Gelation and microstructure of dilute gellan solutions with calcium ions

Food Hydrocolloids 28 (2012) 291e300

Contents lists available at SciVerse ScienceDirect

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

Gelation and microstructure of dilute gellan solutions with calcium ions Sixto J. Pérez-Campos a, Norberto Chavarría-Hernández a, Alberto Tecante b, Mariana Ramírez-Gilly b, Adriana I. Rodríguez-Hernández a, * a

Cuerpo Académico de Biotecnología Agroalimentaria, Instituto de Ciencias Agropecuarias, Universidad Autónoma del Estado de Hidalgo, Av. Universidad km 1, Rancho Universitario, Tulancingo de Bravo, Hidalgo, CP 43600, Mexico b Departamento de Alimentos y Biotecnología, Facultad de Química “E”, Universidad Nacional Autónoma de México, Cd. Universitaria, 04510, Mexico

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 February 2011 Accepted 6 January 2012

The viscoelasticity at 25
C and microstructure of 0.02e0.07 wt% of low acyl gellan aqueous media were investigated for ratios of Ca2þ to gellan in the range of 0e38.8, using small amplitude oscillatory shear rheometry and confocal laser scanning microscopy (CLSM), respectively. The total ionic concentration (CT ¼ g CP þ CS, being CP and CS the gellan and calcium concentrations, respectively, and g the mean activity coefficient) of the systems was found to be the triggering and critical factor for the gelation and elasticity of gellan systems. The gel point (Tgel) and storage moduli (G0 ) increased upon increasing CT. However, G0 showed a maximum for CT ¼ 9.3  1.2 meq/L, followed by a progressive reduction as CT increased; this was primarily due to further addition of calcium, as CP had a low contribution to CT of the systems. CLSM demonstrated that the level of counter-ions was enough to induce the formation of a network, whose connection depended on CP and whose reinforcement was ion dependent. Therefore, even at very low levels of gellan, it is possible to create a wide spectrum of viscoelastic behaviors going from structured liquids to strong gels through the specific combinations of gellan and cation concentrations. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Gellan Solegel transition Confocal microscopy Rheology

1. Introduction Gellan is a bacterial polysaccharide from Sphingomonas elodea (Sworn, Sanderson, & Gibson, 1995). Its deacylated form is an anionic polysaccharide consisting of a linear tetrasaccharide repeating unit of [/ 3)-b-D-Glcp-(1 / 4)-b-D-GlcpA-(1 / 4)-b-DGlcp-(1 / 4)-a-L-Rhap-(1 /] (Jansson, Lindberg, & Sandford, 1983). In the food industry, gellan is used as a stabilizing, gelling, film-forming and encapsulating agent (Kiani, Mousavi, Razavi, & Morris, 2010; León, Lamanna, Gerschenson, & Rojas, 2008; RojasGraü, Tapia, Rodríguez, Carmona, & Martin-Belloso, 2007; SosaHerrera, Berli, & Martínez-Padilla, 2008). In pharmaceutical technology, it is widely adapted as a drug delivery vehicle (Coviello, Matricardi, Marianecci, & Alhaique, 2007; Sunil, Sheetal, & Tejraj, 2006). These multifunctional properties are derived from its reactivity to cations and its gel-forming ability at concentrations remarkably lower than other hydrocolloids such as carrageenans, alginate, pectin or gelatin.

* Corresponding author. Tel.: þ52 771 717 2000x4641; fax: þ52 771 717 2125. E-mail address: [email protected] (A.I. Rodríguez-Hernández). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2012.01.008

The gellan gelation mechanism has been widely studied. It has been reported that gellan shows a thermoreversible solegel transition produced by a conformational change from a random coil or disordered structure to a double helix or ordered structure (Crescenzi & Dentini, 1987). This transition is influenced by the physicochemical properties of the solvent and takes place when the temperature decreases or the ionic concentration increases. Gelation of gellan is generally explained as a two-step thermoreversible process in which helix ordering is followed by the association between stiff double helices through intermolecular interactions. In the presence of cations, the inter-helical associations are enhanced through hydrogen bonds leading to gel formation with increased thermal stability (Noda et al., 2008). It has been suggested that the gelation mechanism of low acyl gellan in the presence of divalent cations is substantially different from that promoted with monovalent cations (Miyoshi & Nishinari, 1999; Nitta, Takahashi, & Nishinari, 2010). In the presence of the latter, gelation occurs with the subsequent aggregation of double helices mediated by cations, and the solegel transition appears at temperatures lower than the coilehelix transition. In contrast, it is assumed that the divalent cations immediately interact with gellan chain segments as cooling takes place, forming specifically ordered structures at temperatures higher than the coilehelix transition. These ordered

