Gelation behaviour of gelatin and alginate mixtures

Food Hydrocolloids 23 (2009) 1074–1080

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Gelation behaviour of gelatin and alginate mixtures Maud Panouille´ 1, Ve´ronique Larreta-Garde* Laboratoire ERRMECe, UFR Sciences et Techniques, Universite´ de Cergy-Pontoise, 2 avenue Adolphe Chauvin BP222, 95302 Pontoise cedex, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 May 2008 Accepted 30 June 2008

Mixtures of alginate and gelatin were studied by rheology as a function of different parameters, such as temperature, biopolymer concentrations, calcium concentration and ionic strength. In particular conditions, the formation of a mixed gel of alginate and gelatin is obtained. A slow release of calcium ions leads first to an irreversible alginate gel and cooling results in a reversible gelatin gel. Depending on experimental conditions, non-linear behaviours upon gelation of alginate occur and a collapse of alginate gel is directly observable by rheology. These trends are favoured between 35 and 45  C, by a high total biopolymer concentration or a high calcium concentration and ionic strength. Different mechanisms could be responsible for this collapse, such as a competition between alginate gelation and phase separation in the biopolymer mixture or an over-association of alginate chains at high Ca2þ concentration, favoured by the presence of gelatin. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Alginate Gelatin Mixed gel Gelation Phase separation Rheology

1. Introduction Biopolymer mixtures have been extensively studied for the last 20 years because of their importance in food properties and formulation (Plucknett, Normand, Pomfret & Ferdinando, 2000; Norton & Frith, 2001; Tolstoguzov, 2003). Despite many studies carried on various mixtures, it is still difficult, if not impossible, to predict the behaviour of these mixtures. When two biopolymers (proteins and/or polysaccharides) are mixed together, three different behaviours can occur (Piculell, Bergfeldt & Nilsson, 1995; Doublier, Garnier, Renard & Sanchez, 2000; de Kruif & Tuinier, 2001; Turgeon, Beaulieu, Schmitt & Sanchez, 2003). In some rare cases or for low polymer concentrations, the biopolymers are miscible and co-exist in a single phase. Their mixture is thus thermodynamically stable (Tolstoguzov, 1992). However, in most cases, mixing two or more biopolymers results in a phase separation, which can be associative (the first phase being enriched in both polymers, the second one in solvent) or segregative (each phase being enriched with one of the two biopolymers). Segregative phase separation, also called thermodynamic incompatibility, is the most frequent and is mainly due to excluded volume effects

* Corresponding author. Tel.: þ33 1 34 25 66 05; fax: þ33 1 34 25 66 94. 1 Present address: AgroParisTech, UMR 782 Ge´nie et Microbiologie des Proce´de´s Alimentaires, F-78850 Thiverval-Grignon, France E-mail addresses: [email protected] (M. Panouille´), [email protected] (V. Larreta-Garde). 0268-005X/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2008.06.011

and repulsion between the two biopolymers (Tolstoguzov, 1992; Doublier et al., 2000). The situation becomes more complex if one or two polymers undergo a sol–gel transition. In that case, a competition between gelation and phase separation appears and the kinetics of each phenomenon determines the final state of the mixture. For example, a fast gelation can freeze a phase-separated system (Kasapis, 1995; Anderson & Jones, 2001; Tromp, van de Velde, van Riel & Paques, 2001). With more than one gelling agent, three groups of multicomponent gels can be obtained: complex, mixed and filled gels (Tolstoguzov, 1995). Critical parameters are the limit of co-solubility of the two biopolymers and their critical gelation concentration. In complex gels, both polymers gel and interact with each other. Mixed gels consist in two relatively independent 3-dimensional networks and occur when the concentrations of each polymer are higher than the critical gelation concentration. Finally, filled gels are constituted by a network filled with another non-gelling polymer. Synergistic or antagonist effects can exist between the two biopolymers. Synergy is generally related to excluded volume effects and water distribution between the two phases (Tolstoguzov, 1995). Gelatin is a protein obtained by the denaturation of the triple helix of collagen. In solution at moderate temperature, gelatin chains are random coils, which associate into helices and gel when temperature decreases (Joly-Duhamel, Hellio, Ajdari & Djabourov, 2002; Joly-Duhamel, Hellio & Djabourov, 2002). The behaviour of mixtures of gelatin and various polysaccharides, such as maltodextrin (Loren & Hermansson, 2000; Plucknett et al., 2000; Loren et al., 2001; Norton & Frith, 2001; Butler & Heppenstall-Butler,

