshahili, & Ramaswamy, 2001). Mixtures of carbohydrates of different properties offer an immense potential to function as replacer for unfavourable food ingredients that do not conform well to cultural, ethical, nutritional and health claims. Currently, alternative gelling agents to gelatine are of special interest for the food industry. However, a detailed knowledge about the physicochemical properties of other gelling additives is essential to fulfil the demands regarding the functional

* Corresponding author. Tel.: þ49 6131 379 149. E-mail addresses: [email protected] (S. Maurer), [email protected] (A. Junghans), [email protected] (T.A. Vilgis). 1 Tel.: þ49 6131 379 513. 2 Tel.: þ49 6131 379 143. 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2012.03.002

characteristics. In this study, the emphasis is placed on the rheological behaviour of a ternary system composed of different carbohydrates unifying three elementary functional properties: gelation (agarose), thickening (xanthan gum), and sweetening (fructose and sucrose). The usage of agarose, the main gelling agent of agareagar extracted from red algae (Rhodophyceae), in this study is twofold: First of all, due to its practical relevancy in the food and non-food industry and secondly due to its relatively simple and neutral molecular ordered structure that alleviates the modelling of the gelation mechanism of carbohydrates. The chemical structure of agarose is composed of alternating repeating agarbiose units, connected through C-1 and C-3 of b-D-galactopyranose (1e4) linked to 3,6-anhydro-a-L-galactopyranose. The physical gelation of such polysaccharides is a complex process of self-assembly accompanied by biomolecular conformation changes, solution demixing and molecular cross-linking (Manno et al., 1999; Morris, 2009). Polymer concentration, presence of co-solutes, temperature history, mixing rate, as well as solvent properties further determine the mechanism of gelation and the final gel properties. The precise gelation mechanism of agarose is not yet fully understood. The investigation of kinetic and diffusion processes, as well as concentration flocculation involved in the gelation mechanisms yield different models being based on e.g. spinodal decomposition

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and nucleation and growth. In general, the gelation process is understood in terms of liquideliquid phase separation. Agarose dissolves in hot water (60e70  C) and gels upon cooling (35e40  C). Pure agarose gels are highly elastic, hard, clear, thermoreversible, relatively prone to synaeresis and provide a poor mouth feeling (Nordqvist & Vilgis, 2011; Rinaudo, 2008). To modify these properties, Nordqvist and Vilgis combined agarose with a, in the physical sense, non-gelling polysaccharide. They reported from rheological, microscopic and synaeresis measurements that combinations with xanthan gum, a bacterial hydrocolloid with unique thickening properties, result in less elastic, softer gels with a higher water holding capacity and a better mouth feeling. The plasticising effect of xanthan gum molecules can be referred to the significant impact of highly negatively charged stiff rod-like xanthan molecules on the formation of flexible agarose chains to aggregates. In absence of co-solutes, the gelation process of agarose is a two-stage process with the formation of agarose helices followed by their aggregation into bundles as it has been discussed recently in detail by Nordqvist and Vilgis (2011). Xanthan molecules, as relatively stiff polyelectrolytes, however undergo a socalled jamming transition, which is a very general phenomenon in nature (Liu & Nagel, 1998). The jamming transition of xanthan gum solutions is induced by random blocking of the dynamics of the rigid molecules. Indeed such a logjam transition of rigid rods has been proposed already by Edwards and Vilgis (1986) earlier. The jamming transition of xanthan solutions can be modified by the ionic strength. At a critical polyelectrolyte concentration, which is determined by the chain length and the charges, xanthan solutions thicken by the jamming process. Combining xanthan and agarose hinder the aggregation of the agarose helical structure into bundles. Thus, gelation is reduced to a single-stage process by the presence of the randomly oriented xanthan molecules (Nordqvist & Vilgis, 2011). Besides the texturizing components, jelly gums contain a considerable amount of sugar. In the case of gelatine, the used sugars have a synergistic effect on the protein gelation properties (Kasapis, Al-Marhoobi, Deszczynski, Mitchell, & Abeysekera, 2003). The purpose of this study was to analyse how different concentrations of fructose and sucrose affect the rheological and water holding properties, as well as the solegel transition of agarose, respectively agaroseexanthan hydrogels. The experimental results are underlined with a simple gelation model to emphasis the special role of water molecules and their involvement in intra- and intermolecular interactions during the gelation. Finally, a remark is needed on the choice of the different constituents. Indeed the choice of agarose, xanthan, and the two types of sugar has different physical aspects. First, the molecules show different conformations and physical properties in solution. While agarose appears to be a relatively flexible macromolecule, xanthan is much stiffer. The persistence length of xanthan is of the order of the total length of the molecule (Song, 2006). Another difference comes from the charges of the molecules. Xanthan is highly charged whereas agarose is practically neutral. These differences on a molecular level allow studying the interplay with the low molecular weight sugars, such as the monosaccharide fructose and the disaccharide sucrose, more systematically. 2. Materials and methods All materials used in this study are shown in Table 1: 2.1. Gel preparation A 1% w/v solution of agarose, dispersed in distilled water or in the appropriate sugar (fructose, sucrose) in water concentration

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Table 1 Used chemicals (all chemicals were used as received.). Chemical

CAS (chemical abstract service)