292

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

structures become extremely stable to temperature upon progressive addition of divalent cations. Therefore, divalent ions may be more efficient to screen charged sites than monovalent ions (Nickerson & Paulson, 2004). Investigations on the chain aggregation mechanism of gellan during gelation and the rheological properties of its aqueous solutions and gels have been conducted to elucidate the structureefunction relationship of this macromolecule. Earlier rheological and confocal laser scanning microscopy studies of 0.005e0.05 wt% low acyl gellan (Rodríguez-Hernández, Durand, Garnier, Tecante, & Doublier, 2003) proved that at these low gellan concentrations, the presence of 10 mM CaCl2 induced the formation of a network. At the lowest gellan concentration, the mechanical spectrum showed a gel behavior while microscopy observations revealed the formation of a three-dimensional network in spite of the physical appearance of a ‘pourable liquid’. This study proved that the progressive increase of the gellan concentration enhances the elastic character of the obtained systems, but the magnitude of the solid-like properties also depends on the cation content. Furthermore, additional studies using different calciumegellan ratios are necessary to provide more information about the gelation process of those dilute gellan systems. Some procedures have been proposed to determine the gel point of polymers. Winter and Chambon (1986) found that at the gel point, the storage modulus (G0 ) and the loss modulus (G00 ) exhibited the same power law dependence on the angular frequency (i.e., G0 and G00 w un). They also showed that the relaxation modulus, G(t), is given by G(t) ¼ Stn, where t is time, S the gel strength and n the relaxation exponent. This implies that at the gel point the phase angle between stress and strain, d, is independent of frequency and proportional to n (i.e., tan (d) ¼ tan (np/2)). Thus, the independence of the angular frequency from tan d provides the gel point and the relaxation exponent, n. This method has been used to determine the time, temperature and polysaccharide concentration, during the gelation of i-carrageenan (Doublier & Cuvelier, 1996) xanthanegalactomannan mixtures (Goycoolea, Milas, & Rinaudo, 2001), tamarind seed xyloglucanesodium gellan mixtures (Nitta, Kim, & Nishinari, 2003) and k-carrageenan (Núñez-Santiago & Tecante, 2007). However, only a few studies concerning gel point determination in aqueous gellan solutions have been reported (Dai, Liu, Liu, & Tong, 2008; Dai, Liu, & Tong, 2010). Considering that many industrial applications of gellan concern its gelation in aqueous media, in the present study we discuss the effect of calcium concentration on gelation of dilute gellan solutions upon cooling, through the determination of the gel point which was determined by two procedures on the basis of the work of Winter and Chambon (1986). A gelation diagram of gellan is reported and the viscoelastic properties and microstructure of the gellan systems at 25
C were also examined. The obtained results facilitate a better understanding of the functionality of gellan in dilute solutions. Reports for dynamic viscoelastic properties of very dilute gellan systems are scarce (i.e., involving gellan concentrations below 0.07% w/w). Moreover, these systems might be good candidates for exploring new and promising gellan food applications as delivery vehicle for bioactive agents or others where permanent networks or structured liquids are required. 2. Materials and methods 2.1. Materials The low acyl commercial form of gellan was used (Kelcogel, CPKelco, San Diego, USA). Its cation content, determined by atomic

absorption spectroscopy, was as follows (meq/g): Naþ ¼ 0.205, Kþ ¼ 1.015, Ca2þ ¼ 0.146 and Mg2þ ¼ 0.086. According to its manufacturer, the molecular weight of low acyl gellan was 2e3  105 g/mol. The commercial form of low acyl gellan without further purification was used to give a true representation of its behavior in industrial applications. Calcium chloride (J. T Baker, Mexico) was used as an external counter-ion source. Chemicals for gellan labeling included fluoresceinamine (SigmaeAldrich 201626, Mexico), dimethyl sulfoxide (SigmaeAldrich 471267, Mexico), cyclohexyl isocyanide (SigmaeAldrich 133302, Mexico), acetaldehyde (SigmaeAldrich 402788, Mexico), acetone (J.T. Baker, Mexico) and ethanol (J.T. Baker, Mexico). Distilled deionized water was used throughout the experiments. 2.2. Gel preparation Gellan solutions (0.02, 0.05 and 0.07 wt %) were prepared by dispersing the polysaccharide in water at 25
C under magnetic stirring and then heating the solution to 90
C. Different concentrations of CaCl2 were added before cooling to 25
C. The proportion of the external divalent ion and the polysaccharide in the gellan solutions was expressed by R, defined as the ratio of calcium ion concentration to low acyl gellan concentration (LAG), i.e., R ¼ [Ca2þ]/[LAG], both in meq/L. The denominator in R was calculated considering a molecular weight of 646 g/mol for the tetrasaccharide repeating unit of gellan. Ratios of 0, 1.29, 2.58, 3.88, 7.75, 12.9, 25.8 and 38.8 were prepared by adding the necessary amount of a 0.09 M CaCl2 stock solution. The systems studied are shown in Table 1. 2.3. Dynamic rheology Small amplitude oscillatory shear tests were conducted in an AR2000 rheometer (TA Instruments, USA) using the concentric cylinders fixture (bob diameter ¼ 28 mm, cup diameter ¼ 30 mm, immersion length ¼ 42 mm) and the double-wall coaxial cylinders fixture (cup diameter ¼ 44.7 mm, stator outer diameter ¼ 40 mm, bob inner diameter ¼ 40.76 mm, bob outer diameter ¼ 43.92 mm, immersion length ¼ 59.5 mm). The most diluted samples were tested in an ARES-RFS III rheometer (TA Instruments, USA) using the double-wall coaxial cylinders fixture (cup diameter ¼ 34 mm, stator outer diameter ¼ 27.94 mm, bob inner diameter ¼ 29.5 mm, bob outer diameter ¼ 32 mm, immersion length ¼ 31.98 mm). All measurements were performed using a solvent trap to prevent any evaporation. Temperature sweeps were run at 6.28 rad/s to obtain the variation of G0 and G00 with temperature; for such purposes, the hot samples (80e85
C) were poured into the preheated fixture (65e60
C) within 2 min after preparation and cooled (0.5
C/min)