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2003), pectin (Antonov, Lashko, Glotova, Malovikova & Markovich, 1996; Gilsenan, Richardson & Morris, 2003a, 2003b, 2003c), or alginate (Tolstoguzov, 1995; Antonov et al., 1996; Voron’ko, Derkach & Izmailova, 2002; Doume`che, Picard & Larreta-Garde, 2007), has been studied (Tolstoguzov, 1992; Tolstoguzov, 1994; Fonkwe, Narsimhan & Cha, 2003; Harrington & Morris, 2008). Alginate is a linear polysaccharide, whose monomers are mannuronic and guluronic acids. Alginate gelation can be initiated by the release of Ca2þ ions, which form egg-box structures between alginate chains (Draget, Ostgaard & Smidsrod, 1990; Stokke et al., 2000; Siew & Williams, 2005). The formation of mixed gels of gelatin and alginate has been previously reported. In that aim, Tolstoguzov first cooled the mixture to induce gelatin gelation, and placed this gel in a calcium-citrate bath to stimulate the gelation of alginate (Tolstoguzov, 1995). More recently, an inverse strategy was adopted by Doume`che et al. (2007). The alginate gel was first obtained by a slow release of Ca2þ ions at 27  C and then cooling resulted in the gelation of gelatin. In both cases, the gelatin gel is almost completely reversible upon temperature cycles and is thus not very dependent on the alginate gel. Depending on the pH value, the formation of a complex between alginate and gelatin was also suggested (Bush & Hill, 1983; Voron’ko et al., 2002). The aim of this paper is to investigate the influence of experimental conditions, such as temperature, biopolymer concentrations, ionic strength and calcium concentration, on the formation of a mixed gel of gelatin and alginate. Rheological and polarimetric studies were performed on the mixtures and constitute complementary methods to highlight the contribution of both polymers and microenvironment during the gelation process. 2. Materials and methods

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2.4. Optical rotation Formation of gelatin triple helices was monitored by polarimetry (Joly-Duhamel, Hellio & Djabourov, 2002). Optical rotation was measured on a Jasco P-1010 polarimeter at 435 nm. Temperature control was performed by a Julabo F25 bath. Cooling and heating ramps of 0.5  C min1 were applied. 3. Results 3.1. Alginate and gelatin co-gel formation In some conditions the formation of a co-gel of alginate and gelatin is possible, as described by Doume`che et al. (2007). The alginate gel is first obtained by complexation of alginate with calcium ions, and then the gelatin gel is formed by cooling the mixture (Fig. 1). More precisely, the addition of D-glucono-d-lactone (GDL, 180 mM) to a solution containing alginate, gelatin and calcium ions chelated by EDTA (CaEDTA, 180 mM) leads to a slow release of Ca2þ ions, which allows the formation of a homogeneous alginate gel. In the meantime, the pH decreases from 8 to 4.5. The alginate gel is kept for 8 h at 27  C, a temperature where the helix amount of a 1.5% gelatin solution is too low to lead to the protein gelation. The small decrease of the optical rotation angle during the first 8 h is also observed for alginate gelation alone (without gelatin) and therefore cannot be related to the formation of gelatin helices. The mixture is then cooled at 10  C. During cooling, the shear moduli G0 and G00 show a significant increase (from 75 to 400 Pa for G0 and from 8 to 40 Pa for G00 ) and the optical rotation angle, measured by polarimetry at 435 nm, decreases sharply. This decrease is

2.1. Materials 30

1000

100

G', G'' (Pa)

25 10 20 1

2.2. Biopolymer mixture preparation

15

0,1

Rheological measurements were performed with a Rheostress 150 (Haake, Courtaboeuf, France) operating in the oscillatory mode, with a strain of 5% and a frequency of 1 Hz. These conditions were checked to stand in the linear viscoelastic region. Storage modulus G0 and loss modulus G00 were recorded as a function of time. Temperature was controlled by a Cryostat-F6 (Haake, Courtaboeuf, France). A cone/plate geometry with a cone of 60 mm/2 was used and water evaporation was prevented by the use of an adapted water trap. Temperature ramps of 0.5  C min1 were applied.