Provider

Source

Agarose

9012-36-6

Schwerte, Germany

Xanthan gum

11138-66-2

D-()-fructose

57-48-7

Sucrose

57-50-1

Fisher scientific GmbH Carl Roth GmbH & Co. KG SigmaeAldrich Chemie GmbH CalbiochemÒ Merck KGaA

Karlsruhe, Germany Steinheim, Germany Darmstadt, Germany

(20, 40, and 60% w/w), was heated up to 68  2  C and kept there for 1 h while stirring (120 rpm). For 1% w/v agaroseexanthan solutions (ratio 1:1), agarose and agaroseesugar solutions were first stirred for 30 min prior adding to the already prepared xanthan gum solutions and subsequently further stirred for 30 min at 120 rpm. The solvent evaporation during stirring was minimised by covering the beaker glass with a glass cover. All hydrogels have been prepared in 20 ml snap-cap bottles. Mono- and multicomponent hydrogels have been produced by pouring the solutions into moulds (PVC) of defined dimensions (ø 25 mm, h ¼ 3 mm) and allowing the solution to cool down-to room temperature. Samples were trimmed to the surface of the mould to obtain samples of a defined height. Due to the relatively unstable texture of agaroseexanthanesugar hydrogels, such gels were directly poured onto sand paper being placed under the mould to prevent structural rupture during the transfer to the rheometer. In general, sand paper was used for all samples, but usually already stuck onto the measurement device, to avoid slippage during the rheological measurements. To achieve a homogeneous hardening of the agarose gels of different composition, the samples were cooled at 4  C for at least 24 h. All samples were sealed with ParafilmÒ to avoid water evaporation during storage. To adapt to room temperature, the hydrogels were taken out of the fridge one hour before measurements. Xanthan gum solutions of 1% w/v were prepared by dissolving the xanthan gum in distilled water. Samples were stirred at room temperature in 20 ml snap-cap bottles at a constant stirring rate of 200 rpm for at least 24 h. 2.2. Dynamic viscoelastic measurements Dynamic viscoelastic deformation tests of all prepared hydrogel compositions were performed on a Gemini 200 Advanced Rheometer (Bohlin Instruments, Malvern, Worcestershire, UK) by applying parallel plates (PP 25) and the amplitude-sweep mode. For the delineation of molecular models and pictures of the gelation process of the agarose hydrogels of different composition, the storage modulus (G0 ) has been analysed as a function of strain. All amplitude-sweep tests were performed at constant frequency of 1 Hz under isothermal conditions (T ¼ 25  C). To avoid slippage during measurement, the plates of the rheometer were pasted up with sandpaper (Flexovit K 500, Wollschläger, Bochum, Germany). After each run, this paper has been renewed. 2.2.1. Amplitude-sweep tests For the amplitude-sweep tests, the gap size was set between 2300 and 2800 mm. This range results from the gel nature of the samples and from the varying water evaporation during storage of the used components while hardening and systematic errors during preparation. To avoid slippage or rupture of the sample during measurement, an appropriate pressure had to be applied on the sample between the plates. This was monitored by the normal

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force displayed on a scale bar of the rheometer. As the height of all samples was approximately 3 mm (the height of the mould used for the sample preparation), the setting of the gap size was started from 2800 mm and lowered bit by bit until the force rose and thus indicating an appropriate pressure on the sample in dependence on the resistance of the sample. The setting has been stopped and the chosen gap size was proven in a trial measurement as the obtained G0 values could indicate an inadequate gap. The validated gap has then been used for the measurements of gels of the same composition. Samples used for this setting were not included in the measurement evaluation. This procedure, however, did not work for soft samples. Here, a defined amount of 2.10 g was weighed in moulds, already prepared with sandpaper, and analysed by using a gap of 2800 mm (approx. consistent with the mould height of 3 mm), sufficient to satisfy the criteria mentioned previously. This had been the case for samples composed of agarose, xanthan and sugar. The default strain range was from 0.0005 to 50 for samples composed of agarose and agarose with sugar. However, this strain had to be changed for the hydrogels additionally with xanthan to 0.0005 to 0.05, as a higher strain caused structural damages of the samples. The descriptive statistical evaluation is based on a fifteenfold determination, which is made up of three replicates of five samples of the same hydrogel composition. Thus, the data set of each G0 (G0 at a strain of 0.01 being within the LVE-range) results from the calculated arithmetic mean value (x) of 15 replicates (n), P x ¼ 1=n ni¼ 1 xi while the associated error bars indicate the standard deviation (s), s ¼ ðx2  x2 Þ1=2 . 2.2.2. Temperature-sweep tests For the temperature-dependent measurements, the solutions were prepared as usual but the samples were not cooled to form gels but placed in their sol-state, here the sample amount was 750 ml, on the pre-heated plate (80  C). The transition of the gelphase was initiated by cooling down at a rate of 1  C/min to 20  C. The upper plate has been cranked by hand to achieve a homogenous distribution free of air entrapments of the sol between the plates. By covering the sample properly with silicon oil and additionally using a solvent trap, the drying out of the sols/gels has been slowed down during the cooling run. Also here sand paper was used to prevent slippage. The gap was set to 1000 mm for each sample. 2.3. Moisture analysis A halogen moisture analyser (Mettler Toledo HR73, Giessen, Germany) has been applied to qualitatively determine the water holding capacity of pure agarose hydrogels and of agarose hydrogels with either xanthan or sugar (fructose, sucrose) or both components. Sugar concentrations of 20, 40, and 60% w/w were used. Samples were prepared as already described in paragraph 2.1 but pouring 2  0.2 g of sol into moulds of 50 mm diameter and 1 mm height. Three samples of each hydrogel composition were produced. In contrast to previous studies (Nordqvist & Vilgis, 2011) a different analysis for the water holding capacity is used. Nevertheless, for a relative comparison between the samples investigated here, it is appropriate to use the halogen moisture analyser to describe the different hydrogel compositions, especially when the hydrogen bonding of water to the different components of the hydrocolloids and co-solutes is discussed (see below). The statistical evaluation of the obtained moisture contents of the different hydrogel compositions is based on the determination of the means and the appropriate standard deviation according to the equations in Section 2.2.1.