Table 1 Gellan systems studied in this work. R

Gellan concentration, wt% (meq/L) 0.02 (0.3096)

0.05 (0.7740)

0.07 (1.084)

0.00 1.00 2.00 3.00 6.00 10.00 20.00 30.00

0.00 1.40 2.80 4.20 8.40 14.00 28.00 42.00

Ca2þ (meq/L)a 0.00 1.29 2.58 3.88 7.75 12.90 25.80 38.80 a

0.00 0.40 0.80 1.20 2.40 4.00 8.00 12.00

Ca2þ concentrations in meq/L are twice those in mmol/L.

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

to 25
C. Subsequently, time sweeps or gel cure tests were run at 6.28 rad/s and 25
C to determine the equilibrium values of the dynamic moduli. After the temperature and time sweeps, the frequency sweeps at 25
C from 102e102 rad/s were run at a constant stress or strain. Strain sweeps were performed to verify that all measurements were completed within the region of linear viscoelasticity. For temperature sweeps in the AR2000 rheometer, the torque was appropriately changed manually to obtain strains lower than 0.5% in the gel phase and to ensure the measurements fell within the region of linear viscoelasticity. In the ARES-RFS III rheometer, strain sweeps were run before each frequency sweep to determine the zone of linear viscoelasticity. The solegel transition was examined by measuring the variation of dynamic moduli with angular frequency at different temperatures. The range of temperatures was narrow and approximately the same as the crossover temperature of the moduli given by the temperature sweeps. The temperature was decreased stepwise and the samples equilibrated to each temperature setting for 10 min before the corresponding frequency sweep was conducted. All tests were performed in duplicate.

293

As an approximation to the solegel transition, the continuous evolution of G0 , G00 and tan d (¼ G00 /G0 ) throughout the cooling process of gellan solutions was examined at 6.28 rad/s. The temperature at which G0 ¼ G00 (i.e., tan d ¼ 1) was identified as the crossover temperature, TC. Fig. 1 shows the change with temperature of the dynamic moduli and tan d during the cooling process of two gellan media containing CaCl2. This change was characterized by an initial region where G00 > G0 (T > TC), followed by a zone where G0 exceeded G00 , close to TC, and finally a remarkable increase in G0 at high R values (see Table 1). A slight increase of G0 over G00 was observed for 0.07% and 0.05% gellan with R ¼ 1.29 and 2.58. The systems with 0.05% gellan (R ¼ 1.29 and 2.58) did not show a plateau in G0 and G00 for T < TC, which showed that aggregation of double helices in the ordered conformation of gellan was not energetically favored at temperatures close to 25
C and lower