Optical rotation 435nm (°)

2.3. Rheological measurements

a -0,3 25 -0,4 -0,5

20

-0,6 15

-0,7

Temperature (°C)

CaEDTA solutions were first prepared by dissolving Na4EDTA, (H2O)2 together with CaCl2 in ultrapure (Millipore) water. Alginate solution was obtained by dispersing powder in a CaEDTA solution by slow magnetic stirring for at least 1 h. Gelatin was swelled at 4  C in water and then solubilised at 40  C for 15 min under magnetic stirring. Alginate and gelatin solutions were mixed at 40  C in quantities necessary to obtain the final desired concentrations. A freshly prepared GDL solution was finally added to allow the release of calcium ions.

Temperature (°C)

Alginate from Macrocystis pyrifera and gelatin extracted from porcine skin (type A) were obtained from Sigma Aldrich (StQuentin en Fallavier, France). D(þ)-gluconic acid d-lactone (GDL), Ethylenediamine tetracetic acid tetrasodium salt dihydrate (Na4EDTA, (H2O)2) and calcium chloride (CaCl2) were respectively purchased from Sigma Aldrich (St-Quentin en Fallavier, France), Research Organics (Cleveland, Ohio, USA) and Prolabo (Paris, France).

-0,8 -0,9

b 0

10 200

400

600

800

1000

1200

Time (minutes) Fig. 1. Kinetics of formation and evolution of a mixed gel of alginate and gelatin followed by (a) rheology and (b) polarimetry, for a mixture with alginate 1%, gelatin 1.5%, CaEDTA 180 mM, GDL 180 mM. (a) G0 (closed circles), G00 (open circles) and temperature (solid line); (b) optical rotation angle (triangles) and temperature (solid line).

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characteristic of the formation of triple helices of gelatin and provides evidence for the formation of a gelatin gel, together with the pre-existing alginate gel. This gelatin gel is almost completely reversible upon temperature cycles, whereas the alginate gel is irreversible. The shear moduli of the co-gel are higher than the sum of those of individual gels at the same temperature, which shows a synergistic interaction between the two biopolymers. At 10  C, G0 and G00 values are, respectively, 135 and 6 Pa for the alginate gel (1%) (see Fig. 5), 130 and 2 Pa for the gelatin gel (1.5%) (data not shown) and 400 and 40 Pa for the mixture (1% alginate þ 1.5% gelatin). However, tan d is slightly higher for the mixture (0.1) than for alginate or gelatin gels (respectively, 0.044 and 0.015), suggesting a weakening of the gel structure in the mixture. 3.2. Alginate gelation The formation of the alginate/gelatin co-gel is very sensitive to experimental conditions, such as temperature, polymer or calcium concentrations and ionic strength, which can modify the behaviour of both polymers. Alginate gelation can be hampered by varying experimental conditions. 3.2.1. Effect of temperature In the presence of gelatin, the formation of alginate gel is dependent on temperature, as described in Fig. 2. At 27  C (Fig. 2b) the results are the same as in Fig. 1. At 40 and 45  C (Fig. 2c and d), G0 and G00 begin to increase up to, respectively, 5 and 1 Pa (40  C) or 6 and 1 Pa (45  C), but then G0 decreases sharply to values close to