The moisture content loss of the hydrogels at constant temperature of 100  C has been determined in per cent as a function of time. After 10 min, the measurement was stopped and the obtained values for the moisture loss during this time have been used for the qualitative comparison between the hydrogel compositions. Based on the results, it was assumed that the loss of water during drying indicates, to some extent, the ability of different composed gel networks to hold water over a certain period of time. In this study, the term “water holding capacity” will be synonymical used for the ability of the hydrogel network to hold water when being exposed to a defined temperature. Of course the values obtained here can be viewed as “apparent water holding capacity”, since the absolute values depend on the temperature which is exposed to the gel samples. 3. Results and discussion The impact of different sugar types (fructose and sucrose) of different concentrations (20, 40, and 60% w/w) on the gelation process and the gel properties of 1% agarose and 1% agaroseexanthan (1:1) hydrogels have been studied by applying three different analytical methods. Oscillation amplitude sweeps have been performed to evaluate the modification of the hydrogel viscoelastic properties when sugar is added. As the viscoelastic behaviour of agarose gels is affected by the gelation process, temperaturedependent measurements have been carried out to analyse the solegel transition. Additional, the loss of water during a defined time period has been determined to study the water holding capacity of agarose hydrogels, respectively agaroseexanthan hydrogels containing different concentrations and types of co-solutes. 3.1. Viscoelastic properties Comparing the elastic modulus of agaroseesugar hydrogels with the ones of agaroseexanthanesugar gels, distinct differences are observed (Fig. 1). The mixture of agarose with 20% and 40% of fructose, respectively sucrose provokes that the resulting gels exhibit a stronger elasticity. Already 20% of sugar is sufficient to increase the elasticity of the pure agarose gel (G’ ¼ 5.3  103  7.9  102 Pa) of about 30% in the case of fructose, and almost 50% for sucrose. A slight, but not statistical significant difference is obtained for 20% and 40% of sucrose (G’ ¼ 9.9  103  5.9  102 Pa), and 40% of fructose with G’ ¼ 8.9  103  1.5  103 Pa, similar to the value of sucrose. However, the more sugar is implemented in the agarose gels the more the particular agaroseesugar compositions differ from each other. While 60% of fructose results in a gel of an elasticity that is almost three times higher (G’ ¼ 1.4  104  1.6  103 Pa) than the initial one, a significant decrease of G0 of about 33% (G’ ¼ 1.8  103  4.9  102 Pa) is observed when 60% of sucrose is added. Thus, high amounts of sucrose reduce the elastic modulus significant, evolving less elastic agarose gels. However, the effect of the sugar concentration on the viscoelastic properties diminishes rigorously when xanthan gum is also part of the mixed system. Looking only at the hydrogel samples devoid of any sugar, the elastic modulus of the binary hydrogel (G’ ¼ 1.8  103  4.1  102 Pa) is almost three times lower than G0 of the pure agarose gel. In the case of agaroseexanthan gels with 20% of fructose, G0 decreases (G’ ¼ 1.3  103  1.4  102 Pa) but exhibits a reverse effect when 40% (G’ ¼ 2.2  103  3.0  102 Pa) and 60% (G’ ¼ 2.3  103  2.0  102 Pa) are incorporated, whereby 60% does not differ significantly from 40% of fructose. Such a trend cannot be observed for the different concentrations of sucrose. No change in G0 is observed for 20% of sucrose and 40% provokes a decrease to 1.4  103  1.2  102 Pa. A pronounced drop of G0 is revealed at a sucrose concentration of 60%. With 8.1  102  1.0  102 Pa, it is