a

2.4. Confocal laser scanning microscopy (CLSM) Gellan was covalently labeled with fluoresceinamine (FA) using a modified procedure by De Belder and Wik (1975). Gellan (2 g) was dispersed in water (300 mL) under magnetic stirring at room temperature. Dimethyl sulfoxide (75 mL) was added and mixed by gentle stirring. Then fluoresceinamine (0.028 g), dissolved in a small volume of dimethyl sulfoxide, cyclohexyl isocyanide (0.0281 mL) and acetaldehyde (0.0281 mL) were added. After 5 h at room temperature with gentle agitation, the reaction mixture was poured into two volumes of cold acetone to precipitate the FAegellan. The labeled polymer was washed several times with acetone until dehydrated. The FAegellan was dried overnight in a desiccator at room temperature, then solubilized in water. Subsequently, the FAegellan was dialyzed in a membrane of cellulose with a molecular weight cut-off of 12e14 kDa, against deionized water until no free dye was detected. Finally, the FAegellan was precipitated and dried again as previously mentioned. The gelling ability of labeled gellan in the presence of external calcium ions was verified by comparing it with the mechanical spectra of the untagged material. CLSM observations were done in a confocal microscope (Fluoview 1000, Olympus, Japan) in fluorescence mode with a 60 objective. The fresh hot samples were placed between a preheated slide and cover slip and sealed to avoid dehydration. Observations were performed at room temperature 24 h after sample preparation. A laser beam at a wavelength of 488 nm was used to excite the FA and the emission of fluorescence was recorded between 510 and 565 nm.

b

3. Results and discussion 3.1. Solegel transition temperature The solegel transition is one of the most relevant phenomena in food processing, but its experimental detection in physical gels is not necessarily easy. From the classical theory, the attributes of the gel point are an infinite steady-shear viscosity and a zero equilibrium modulus at zero frequency (Lopes da Silva & Rao, 2007). These criteria, which have been widely used to detect the gel point of some chemical gels, have not been useful in physical gels because continuous shearing affects gel formation. Herein, measurements of the variation of the dynamic moduli during gelation were used to identify the gel point of gellan in aqueous media.

Fig. 1. Temperature evolution of G0 (squares), G00 (circles) and tan d (triangles) during cooling (0.5
C/min) at 6.28 rad/s of: (a) 0.07% gellan with 0.7 mM CaCl2 (R ¼ 1.29) and (b) 0.07% gellan with 21 mM CaCl2 (R ¼ 38.8). The applied stress was a) 0.036 Pa and b) 0.18e0.34 Pa; g  0.5% in the gel state. The dotted lines are included as a guide to locate the crossover temperature (TC) where G0 ¼ G00 (i.e., tan d ¼ 1).

294

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

temperatures were required to overcome the conformational entropy (Nickerson, Paulson, & Hallet, 2008). It was worth noting that TC could not be detected by this method on media without CaCl2 and for 0.02% gellan systems with R < 7.75. The addition of CaCl2 shifted the crossover of dynamic moduli to higher temperatures (Fig. 2). In agreement with the gelation mechanism of gellan, gelation from hot solutions in the presence of high calcium concentrations occurred abruptly once TC was reached in spite of the low gellan concentrations (0.02e0.07%). Therefore, it can be assumed that as the temperature decreased, calcium ions screen carboxyl groups in the gellan backbone, which could help overcome the entropy barrier. Additionally, the polymerepolymer interactions were enhanced at higher temperatures as the concentration of calcium ions increased. It has been discussed that the temperature or time for the G0 and G00 crossover to occur could depend on angular frequency for synthetic and natural polymers (Lopes da Silva & Rao, 2007). Consequently, TC could not be taken as the solegel transition temperature (Tgel) because it was obtained at a single angular frequency. However, TC was expected to be close to Tgel. The Tgel values of gellan upon cooling was determined by two procedures based on the work of Winter and Chambon (1986): a) the power law dependence of G0 and G00 on the angular frequency, and b) the convergence of tan d at various angular frequencies, hereinafter called the critical phase angle procedure. Although both procedures arise from the same principle, the handling of rheological data could render different results. Some authors have reported that the procedure based on the power law dependence of G0 and G00 on the angular frequency is not a straightforward way for Tgel determination in physical gels due to the important changes of the power law exponent within a narrow temperature range (Núñez-Santiago & Tecante, 2007). Fig. 3(a) shows the variation with frequency of the dynamic moduli from 26.0 to 25.2
C for 0.05% gellan with 1 mM CaCl2 (R ¼ 2.58); data in this figure were vertically shifted by a factor of 10a to avoid overlapping. At 26.0
C, the gellan system showed a liquid-like viscoelastic behavior with

a

b

50

Tc (°C)

40

30

20

10

Fig. 3. Variation of G0 (black symbols) and G00 (white symbols) with angular frequency for: a) 0.05% gellan with 1 mM CaCl2 and b) 0.07% gellan with 0.7 mM CaCl2 at temperatures near the solegel transition. The data were vertically shifted by a factor of 10a with given a value to avoid overlapping. Values were obtained within the linear viscoelastic zone (s ¼ 0.036 Pa). Insets show the exponents n and the solegel transition temperature of gellan according to Winter and Chambon (1986).

Gellan (%) 0.02 0.05 0.07

0

4

8

12

16

20

24

CaCl2 (mM) Fig. 2. Variation of TC during cooling (0.5
C/min) of gellan solutions at 6.28 rad/s with CaCl2 concentration (R values are given in Table 1) for different gellan concentrations. Measurements were run in duplicate. Error bars show the standard deviation.