100

1 Pa. The elastic modulus G0 remains higher than the viscous modulus G00 , suggesting that the mixture is in a weak gel state. Visually, the mixtures are viscous, heterogeneous and sticky solutions. At 50 and 60  C (Fig. 2e and f) the alginate gel formation becomes possible again. The value of G0 at 50  C is lower than those at 27 and 60  C, suggesting that 50  C constitutes an intermediate situation between the collapse of alginate gel around 40  C and the optimal gel formation. The values of G00 above 50  C are smaller than that at 27  C, perhaps because of a smaller viscosity of gelatin at these high temperatures. At 20  C (Fig. 2a), the shear moduli are slightly higher than those at 27  C, probably because of the formation of a small quantity of gelatin helices. Indeed, visual observations were performed on mixtures placed successively at different temperatures, as shown in Table 1. Alginate and gelatin solutions alone were also prepared as control. Alginate solutions without GDL do not evolve whatever be the temperature cycles. Addition of GDL to alginate solutions leads to gel formation for all tested temperatures, but alginate gelation rate increases with temperature, probably because of an increase of GDL hydrolysis rate. Gelatin solutions only gel at 4  C and a very weak gel is observed after one night at 20  C. Addition of GDL does not influence the final state of gelatin solutions. For alginate (1%) and gelatin (1.5%) mixtures, at 4 or 20  C, a gel is formed after respectively 30 min and 3 h, with or without GDL. This suggests that this gel is a result of the formation of a gelatin network. By comparing with the gelatin control at 20  C, the presence of alginate seems to favour gelatin gelation. On the other hand, when the mixture is thereafter placed at 40  C, a gel state is still observed,

a

b

c

d

e

f

G', G'' (Pa)

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1

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G', G'' (Pa)

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G', G'' (Pa)

10

1

0,1

0,01

0

50

100

Time (minutes)

150

50

100

150

200

Time (minutes)

Fig. 2. Effect of temperature on the gelation of alginate, for a mixture with alginate 1%, gelatin 1.5%, CaEDTA 180 mM, GDL 180 mM. (a) 20  C; (b) 27  C; (c) 40  C; (d) 45  C; (e) 50  C; (f) 60  C. G0 (closed circles), G00 (open circles).

M. Panouille´, V. Larreta-Garde / Food Hydrocolloids 23 (2009) 1074–1080 Table 1 Visual state of mixtures observed after different temperature cycles

GDL

a

After 3 h

4 C

Gelm (30’)

4 C 40  C

Gelm Gela

40  C 4 C

Gela Gelm

20  C

Gelm (3 h)

4 C 40  C

Gelm Gela

40  C 4 C

Gela Gelm

40  C

Sticky, viscous

4 C 40  C

Gelg Sticky, viscous

40  C 4 C

Liquid Gelg

4 C

Gelg (30’)

4 C 40  C

Gelg Liquid

40  C 4 C

Liquid Gelg

20  C

Gelg (3 h)

4 C 40  C

Gelg Liquid

40  C 4 C

Liquid Gelg

40  C

Liquid

4 C 40  C

Gelg Liquid

40  C 4 C

Liquid Gelg

Alginate (1%) and gelatin (1.5%) mixtures (CaEDTA 180 mM) were observed after addition or not of GDL (180 mM) at different temperatures. First mixtures were placed for 24 h in a water bath at 4  C, 20  C or 40  C, and then successively for 3 h at 4  C (or 40  C) and 3 h at 40  C (or 4  C). ‘‘Gel’’ is defined as a state that does not flow under its own weight. As a function of experimental conditions, we can assume that the observed gel is an alginate gel (a), a gelatin gel (g) or a mixed gel (m).