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a certain viscosity of the embedded liquid, which acts as a swelling agent, in this context, a viscosity increase caused by 60% of fructose. The higher viscosity in the pores presumably acts as a “cushion” without affecting the coil-to-helix transition of agarose gels. Similar effect on the network topology is observed in mixtures of sugar and agarose gels of up to 40% of sucrose. But in addition, 60% of sucrose causes a structural breakdown, which will be discussed later in this section. The discrepancy between both sugar types might refer to the different impact on the solution viscosity, e.g. based on the hydration number and the ability of the sugar to change the structure of water (Galema & Hoeiland, 1991; Uedaira & Uedaira, 1985). These assumptions, concerning the stabilisation of agarose gels associated with increasing sugar concentration, coincide well with the results obtained by several scientists (Deszczynski, 2003b; Normand, 2003; Watase, Nishinari, Williams, & Phillips, 1990). They suggested that the exclusion of sugar from the surface of the structure is responsible for this phenomenon. Further congruence could be found with the results obtained by Kasapis et al. (2003), who investigated gelling polysaccharides such as kappacarrageenan and deacylated gellan gum (in a concentration range of 0.5e12%). However, the formation of stronger and thermal more stable gels has only been valid for sucrose, respectively fructose concentrations up to 40%. High concentrations of sugar (60% of sucrose) provoke a drastic drop of the elastic modulus revealing a reverse trend as for the concentration up to 40% (Fig. 1). The high level of co-solutes in the agarose solution probably affects the coil-to-helix transition so that the formation of aggregates is inhibited. This assumption is supported by results of investigated agarose gels obtained from optical (Deszczynski, 2003a), calorimetric (Normand, 2003), microscopic (Nordqvist & Vilgis, 2011; Normand, Lootens, Amici, Plucknett, & Aymard, 2000), and deformational measurements (Watase et al., 1990). Accordingly, during the preparation of agarose and agaroseesugar gels, it was observed that the degree of turbidity decreases the higher the concentration of sugar is, thus producing clearer gels (Fig. 2). This change in turbidity is presumably due to Fig. 1. Average elastic modulus of 1% agarose hydrogel (-) and agaroseexanthan hydrogel (1:1) (6) with different fructose (A) and sucrose (B) concentrations measured at of strain of 0.01.

significant lower than the value obtained for the pure agaroseexanthan hydrogel. In general, the differences between the several concentrations, as well as the different sugar types used are not as distinct as for hydrogels devoid of xanthan gum. It seems that the magnitude to be able to affect the viscoelastic properties of agarose gels by sucrose respectively fructose is diminished by the addition of xanthan gum. Agarose hydrogels with xanthan exhibit a pronounced turbidity, which does not change macroscopically with increasing sugar concentration. As the rheological properties are based on the network characteristics, the impact of the different sugar types on the gelation process of agarose and thus its structural properties is crucial. Upon cooling, coils of flexible agarose chains transform to helices, stabilised through intra- and intermolecular interactions, with subsequent aggregation of such helices to bundles. Due to the high degree of aggregation, agarose hydrogels appear to be turbid and distinct elastic (Deszczynski, 2003a). Regarding the mechanical strengthening, low to relatively high levels of fructose seem to stabilise the gel structure and the binary mixture evolves into a stronger, more stress resistant hydrogel. To explain this rheological behaviour with a schematic but speculative model, helical fibrils of agarose form a cohesive, continuous network that extends over the entire solution and thus embeds the liquid phase. The network topology reveals a pore, or mesh size, which tolerates

Fig. 2. Observed changes in turbidity of 1% agarose hydrogels with increasing concentration of sugar.

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solutions with high sugar concentrations exhibit a fluid-like rather than “weak-gel”-like behaviour. However, the effects of sugar on the one-component systems (1% xanthan solution and 1% agarose gel) and on the multicomponent system (1% agaroseexanthan gel (1:1)) have to be regarded separately as the physical nature of these two systems is different.

the decreasing part of aggregation and to the smaller correlation length (average size of the pores). Subsequently, an evolution from a coarse structure to a fine-stranded gel of homogeneous structure is suggested (Normand, 2003). Oscillation measurements of the solegel transition of agarose gels by Nordqvist and Vilgis (2011) revealed that the gelation mechanism, original being a two step process, is reduced to a one-step process. As this is based on the impact of xanthan gum, a similar effect can be attributed to sugar. It is assumed that xanthan gum has a pronounced impact on the physical behaviour when immersed in agarose gels. A brief consideration of the molecular structure of xanthan gum might be necessary to explain this phenomenon. Due to their chemical configuration, xanthan molecules are assumed to be highly charged, stiff rods in absence of salt ions. Such rods tend to undergo a so-called jamming transition at increasing polymer concentration due to strong repulsion and the incapability of forming liquid crystalline phases (Nordqvist & Vilgis, 2011). Therefore, agarose chains have only restricted space to form bundles of helices as the diffusion process is prevented by the jammed xanthan rods. Consequently, a looser gel network with a lower modulus evolves. Additionally, the high negative charge of xanthan molecules dominates the effect of sugar molecules to interact with water molecules. It seems to be reasonable to assume that the reduction of the modulus is a logical consequence of the water shortage as there are three components present competing for water. Extensive studies regarding xanthanesugar solutions of different concentration (20, 40, and 60% w/w) have also been conducted. In general, xanthan gum does not form gels in a physical sense but can be regarded as a “weak gel” (Voragen & Challen, 2000). The addition of moderately concentrations of up to 60% of sugar (fructose or sucrose) shows a significant impact on the solution behaviour of 1% xanthan gum solutions. High amounts of fructose provoke a significant reduction of the elasticity modulus, whereby the increasing addition of up to 60% of sucrose does not have any further impact (Fig. 3). Fructose and sucrose concentrations of 60% provoke a slight increase in the viscosity modulus (data not shown) of 1% xanthan gum solutions. At such high sugar concentrations, the viscosity of the continuous phase increases significantly, thus affecting the distribution of the stiff xanthan molecules in the sugar in water solution. Concluding, it is assumed that 1% xanthan