G00 > G0 and both moduli were dependent on frequency, G0 w u1.13 (at u > 1 rad/s) and G00 w u0.80. At 25.8
C, G00 was slightly higher than G0 and both moduli were parallel on the logarithmic scale (G0 and G00 w u0.64). Nonetheless, at 25.7
C, G0 became higher than G00 , and at lower temperatures these differences became more pronounced; also, a plateau in the G0 curve tends to appear, indicating a transition from liquid-like to solid-like behavior. These results showed that the solegel transition of 0.05% gellan with 1 mM CaCl2 took place around 25.8 to 25.7
C. A similar behavior for the solegel transition of 0.07% gellan

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300 Table 2 Solegel transition temperatures of gellan with added calcium chloride. Gellan/CaCl2 (wt%)/(mM)

R

TCa (
C)

Tgelb (
C)

0.07/0.7 0.05/1.0 0.05/3.0 0.07/14.0 0.02/6.0 0.05/15.0

1.29 2.58 7.75 25.8 38.8 38.8

19.0 25.0 34.0 42.5  0.50 35.5  0.50 42.5  0.50

20.8 25.7 31.4 46.5 35.2 45.6

0.12 0.04 0.04 0.01 0.20 0.01

a

G0 w G00 w un Tgeld (
C)

nc      

0.54 0.63 0.88 0.32 0.53 0.49

295

     

0.06 0.11 0.11 0.01 0.11 0.01

20.6 25.7 31.6 46.4 35.8 45.7

     

0.01 0.13 0.07 0.01 0.59 0.01

a

Transition temperature. Gelation temperature determined from the ‘critical phase angle method’. c Exponents determined from the power law dependence of G0 and G00 on angular frequency (Winter & Chambon, 1986). d Gelation temperature determined from the power law dependence of G0 and G00 on angular frequency (Winter & Chambon, 1986). b

with 0.7 mM CaCl2 (R ¼ 1.29) is shown in Fig. 3b; at 20.6
C both moduli were practically equal, indicating the existence of a solegel transition upon cooling from 20.8 to 20.4
C. The exponent n for the solegel transition of gellan in aqueous solutions was obtained from the power law dependence of G0 and G00 on angular frequency, G0 w G00 w un (Winter & Chambon, 1986). The entire frequency range was considered to carry out the linear regression fits with the exception of the notoriously non-linear regions. For instance, the values at frequencies higher than 1 rad/s were used at 26.0
C and 21
C for 0.05% and 0.07% gellan concentration systems, respectively. The n values for both gellan concentrations in Fig. 3a and b are shown in the insets. The exponent n increased with temperature and the dynamic moduli crossover sets the value of n and Tgel for each system. The n and Tgel values for the examined gellan systems are summarized in Table 2. Fig. 4a and b show the variation of tan d with temperature for different angular frequencies for 0.05% gellan with 1 mM CaCl2 and 0.07% gellan with 0.7 mM CaCl2, respectively. Data correspond to the mechanical spectra shown in Fig. 3. The point of convergence of all frequencies provided the gelling temperature (Tgel) because at this point the critical phase angle, d, became independent of frequency. The difference in Tgel values between the two procedures was less than 2%. This difference was not surprising considering that both procedures were based on the same principle. Furthermore, the potential discrepancy was based only on the ease in handling the rheological data. The results are summarized in Table 2. The Winter and Chambon’s method was proposed for chemical gels wherein the value of n is fixed at 0.5. However, the different values of this exponent in physical gels reflect the fact that n is not a universal parameter for gelation; it was found to be dependent on the nature of the system (i.e., stoichiometry, molecular weight, concentration, cross-linking mechanism, and thermal history) (Lopes da Silva & Gonçalves, 1994; Lu, Liu, Dai, & Tong, 2005). A range of values was obtained for the exponent n in physical gels (i.e., 0.71 for low methoxyl pectin/calcium systems (Axelos & Kolb, 1990), 0.5 for xanthan/carob (Cuvelier, PeigneyNoury, & Launay, 1990), 0.22 to 0.71 for gelatin (Michon, Cuvelier, & Launay, 1993), 0.41 to 0.65 for high methoxyl pectin/sucrose aqueous gels (Lopes da Silva & Gonçalves, 1994), 0.31 to 0.42 for tamarind seed xyloglucanesodium gellan mixtures (Nitta et al., 2003), 0.37 to 0.72 for calcium alginate (Lu et al., 2005), 0.62 for k-carrageenan (Núñez-Santiago & Tecante, 2007), 0.38 to 0.77 for deacetylated potassium-type gellan without added ions (Dai et al., 2008) and 0.37 to 0.63 for deacetylated potassium-type gellan with calcium ions (Dai et al., 2010). The n values obtained in this work ranged from 0.32 to 0.88, which were in agreement with those obtained by Dai et al. (2008, 2010) for gellan at higher concentrations (1e2.5 wt%). Dai and coworkers reported n values in the range of 0.37e0.77 and n decreased with increasing gellan (Dai et al.,