despite the melting of the gelatin gel, and is probably constituted by an alginate network. We can therefore assume that at 4 or 20  C, both alginate and gelatin networks are formed at the same time. 3.2.2. Effect of biopolymer concentrations Both alginate and gelatin concentrations were modified to investigate the effect of polymer concentrations on alginate gelation at 40  C. First the alginate concentration was kept constant (1%) and the gelatin concentration varied from 0 to 2% (Fig. 3). Without gelatin, alginate gels are formed in 20 min and the G0 value reaches 65 Pa. The gel time is about the same for all gelatin concentrations, but the further evolution of the gel is dependent on gelatin concentration. With 1% gelatin, alginate gels have lower moduli. Above 1.25% gelatin, the alginate solutions begin to gel (G0 and G00 increase) but gels seem to be destabilized and G0 and G00 decrease. The higher the gelatin concentration, the faster the collapse of the gel. For 2% gelatin, G0 does not even reach 1 Pa before decreasing and is finally lower than G00 . The same experiment was done with a constant gelatin concentration (1%) and increasing concentrations of alginate (from 1% to 1.5%), see Fig. 4. As previously, the increase of alginate concentration leads to a collapse of alginate gel and to a decrease of shear moduli after the beginning of gelation. By comparing these results, it appears that the total biopolymer concentration is the key parameter in the alginate gel break. If the total concentration is lower than 2%, the alginate solution can gel, but above 2.25% we observe a further evolution of the gel into a viscous heterogeneous solution. 3.2.3. Effect of calcium concentration and ionic strength The salt concentration also influences the gelation behaviour of alginate. In the following experiment, the molar concentration ratio between CaEDTA and GDL was kept constant and equal to 1. Their concentrations were varied from 20 mM to 180 mM, for alginate and gelatin concentrations, respectively, 1% and 1.5% at 40  C (first 200 min in Fig. 5; longer experimental times will be described in Section 3.3.2). For 180 mM, the alginate gel collapses, as shown previously. For 135 mM, G0 increases up to 20 Pa, but then decreases slowly to a plateau value of 2 Pa. By decreasing the salt concentration to 90 mM, the alginate solution gels but G0 reaches a maximum value (150 Pa) after 250 min and then decreases slowly (95 Pa after 480 min). For 45 mM, we observe a typical gelation curve with G0

100

G' (Pa)

þGDL

After 3 h

1000

10

1

0,1

b 1

G'' (Pa)

After 24 h

1077

0,1

0,01

0

50

100

150

200

Time (minutes) Fig. 3. Effect of gelatin concentration on the gelation of alginate at 40  C, for a mixture with alginate 1%, CaEDTA 180 mM, GDL 180 mM. (a) G0 ; (b) G00 . Gelatin 0% (circles), 1% (squares), 1.25% (up triangles); 1.5% (down triangles); 2% (diamonds).

and G00 increasing up to plateau values of 120 and 5 Pa, respectively. For 20 mM, the release of a sufficient Ca2þ is slower and gelation occurs only after 130 min. In all these cases, the time evolution of pH is the same, showing a slow decrease from 8 to 4.5. Differences in pH are thus not responsible for the various behaviours described here. In order to determine if alginate gel collapse by increasing Ca2þ concentration is only related to ionic strength or is a specific effect of calcium ions, complementary experiments were performed by adding NaCl in a mixture containing 1% alginate, 1.5% gelatin and 45 mM CaEDTA, see Fig. 6. Addition of NaCl also influences alginate gelation, but in a different way from Ca2þ (Fig. 5). Contrary to calcium ions, which destabilise alginate gel after the beginning of its formation, a high concentration of NaCl increases gelation time and decreases final alginate gel strength. As a consequence, it seems that calcium ions play a specific role in the collapse of alginate gel. High concentrations of Ca2þ could lead to an overassociation of alginate chains, resulting in a weakening of the gel.

3.3. Gelatin gelation The same parameters also influence the gelation of gelatin when the mixture is cooled. For illustration we will only consider the influence of gelatin and calcium concentrations. 3.3.1. Effect of gelatin concentration Fig. 7 shows the effect of gelatin concentration for an alginate concentration of 1% as Fig. 3, but for longer experimental times and temperature cycles from 40 to 27 and 10  C. For 1% gelatin, cooling from 40 to 27  C leads to a decrease of G0 from 35 to 20 Pa. This result is quite surprising because biopolymer

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1000

100

a

a

40

100

G' (Pa)

1

0,1

30

10

1

20

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G' (Pa)

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0,1 10

b

b

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100

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G'' (Pa)

30

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20

0,1

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1

0,1 10 0,01

0,01 0

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0

Fig. 4. Effect of alginate concentration on the gelation of alginate at 40  C, for a mixture with gelatin 1%, CaEDTA 180 mM, GDL 180 mM. (a) G0 ; (b) G00 . Alginate 1% (squares), 1.25% (up triangles); 1.5% (circles).