Temperature-dependent measurements have been performed to analyse the solegel transition of agarose and agaroseexanthan gels with varying fructose or sucrose concentration. Figs. 4 and 5 show the solegel transition of agarose, respectively agaroseexanthan hydrogels of 20, 40, and 60% w/w sugar added. Representative curves have been chosen and slight variations of absolute values of the elastic modulus might be possible. For the present, the focus is placed on the curve of the pure agarose gel as it reflects the characteristic gel mechanism of this gelling polysaccharide. The sigmoidal curve progression suggests a two-stage process that has already been suggested by several other scientists (Fernandez et al., 2008; Mohammed, Hember, Richardson, & Morris, 1998). Two pronounced rises (“steps”) in the curve indicate conformational changes of agarose molecular chains upon cooling. The first step of increase of the elastic modulus (G0 ) by lowering the temperature is referred to the formation of helices, which act as cross-links for the further formation of such helices to bundles. As shown in Fig. 4, the first rise takes place in a temperature range of 76  Ce60  C, where G0 increases from 2 to 38 Pa. Further cooling to 50  C reveals that the gradient slightly diminishes until exhibiting a significant, second rise when a temperature of 30  C is achieved. For a better illustration the two step process has been indicated and the corresponding measured temperatures are given (Fig. 4). In Nordqvist and Vilgis (2011) the physical interpretations of the two steps and the interplay between the gelling and thickening agents have already been discussed in detail. The rigorous change of G0 from 1.1  102 to 1.1  104 Pa represents the aggregation of agarose helices to bundles, thus the second step, driven by entropic restrictions (Nordqvist & Vilgis, 2011).

Fig. 3. Average elastic modulus of 1% xanthan solutions of different sugar type (fructose (-) and sucrose (6), zero sugar (,)) and concentration measured at a strain of 0.01.

Fig. 4. Temperature-dependent measurements of 1% agarose hydrogels with 0 (-), 20 ( ,B), 40 ( ,6), and 60 ( ,>) % w/w of fructose (half-filled symbols) or sucrose (empty symbols) added.  and - - - indicate the two step process of the gelation mechanism of pure agarose and the corresponding temperatures.

3.2. Solegel transition

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Fig. 5. Solegel transition of 1% agaroseexanthan (1:1) hydrogels supplemented with 0 (-), 20 ( ,B), 40 ( ,6), and 60 ( ,>) % w/w of fructose (half-filled symbols) or sucrose (empty symbols).

As a side remark, it is mentioned that the gelling transition is a quite general phenomenon, which has universal features as all phase transitions. Universal critical exponents of the percolation theory (rather than the mean-field Cayley tree and graph approximations) rule the physics around the gel point. However, to find such features here appears quite difficult and is not the scope of the present study. The reasons can be summarized as follows: Critical exponents for percolation, e.g. those for correlation length, cluster size etc. show up clearly in thermodynamic equilibrium processes, i.e. at very low cooling rates, at infinite systems. Alone from the here present experimental situation (avoiding the drying out of the samples) it is not possible to reach that state. More down-to -earth difficulties regard the characterization of the gelling agent (agarose). Its molecular weight (distribution) is not known. Indeed the dynamics of the gelation process are relatively slow (chain length, helix formation) so that agarose gels are not the best system for the search of exponents. Regarding the critical exponents of the modulus as defined by Gf(Tg  T)t (Tg being the equilibrium gelation temperature, which cannot be measured within the framework of the experiments carried out in this study) similar remarks can be made. The exponent t depends strongly on the nature of the polymer chains. For totally flexible chains for example, t is identical to the exponent of the electrical conduction of percolating conducting networks. In contrast for stiff (more realistic) chains bending forces and torque forces are involved (as already mentioned by de Gennes, Pincus, Velasco, & Brochard 1976), which change the universality class. The latter statements have been investigated by a number of papers starting by Kantor and Webman (Kantor & Webman, 1984) and many others; see e.g. (Stauffer & Aharony, 1994). For the present system that means, depending on the stiffness, which might change during increasing network connectivity by the formation of helices in the agarose network, it is expected to see a crossover behaviour between the two limiting cases. Again, there is no strict thermal equilibrium during the measurements, which means that it is unclear how to assign values for critical exponents in the framework of such connectivity (phase) transition. For the remarks made in the last paragraph, see e.g. Vilgis, Klüppel, and Heinrich (2009) and references therein, where the situation for polymer network formation has been summarized. Indeed in the gelation process of pure agarose, the gelling temperature is not clearly defined according to the rheological

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Fig. 6. Moisture content in per cent of agaroseefructose (-), respectively agaroseesucrose (6) hydrogels being exposed to 100  C over a time period of 600 s ((,) zero sugar).

measurements shown in Fig. 4. Given the two step process the rise of the modulus appears in two steps upon cooling. The first step, already at relatively high temperatures, around 70  C has been assigned by the formation of helices of the agarose molecules (being presumably completed at a temperature of 61  C as indicated in Fig. 4). The molecular connectivity of the sample enhances, the modulus rises about two orders of magnitude. The second step appears at about 40e45  C (completed around 30  C as shown in Fig. 4). Here some of the helices aggregate and form microstructures (as indicated in Fig. 8). Agarose gels are temperature stable up to 70  C, which is within the same range as the melting of the helices. Looking at the curve progression of the binary hydrogel composition in Fig. 4 and of the ternary hydrogel composition in Fig. 5, the addition of further fructose or sucrose has a pronounced but different impact on the solegel transition of agarose hydrogels. Regarding agaroseefructose, respectively agaroseesucrose hydrogels, two distinct effects are observed: First, the aggregation step is shifted to lower temperatures. Secondly, the first rise in G0 is less

Fig. 7. Moisture content of agaroseexanthan hydrogels with fructose (-) and sucrose (6) of different concentration ((,) zero sugar).