b

Fig. 4. Variation of tan d with temperature for different angular frequencies: a) 0.05% gellan with 1 mM CaCl2 and b) 0.07% gellan with 0.7 mM CaCl2 from which the gel points (Tgel) were determined according to the critical phase angle procedure. Values correspond to the mechanical spectra shown in Fig. 3.

2008) and CaCl2 concentration (Dai et al., 2010). In the present work, the lowest n values were obtained with the highest calcium concentrations, and the Tgel values of those systems were the highest. The concept of a universal critical exponent fails for the solegel transition of gellan because its gelation involved a crosslinking mechanism (Dai et al., 2008, 2010). Additionally, the transient nature of physical gelation of gellan at low concentrations made it difficult to determine the gel point by rheological methods. It was possible that the differences in the thermal history applied to the samples (i.e., rate of cooling and time for thermal stabilization of the samples) had a strong effect on the determination of the gel

296

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

point because of the rapid gelation of gellan in the presence of divalent cations, which cannot be stopped once it gets started. Similar findings were reported for the solegel transition of kcarrageenan (Núñez-Santiago & Tecante, 2007). Table 2 shows that Tgel increased with increasing R values. Therefore, it was likely that Ca2þ triggered the solegel transition of gellan; therefore, when Ca2þ was in excess, the ions reinforced the aggregation of double helices and elastic gels were formed. Conversely, when the binding effect was not strong enough (e.g., for low values of R), the gel point was observed at lower temperatures. Fig. 5 shows the gelation diagram for gellan obtained in this investigation. The diagram shows the relationship between the total ionic concentration (CT) and the inverse absolute temperature (T). CT was calculated as g CP þ CS, being CP the gellan concentration (meq/L), CS the calcium concentration added to gellan solutions (meq/L) and g the mean activity coefficient (i.e., 0.53 for Ca2þgellan; calculated from Milas & Rinaudo, 1996 and Milas, Shi, & Rinaudo, 1990). The total concentration, CT (meq/L), for the solegel transition of gellan at 25
C, was calculated from the data in Fig. 5. There were obtained 2.94 and 2.85 meq/L using the critical phase angle procedure and the method based on the power law dependence on angular frequency of modulli, respectively. Fig. 5 also includes some of the gellan systems studied in this work, primarily those near the solegel border line at 25
C. 3.2. Viscoelastic behavior of gellan at 25
C Fig. 6 shows the mechanical spectra of some of the gellan systems examined in this work. All of the mechanical spectra were collected after a gel cure test at 25
C and a constant angular

GEL

-1

CT= Cp+Cs (meq L )

102

101

100

10-1 3.0

SOL

3.1

3.2

3.3

3.4

3.5

-1

1/T (mK ) Fig. 5. Solegel transition diagram for low acyl gellan in the presence of external calcium counter-ions. Data obtained on cooling from the critical phase angle procedure (white inverted triangles) and from the method based on the power law dependence on angular frequency of G0 and G00 (white circles). Some of the gellan systems studied at 25
C are also included: 0.02% gellan without calcium (triangle) (CT ¼ 0.16 meq/L), 0.07% gellan with 0.7 mM CaCl2 (inverted triangle) (CT ¼ 1.97 meq/L), 0.02% gellan with 1.2 mM CaCl2 (square) (CT ¼ 2.56 meq/L), 0.07% gellan with 1.4 mM CaCl2 (diamond) (CT ¼ 3.37 meq/L) and 0.02% gellan with 2 mM CaCl2 (circle) (CT ¼ 4.16 meq/L). The solid and long dash lines correspond to the linear regression of data from the critical phase angle procedure and the procedure based on the power law dependence on angular frequency of G0 and G00 , respectively.