gels generally exhibit an increase of shear moduli after cooling, as observed for alginate alone. When cooling the mixture to 10  C, an increase of G0 up to 60 Pa is observed, due to gelatin gelation. In this case, the moduli values are a combination of alginate and gelatin co-gel. For higher gelatin concentrations, the alginate gel collapses and cooling leads to the gelation of gelatin. When cooling to 27  C, then 10  C, the shear moduli increase successively. The higher the gelatin concentration, the higher the G0 and G00 values. The effect is particularly significant for 2% gelatin at 10  C, where G0 reaches 425 Pa (instead of 49 Pa for 1.5% and 30 Pa for 1.25%). This could be explained by the higher gelatin concentration, but also by a looser alginate network, allowing a better development of gelatin helices and network. 3.3.2. Effect of calcium concentration and ionic strength The same trend is observed by increasing calcium concentration, see Fig. 5. The behaviour obtained for 180 mM has been described previously (Fig. 7). For a salt concentration of 90 mM, cooling to 27  C results in a sharp decrease of G0 and a small increase of G00 . The decrease of G0 is probably related to the progressive decrease already seen at 40  C and could be due to an over-association of alginate chains. The increase of G00 is the result of an increase of gelatin viscosity due to the formation of a few helices. However, at 10  C, G0 and G00 increase again, because of the gelation of gelatin. With 45 mM, the situation is quite similar to that observed in Fig. 7 for 1% gelatin and 180 mM CaEDTA and GDL, i.e. a small decrease of G0 at 27  C and an increase at 10  C. Finally, for 20 mM salt, G0 and G00 increase significantly both at 27 and 10  C. At 27  C, this increase is probably due to the formation of gelatin helices, but also to a reinforcement of alginate gel by cooling. It is interesting to note that the shear moduli values are almost the same at 10  C for salt

200

400

600

800

Time (minutes)

Time (minutes)

Fig. 5. Effect of CaEDTA and GDL concentrations on the gelation of alginate and gelatin upon temperature cycles, for a mixture with alginate 1% and gelatin 1.5% (a) G0 ; (b) G00 . CaEDTA and GDL 20 mM (diamonds), 45 mM (up triangles), 90 mM (squares), 135 mM (circles), 180 mM (down triangles), temperature (solid line).

concentrations ranging from 20 mM to 90 mM, which means that gelatin gelation is not influenced by calcium concentrations in this range. However, for 180 mM, G0 and G00 are much smaller, suggesting that the mixture structure and its heterogeneity limit the formation of helices of gelatin, even at 10  C. Fig. 6 shows the effect of increasing ionic strength (NaCl concentration). Up to 270 mM NaCl, gelation of gelatin occurs in the same way at 10  C, but for higher ionic strength (1 M NaCl), gelatin gel strength decreases. 4. Discussion This paper describes experimental work performed on alginate and gelatin mixtures. The system studied is quite complex, as it contains not only a mixture of two biopolymers, but also many salt components (Na4EDTA, CaCl2, GDL). However, such a complex system is required to obtain a homogeneous alginate gel, because direct addition of calcium ions to an alginate solution leads instantaneously to a heterogeneous alginate gel. The use of CaEDTA/GDL is thus necessary, but generates a dynamic system, in constant evolution. The addition of GDL is responsible for a decrease in the pH value, leading to a slow release of calcium ions. In our point of view, two different mechanisms could explain the collapse of alginate gel. A first possible interpretation is that alginate gel collapse is caused by a phase separation in the biopolymer mixture. Whereas the release of calcium ions tends to cause alginate gelation, the decrease of pH modifies interactions between alginate and gelatin and could initiate a phase separation. In that case, alginate should be confined in a dispersed phase in the gelatin-rich continuous matrix. Even if the dispersed phase of alginate is still able to gel, its

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100

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100

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0,01

Temperature (°C)

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100

Temperature (°C)

G' (Pa)

1079

0

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800

Time (minutes)

10 0,01

0

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600

800

Time (minutes)

Fig. 6. Effect of NaCl concentration on the gelation of alginate and gelatin upon temperature cycles, for a mixture with alginate 1%, gelatin 1.5%, CaEDTA 45 mM and GDL 45 mM (a) G0 ; (b) G00 . Without NaCl (up triangles), NaCl 270 mM (circles), 1 M (squares), temperature (solid line).