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Fig. 8. Gelation model.

pronounced as for the pure agarose gel. Additionally, the different sugar types used affect the solegel transition differently. While 20% of sugar causes slight variations in the formation of agarose chains to helices, 40% of fructose, respectively sucrose reveals two distinct different curve progressions. 40% of sucrose in the agarose sol provokes that G0 moderately increases from 2.1 Pa to 69.3 Pa in a temperature range of 80  Ce37  C, whereby a pronounced rise in G0 of agarose with 40% of fructose is already observed at 50  C. The discrepancy in the gradients of the two previously described curves is also reflected by a final and second rise of G0 . While 40% of sucrose still exhibits the distinct sigmoidal curve as obtained for the lower concentrations, a rather flattened slope can be seen in the curve of 40% of fructose. It seems to be that in the case of the latter not only the strengthening of the elasticity has been shifted to a lower temperature but also the formation of helices to aggregated bundles has been affected. The curve progressions of the jellied agarose sols combined with 60% of fructose, respectively sucrose show that high amounts of sugar have a rigorous impact on the gelation mechanism. First of all, the elastic modulus is significantly reduced, being 1.3  102 Pa at 21  C in the case of fructose and 5.5  102 Pa in the case of sucrose. This is, in comparison to the other concentrations, two orders of magnitude lower. Nevertheless, the relatively flat increase of the elastic modulus during cooling suggests that the aggregation of helices has been hindered due to the high level of sugar present. Regarding these observations, it seems to be that the gel mechanism is more affected by the addition of fructose than by the one of sucrose.

Comparing the temperature-dependent measurements with the oscillation measurements of the cured agarose hydrogels, some correlations between the evaluations of the obtained results can be identified. For both applied analytical methods, high amounts of sucrose reduce the elasticity of 1% agarose gels significantly. In contrast, jelled agaroseefructose mixtures have shown an increase in G0 , while a decrease has been measured in the oscillation temperature sweep. The discrepancy between these rheological measurements might be due to the missing hardening step in the evaluation of the sol-transition. For the oscillation measurements, the samples were cooled at 4  C for 24 h. In the case of agaroseefructose that might have helped to strengthen the elasticity of those gels. Concluding, drastic changes occur when sugar concentrations of 60% are incorporated into agarose hydrogels. Two factors, already proposed in connection with the capability of 60% sucrose to reduce the elastic modulus of the 1% agarose gel (sec. viscoelastic properties), might attribute to this effect: First, the distinct increase in solution viscosity at high co-solute levels and secondly, strong competition for water. Due to the higher solution viscosity, the molecular chains of the dissolved agarose are restrained from diffusing freely by the high amount of present sugar molecules. In terms of a nucleation and growth mechanism, random coil structures of agarose chains would rather tend to helix nucleation and the growth process would be inhibited (Xiong et al., 2005). The transformation from disordered to ordered structures is affected and presumably shifted to lower temperatures, equivalent to a slower gelation process.

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The examination of the solegel transition of agaroseesugar gels has shown that the two step mechanism diminishes further with increasing sugar concentration. Looking at Fig. 5, the shape of all curves displayed reveals only one distinct rise in G0 of the agaroseexanthan hydrogels. This observation coincides well with the results obtained by Nordqvist and Vilgis (2011). Moreover, neither the sugar concentration nor the sugar type used show any significant effect in the gelation process of the agaroseexanthan gel. As only representative measurements have been chosen for the evaluation, precise conclusions according to the impact of the different sugar types and concentrations used on the gel formation mechanism are not possible. Apart from that, it is shown that the elastic modulus of these compositions is one to two orders of magnitude lower in comparison to agaroseesugar gels free of any xanthan. According to the temperature-dependent measurements, it is assumed that the aggregation of agarose helices to bundles is inhibited by the highly negative charged xanthan rods. The physical behaviour of the xanthan molecules seems not to be affected much by the temperature change during the gelation process, thus still undergoing jamming transition under the given environmental conditions. Additionally, the charge of this polyelectrolyte probably dominates over the effect given by the sugar concentration. Due to their high affinity to water molecules, interactions between waterexanthan molecules are presumably favoured so that less water is available for the formation of hydrogen bonds with agarose or sugar. 3.3. Moisture holding Agarose hydrogels are susceptible to synaeresis. The water holding capacity of biopolymers is, additional to the textural properties, of great importance to the mouth feeling. Thus, the percentage loss of moisture content (MC) of simple and mixed hydrogels exposed to 100  C over a time period of 600 s has been quantified, as illustrated in Fig. 6 and Fig. 7. 1% agarose hydrogels devoid of any xanthan or sugar exhibit the lowest moisture content loss (MC ¼ 53  3%) (Fig. 6). The addition of 20% of fructose to agarose hydrogels seems to have no significant impact on the water holding capacity; the moisture content after 600 s is 49  2%. The same concentration of sucrose tends to lose more water by showing a MC of 46  4%. After 600 s the residual moisture in the gel of 40% sugar is 36  2% in the case of fructose and 37  1% for sucrose. Regarding the different sugar types, no significant differences can be observed. The highest capacity to hold water is substantiated for agarose hydrogels with 60% of fructose or sucrose. Accordingly, the MC, after the measurement has been stopped, of fructose is 17  1% and of sucrose is 15  1%, revealing that sucrose holds water slightly stronger than fructose in this concentration regime. The results obtained by the evaluation of the water holding capacity coincide well with the rheological behaviour of the different agarose gels during the solegel transition. Sugar concentrations of 20% show little impact on the gelation mechanism, as well as on the water holding capacity. Increasing incorporation of sugar into the jelled system reveals that water is stronger bound verifying the assumption that the helical structure of agarose has a destabilisation effect due to water shortage provoked by sugar molecules. Progressively increasing the concentration of sugar in agaroseexanthan hydrogels has hardly any impact on the water holding capacity, except for concentrations of 60%. In general, no significant differences exist between 1% agarose gels and those combined with 50% of xanthan (MC ¼ 52  3%) (Fig. 7). This is in contrast to the previous observations made by Nordqvist and Vilgis (2011) who reported that already little amounts of xanthan