frequency of 6.28 rad/s to obtain the viscoelastic behavior once the evolution of dynamic moduli approached equilibrium (DG0  1%/ min was used as an arbitrary criterion for equilibrium). Fig. 6a shows the obtained results for two gellan systems with CT values lower than 1.9 meq/L, that involves gellan systems in the sol state in agreement with Fig. 5. At low angular frequency (u < 10 rad/s), G00 was higher than G0 and both moduli varied sharply with frequency, G0 w u1.72 and G00 w u0.81 for 0.02% gellan without added ions whereas G0 w u1.42 and G00 w u0.80 for 0.02% gellan with 0.4 mM CaCl2. As angular frequency increased, G0 surpassed G00 and a plateau appeared, being a distinctive feature of a macromolecular solution with topological entanglements. This liquid-like behavior was also observed in the other gellan solutions without added ions and 0.05% gellan with 0.5 mM CaCl2. Fig. 6a. also shows the variation of dynamic viscosity with frequency for the two gellan systems mentioned above. At low frequencies, h0 approached to values of 15 and 12 mPa s for 0.02% gellan without calcium and 0.02% gellan with 0.4 mM CaCl2, respectively. In contrast, at higher frequencies (i.e., u > 10 rad/s) where the G00 plateau did appear, h0 decreased monotonically as frequency increased and approached to a limiting value which was nearly the solvent viscosity. That behavior is the theoretical one described for viscoelastic liquids and the frequency region in which the transition of h0 occurs has been related to configurational relaxation times (Ferry, 1980). Jampen, Britt, and Tung (2000) reported a critical concentration value, C*, of 0.064% for low acyl gellan solutions at 25
C. Nickerson, Paulson, and Speers (2003) demonstrated, using steady-shear rheometry, that C* of low acyl gellan decreased when the external cation concentrations increased and that C* depended on temperature. For instance, these authors reported that C* ¼ 0.09 wt% for gellan at 25
C in the presence of 0.5 mM Ca2þ. According to the mentioned published findings, the gellan systems discussed above were in the dilute regime. Therefore, the amount of macromolecules is insufficient to form coil overlaps (Cp < C*), and the addition of CaCl2  0.5 mM could not be enough to produce the aggregation of double helices and a three-dimensional network. Thus, the behavior exhibited in Fig. 6a might be the result of the conformational transition of gellan during the cooling process where electrostatic repulsions overcome along the stiff double helices due to the high dilution of the polyelectrolyte in a medium with low ionic force. Several studies have reported the increase of the hydrodynamic volume of polyelectrolites, without counter-ions, as solution temperature decreases (Ferry, 1980; Launay, Doublier, & Cuvelier, 1986; Milas et al., 1990; Nickerson et al., 2008). This circumstance enhances the surface potential of the polyelectrolyte causing an increase in water associations (Nickerson et al., 2008). Other studies have found that the charge increasing of a polyelectrolyte is associated to a rapidly increasing in its relaxation times (Fujimori, Nakajima, Wada, & Doi, 1975). However, further studies are necessary to clarify this behavior. Conversely, solid-like properties were displayed by 0.07% gellan with 0.7 mM CaCl2 (R ¼ 1.29; CT ¼ 1.97 meq/L) and 0.02% gellan with 1.2 mM CaCl2 (R ¼ 7.75; CT ¼ 2.56 meq/L), as shown in Fig. 6b. The physical aspect of these systems was comparable to homogeneous and pourable liquids, with a linear relationship between stress and strain up to 40% strain. The presence of aggregates was not apparent in the bulk system. The mechanical spectra showed G0 > G00 over the frequency range, G0 exhibited a weak dependence on frequency and G00 depended more on it. As a result, tan d increased with the frequency, ranging from 0.05 to 0.82 for 0.07% gellan and from 0.18 to 0.57 for 0.02% gellan. These features indicated a solid-like behavior that resulted from the existence of a three-dimensional structure formed by the weak intermolecular chain associations. At these gellan and calcium contents, polysaccharide chains were not able to form a permanent and strong

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

a

101

101 a=2

100

a=0

10-1

10-2

' (Pa s)

10-1

10-2

a

a

10 G', 10 G" (Pa)

100

10-3 10-3

10-4

10-5 10-1

b 10

100

10-4 102

101

(rad/s) 1

G'; G" (Pa)

100

10-1

10-2

10-3 10-2

10-1

100

101

102

101

102

(rad/s)

c

104

103

G', G" (Pa)

102

101

100

10-1

10-2 10-2

10-1

100

(rad/s) Fig. 6. Variation of G0 (black symbols), G00 (white symbols) with angular frequency for: a) 0.02% gellan without calcium (R ¼ 0) (inverted triangles) and 0.02% gellan with