Fig. 7. Effect of gelatin concentration on the gelation of alginate and gelatin upon temperature cycles, for a mixture with alginate 1%, CaEDTA 180 mM and GDL 180 mM (a) G0 ; (b) G00 . Gelatin 0% (circles), 1% (squares), 1.25% (up triangles); 1.5% (down triangles); 2% (diamonds), temperature (solid line).

gelation would have little effect on overall rheology, explaining the decrease and low values of moduli. The hypothesis of phase separation is consistent with previous reports in literature and can be supported by the following remarks. The destabilization of alginate gel occurs above a critical total biopolymer concentration and for high ionic strengths. Biopolymer mixtures are known to be stable for low polymer concentrations, but phases separate over a critical polymer concentration (Tolstoguzov, 1995; Semenova & Savilova, 1998; Alves, Antonov & Goncalves, 1999; Tolstoguzov, 2003). For gelatin/linear polysaccharide mixtures, the concentration threshold for phase separation was found between 2% and 4%, and around 1.5% for gelatin/alginate mixture (Tolstoguzov, 2003). In this study we found a little higher phase separation critical concentration, which is 2.25%. This slight difference could be explained by different polymer molar mass or ionic strength. Moreover, alginate is acquiring a gel state at the beginning of phase separation and it is known that polysaccharides exhibit a high co-solubility with other biopolymers after gelation (Tolstoguzov, 2003). Many studies, performed on various biopolymer mixtures, have also shown that an increase in ionic strength promotes and accelerates phase separation if at least one of the biopolymers is charged (Antonov et al., 1996; Vinches, Parker & Reed, 1997; Alves et al.,1999; Nordmark & Ziegler, 2000; Norton & Frith, 2001; Gilsenan et al., 2003a, 2003b, 2003c), which is consistent with our results. The decrease of pH, resulting from GDL addition, modifies interactions between alginate and gelatin chains. The pKa of alginate and gelatin are around 3.4 and 8.8, respectively. GDL addition is responsible for a decrease of pH from 8 (related to CaEDTA) to 4.5. In this whole range of pH, alginate chains carry a global negative net charge, whereas gelatin has a global positive net charge. Attractive interactions thus exist between the two biopolymer chains and the formation of electrostatic complexes has been described (Bush &

Hill, 1983; Voron’ko et al., 2002). A decrease of pH has also been reported to increase the compatibility in solution between gelatin and alginate or oligosaccharide (Antonov et al., 1996; Vinches et al., 1997). This is not contradictory with the effects here observed as the formation of the alginate gel occurs with the fixation of Ca2þ, inducing a decrease in the net negative charge of the polysaccharide. The non-linear effect of temperature on the gelation of alginate is complex, but in agreement with formerly described results. Phase separation would be favoured only between 35 and 45  C, alginate gelation being achieved outside this range of temperature. This result can be related to previous observations on mixed gelatin–alginate gels (Tolstoguzov, 1995). Below 30  C, the mixed gel behaved like a gelatin gel; above 45  C, it behaved like a calcium alginate gel. Between 30 and 45  C, the gel was reported to be anomalously highly deformable. The author explained this fact in terms of viscosity changes in the gel dispersion medium. Calcium and sodium ions have a different influence on alginate gelation, as shown in Figs. 5 and 6. As calcium ions tend to favour alginate gel collapse, sodium ions delay alginate gelation and lead to a weakening of alginate gel. This is consistent with previous results showing that sodium ions have no effect on formation of dimeric calcium alginate junctions, but inhibit further calcium induced association of dimers into larger junctions (Morris, Rees, Thom & Boyd, 1978). This observation could support another interpretation of the results. The collapse of alginate gel could be related to an over-association of alginate due to a high calcium concentration and favoured by the presence of gelatin. Indeed, segregative interactions within single-phase mixtures can promote self-association of the constituent polymers. Such behaviour was already observed for low methoxy pectin and oxidised starch mixture in the presence of high concentrations of calcium (Abdulmola, Richardson & Morris, 2000; Picout, Richardson & Morris, 2000; Picout, Richardson, Rolin, Abeysekera & Morris,