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contribute strongly to the improvement of the water holding on the pure, sugar free gels. The reason for the discrepancy is twofold: On the one hand, the preparation procedure of the agaroseexanthan gels has been different (30 min stirring instead of 1 h, different sample geometries) and on the other hand, the quantification of the moisture content loss during a certain period of time has been determined by means of different methods (Nordqvist & Vilgis (2011) investigated the moisture loss, respectively loss in weight during 2 h of drying at room temperature). In contrast to the observed trend of agaroseefructose gels to enhance the water holding capacity at a sugar level of already 20%, 20% of sucrose actually shows a slight tendency to impair the water holding capacity. Thus, after ten minutes of measurement, 20% of fructose exhibits a moisture loss of 49  2%, while 51  3% is recorded in the case of 20% of sucrose. The water evaporation is even more intense when 40% of sucrose is incorporated in the agaroseexanthan gel. With 55  4% it shows the lowest measured moisture content in comparison to the other gel samples. Regarding the relatively large error bars, it is assumed that no statistical significant difference can be observed. From this it follows that the addition of up to 40% of sucrose has probably no impact on the water holding capacity of agaroseexanthan gels. A mixture of 1% agaroseexanthan gel with 60% sucrose results in a gel network, which is able to hold water more strongly (MC ¼ 41  2%). However, 40% and 60% of fructose exhibit a more pronounced impact on the ability of the gels to hold water, as reflected by a lower moisture loss of 47  3%, respectively 38  2%. Based on the previously discussed results, the water holding capacity is more influenced by sugar concentration and less by xanthan gum. Agarose gels are less susceptible to synaeresis when immersed in solutions of high co-solute content. Xanthan used as additional additive in the agarose gel slightly impinges on the positive impact of sugar. Moreover, it seems that combinations of agarose, xanthan, and sucrose evolve into gels with almost unchanged water holding capacity. It is assumed that the particular components could not be hydrated and mixed properly with one another. During the preparation of the ternary solutions, sucrose crystallisation occurred presumably due to restricted interactions with water molecules. Thus, a rather inhomogeneous solution composed of three separate components is generated, producing a gel whose susceptibility is probably predominately based on the water holding capacity of the gelling agent agarose. 3.4. Gelation and competition for water Regarding the competition for water, a schematic model as illustrated in Fig. 8, has been developed. It is assumed that water molecules are directly attached to the helical agarose chains or arrange themselves in form of hydration shells around the polymeric agglomerates (Fig. 8 A.1-A.2). Hydrogen bonding between the solvent and the solute stabilises the helical structure formation of agarose chains in aqueous solutions thermodynamically (Gekko, Mugishima, & Koga, 1985). Likewise, the formation of hydration shells plays an elementary role in the solvation of xanthan gum and sugar molecules. As outlined in the gelation model, high amounts of sugar molecules in the agaroseesugar solution react preferably with water molecules due to their molecular structure (Fig. 8 B.1 and B.2). Thus, the shortage of water probably provokes destabilisation of the biopolymeric helices and seems to contribute to a lower degree of coil-to-helix conformation. The competition for water molecules used as “tools” for structural stabilisation would also explain the alleviated increase of the elastic modulus of the agaroseefructose, respectively agaroseesucrose hydrogels (sugar concentration 60%) at the beginning of the cooling process (Fig. 4).