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2000). It was explained by a thermodynamic incompatibility between the two biopolymers, causing precipitation of calcium pectinate within the gel network (Abdulmola et al., 2000). The final values of G0 in Fig. 5 (after heating to 27  C to melt the gelatin network) decrease systematically as the concentration of CaEDTA is increased, which is consistent with progressive overassociation of alginate in response to the combined effect of increasing concentration of calcium ions and segregative interactions with disordered gelatin. The reductions in moduli after initial development of alginate gel structure could similarly be explained by over-association, with consequent collapse of network structure. As would be expected from this interpretation, collapse occurs progressively earlier (Figs. 3 and 4) as the concentration of either polymer is increased (causing an increase in segregative interactions) and as the concentration of CaEDTA (Fig. 5) is increased (with increasing concentration of calcium ions acting together with segregative interactions to promote excessive aggregation of alginate). This hypothesis would also be supported by the behaviour of alginate gels (2.5%), which are also subjected to a sharp decrease of shear moduli during a temperature change from 40 to 27  C (data not shown). A loss of gel strength during cooling was reported for mixtures containing low methoxy pectin and oxidised starch, galactomannans or dextran (Giannouli, Richardson & Morris, 2004a, 2004b, 2004c). It was interpreted by an excessive association of pectin into large aggregate bundles, driven by segregative interactions with the polysaccharides (Giannouli et al., 2004a). As pectin and alginate have a similar association mechanism implicating interactions with calcium ions, over-association of alginate chains could also explain alginate collapse. 5. Conclusion This study evidences the complex behaviour of alginate and gelatin mixtures and highlights conditions when the formation of a mixed gel is possible. In some cases (high polymer or calcium concentrations, high ionic strength and a temperature ranging from 35 to 45  C), alginate gel collapses. This collapse could be caused by a phase separation in the mixture or by an over-association of alginate favoured by a high calcium and total biopolymer concentration. In other particular conditions, a mixed gel of gelatin and alginate is formed, the alginate and gelatin gels being, respectively, irreversible and reversible upon temperature cycles. Additional confocal microscopy or scattering experiments could help to better understand the mechanism responsible for the alginate gel collapse and the micro-structure of the mixture. References Abdulmola, N. A., Richardson, R. K., & Morris, E. R. (2000). Effect of oxidised starch on calcium pectinate gels. Food Hydrocolloids, 14(6), 569–577. Alves, M. M., Antonov, Y. A., & Goncalves, M. P. (1999). The effect of structural features of gelatin on its thermodynamic compatibility with locust bean gum in aqueous media. Food Hydrocolloids, 13(2), 157–166. Anderson, V. J., & Jones, R. A. L. (2001). The influence of gelation on the mechanism of phase separation of a biopolymer mixture. Polymer, 42(23), 9601–9610. Antonov, Y. A., Lashko, N. P., Glotova, Y. K., Malovikova, A., & Markovich, O. (1996). Effect of the structural features of pectins and alginates on their thermodynamic compatibility with gelatin in aqueous media. Food Hydrocolloids, 10(1), 1–9. Bush, N. L., & Hill, C. R. (1983). Gelatine–alginate complex gel: a new acoustically tissue-equivalent material. Ultrasound in Medicine & Biology, 9(5), 479–484. Butler, M. F., & Heppenstall-Butler, M. (2003). Phase separation in gelatin/dextran and gelatin/maltodextrin mixtures. Food Hydrocolloids, 17(6), 815–830. Doublier, J.-L., Garnier, C., Renard, D., & Sanchez, C. (2000). Protein–polysaccharide interactions. Current Opinion in Colloid & Interface Science, 5(3–4), 202–214. Doume`che, B., Picard, J., & Larreta-Garde, V. (2007). Enzymatic tailoring of alginate gels and gelatin–alginate IPN. Biomacromolecules, 8(11), 3613–3618. Draget, K. I., Ostgaard, K., & Smidsrod, O. (1990). Homogeneous alginate gels: a technical approach. Carbohydrate Polymers, 14(2), 159–178. Fonkwe, L. G., Narsimhan, G., & Cha, A. S. (2003). Characterization of gelation time and texture of gelatin and gelatin–polysaccharide mixed gels. Food Hydrocolloids, 17(6), 871–883.

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