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Additionally, water shortage might force crystallisation of the sugar molecules involved in the network formation. In the case of sucrose, it could be observed that small crystals settle out of the agarose sol during the gel preparation. Presumably, (nano-) crystallisation also occurred in later stages of the gelation process and might contribute to the clear appearance of the agarose gels. When additional co-solutes like xanthan gum are implemented in the agaroseesugar solution, more water molecules seem to be attracted by the highly negatively charged polyelectrolytes. Thus, the water molecules are predominately arranged around the stiff xanthan rods and are not any more available for the formation of hydrogen bonds between the other co-solutes. In comparison to agaroseesugar hydrogels of high sugar concentration (60%), the resulting gel network is looser, less elastic and less turbid (Fig. 8 C.2). It is likely that the impact of sugar type and concentration used is diminished by the xanthan molecules. 3.5. Effect of water on chain conformation and gelation The experimental results can be understood, at least qualitatively, with some naive physical ideas. The main issues were already stated: the interplay between the chain conformation, mainly agarose chains, which appear as relative flexible molecules, the chain rigidity, which addresses xanthan molecules and the water binding by hydrate shells by polar and charged groups of all constituents. Obviously the different sugars have different impact on the hydrogels. Whereas the modulus of fructose gels rises with increasing concentration up to 60% the sucrose loaded gels show a dramatic decrease of the modulus on larger sucrose concentration. On the other hand, when xanthan is added, both sugars show no influence. The increase of the modulus for fructose in the total range of measured concentrations and sucrose up to 40% may be addressed to the increase of the viscosity of the solution. On the other hand, low molecular weight carbohydrates may interact via hydrogen bonds with the hydrocolloids directly, which may implicate an apparent stiffening of the flexible chains parts, which are responsible for the entropic part of the elasticity. Stiffer chains yield larger moduli (de Gennes, 1979), since additional bending forces are required to deform the flexible part of the chains in the agarose networks. Additionally the helix formation is shifted slightly to lower temperatures. This is caused by two factors: First the viscosity of the solvent is enhanced by the presence of the sugars. The chain segments experience more friction; the dynamics become slower (Doi & Edwards, 1986). In addition the direct interaction of the polar sugar molecules slows down the helix formation between the agarose chains and shifts kinetically the gel formation to lower temperatures. The relatively strong influence of the sugars on the flexible agarose network can be drawn back on the interplay between the sugar molecules and the monomers of the agarose chains, which consist of D- galactose connected with 3,6-anhydro-L-galactose. The monomer unit contains five OH-groups, which may bind water molecules in a hydrate shell. Thus, the polymer is basically surrounded by hydrate water, which has some impact on the conformational properties of the polymer, e.g. its flexibility, which can be measured with the local persistence length of the polymer. When low molecular weight sugars such as fructose and sucrose are added, a competition for water takes place as the low molecular carbohydrates need to be hydrated as well. To understand the nature of the competition better, detailed molecular dynamics on atomistic levels are required. On an intuitive level, it can be concluded that sucrose has a higher capability to form hydrate shells due to a larger number of polar OH-groups (Max & Chapados, 2001). Thus, the strong decrease for sucrose loaded gels at concentrations

between 30% and 40% can be understood by the competition effect since the hydration of sucrose is preferred. The strong drop at 60% is additionally caused by the formation of sucrose crystallites, which prevent the formation of helix aggregates, which in sugar free agarose gels yields a further stiffening of the network. Indeed Figs. 1and 4 indicate that the modulus of the sucrose loaded gel is of the order of magnitude where the helices are formed. When xanthan is added these effects are more or less ruled out. First xanthan dominates the physical behaviour by its high charge degree and secondly by its strong stiffness (Nordqvist & Vilgis, 2011). Therefore sugars have no strong influence on the mechanical behaviour of agaroseexanthan gels, as indicated by the experiments shown in Fig. 5 and summarized in Fig. 8. This model represents the two-stage solidification mechanism of agarose hydrogels and the special role of water involved in this process. The formation of hydrogen bonds is essential for the formation of agarose chains (Fig. 8-A.1) to helices (Fig. 8-A.2) and the subsequent aggregation of them into bundles (Fig. 8-A.3). With the addition of high amounts of sugar (Fig. 8-B.1), both solutes compete for the solvent water whereby sugar molecules seem to dominate over agarose molecules. Thus, restrained water molecules by fructose, respectively sucrose prevent the formation of aggregates and lead to a destabilization of the agarose gel network (Fig. 8-B.2). The resulting gel is less elastic but clearer than gels devoid of any sugar as shown by the photographs in Fig. 2. The water shortage by additional co-solutes in agarose gels becomes even more pronounced when highly negative charged stiff and rod-like xanthan molecules are present (Fig. 8-C.1). Presumably, hydration shells are predominately arranged around the stiff rods and the gel texture is loose and soft. 4. Conclusion In the course of this work, it could be illustrated that the rheological properties of 1 % agarose hydrogels are influenced by the addition of other carbohydrates, such as fructose, sucrose, and xanthan gum, verified by strain-dependent and temperaturedependent oscillation measurements. Thereby, the magnitude of rheological changes depends partial on the type and on the concentration of the carbohydrate used. In general, it could be demonstrated that the addition of high amounts of sugars and/or highly negatively charged polyelectrolytes evolves less elastic, clearer gels of looser network structure and better water holding capacity. The fact, that adding xanthan to hydrocolloids systems seems to lower the influence of the amount of added sugar, could be systematically used for various food applications. In consequence this study shows the detailed interplay between chain flexibility, chain charge fractions and hydrate shells. Acknowledgement The authors thank Dr. Harald Pleiner (MPI for Polymer Research) for valuable discussions and critical reading of the manuscript. Many regards also to Prof. Dr. Dietrich Knorr (TU-Berlin) for supporting this work. Special thanks to the members of the MPIP Food Group for fruitful discussions. References Deszczynski, M. (2003a). Effect of sugars on the mechanical and thermal properties of agarose gels. Food Hydrocolloids, 17(6), 793e799. Deszczynski, M. (2003b). Rheological investigation of the structural properties and aging effects in the agarose/co-solute mixture. Carbohydrate Polymers, 53(1), 85e93. Doi, M., & Edwards, S. F. (1986). The theory of polymer dynamics. Oxford: Oxford University Press. Edwards, S. F., & Vilgis, Th. (1986). The effect of entanglements in rubber elasticity. Polymer Papers, 27, 483e492.

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