297

network. This could be attributed to the proximity of these gellan systems to the solegel transition as shown in Fig. 5. The involved CT values were lower than 2.9 meq/L; consequently, the solegel transition did not take place immediately because it happened at higher ionic concentrations. Nevertheless, the solid-like behavior of these systems could be the result of the formation of double helices stabilized primarily through intramolecular interactions due to the low polymer concentrations. It is likely that during gel curing, this ordered conformation was further stabilized and reinforced by hydrogen bonding and ionic interactions, resulting in macromolecular assemblies that could give rise to a solid-like rheological behavior. Sakurai, Tanaka, and Nakamura (1995) suggested that the formation over time of a pre-gel network structure could be mediated by hydrogen bonding in dilute gellan solutions. Others (Garnier, Axelos, & Thibault, 1993; Nickerson et al., 2008) have also reported the gelation of anionic biopolymers at concentrations lower than C*, presumably because of strong interactions between biopolymer and external counter-ions. The further increase in the total ionic concentration resulted in the formation of gellan gels. Systems with 0.07 and 0.05% gellan with R ratios >2.58 and 0.02% gellan with R ratios >7.75, all of them involving CT values >2.9 meq/L, showed typical viscoelastic behaviors of strong gels (i.e., G0 >> G00 ) with practically no dependence on angular frequency. The corresponding tan d values were lower than 0.1, denoting a permanent network. Fig. 6c shows some of the gellan gels obtained in this work. The variation of G0 with time after quenching from 60 to 25
C for some gellan systems is shown in Fig. 7. The transient nature of viscoelastic properties was evident; the lower the calcium concentrations the higher the transient behavior of G0 . It is worth noting that all systems at gelling conditions (i.e., CT  2.9 meq/L) reached a plateau value of G0 within 2 h of gel curing. However, the pre-gel systems showed a transient behavior in G0 . For instance, although the solegel transition of 0.07% gellan with 0.7 mM CaCl2 was close to 21
C (Table 2), the system, even being in a quasiequilibrium state, exhibited a solid-like behavior only after 3 h of gel curing at 25
C. Other studies have concluded that the kinetics of the helixehelix aggregation of anionic polysaccharides become progressively slower as the polymer concentration decreases (Mohammed, Hember, Richardson, & Morris, 1998). Our results showed that the gel formation is dominated by the kinetics of the cross-linking of double helices of gellan which became progressively slower as CT decreased. Fig. 8 shows the change of G0 with the R ratios (Fig. 8a) and CT values (Fig. 8b) at 6.28 rad/s and 25
C. The plotted data were obtained from the frequency sweeps recorded at the end of the cure test at 25
C. The increase of the R ratio up to 7.75 enhanced the elastic character of gellan systems, but such character remained reasonably constant with further increases of R. The storage modulus reached a maximum R value that depended on the gellan concentration. The maximum value for G0 was observed at R values of 7.95, 12.9 and 25.8 for 0.02, 0.05 and 0.07% gellan, respectively. This behavior did not follow the trend reported by Tang, Tung, and Zeng (1996) who observed a critical R of 0.5; further increasing Ca2þ did not modify the failure strains of the gels, which remained the same regardless of polymer concentration. In contrast, the dependence of G0 on total ionic concentration, CT (Fig, 8b), showed

0.4 mM CaCl2 (R ¼ 2.58) (squares). The G0 and G00 data were vertically shifted by a factor of 10a with given a value to avoid overlapping. Dotted symbols represent the variation of h0 with angular frequency; b) 0.07% gellan with 0.7 mM CaCl2 (R ¼ 1.29) (triangles) and 0.02% gellan with 1.2 mM CaCl2 (R ¼ 7.75) (stars); c) 0.02% gellan with 2 mM CaCl2 (R ¼ 12.92) (diamonds), 0.05% gellan with 1.5 mM CaCl2 (R ¼ 3.88) (circles) and 0.07% gellan with 1.4 mM CaCl2 (R ¼ 2.58) (hexagons). Data were obtained at 25
C within the linear viscoelastic zone. Error bars represent the standard deviation.

298

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

Fig. 7. Time evolution of the storage modulus for 0.05% gellan with 1 mM CaCl2 (circles), 1.5 mM CaCl2 (squares), 15 mM CaCl2 (triangles), 3 mM CaCl2 (inverted triangles) and 0.07% gellan with 0.7 mM CaCl2 (diamonds). Data were obtained after quenching samples from 60 to 25
C (0.5
C/min). G0 was obtained at 25
C and 6.28 rad/s within the linear viscoelastic zone (the applied stress or strain is indicated in each system description).

a maximum in G0 at 9.3  1.2 meq/L for the three gellan concentrations, followed by a progressive diminution as CT increased, primarily because of further addition of calcium ions and the low contribution of gellan to the ionic strength of the systems. Similar observations have been reported for gellan concentrations in the range of 0.1e2% (Sworn et al., 1995). It has been hypothesized that gellan has an optimum requirement for gelling ions and, hence, the gel strength increases up to a maximum; thus, the excess ions force the polymer to self-associate in a progressively less well-formed three-dimensional network (Sworn et al., 1995) or to form nuclei at high cation concentrations, which weaken the gel network (Kasapis et al., 1999). Others (Mao, Tang, & Swanson, 2001) have suggested that the presence of large pores in the microstructure of gellan gels, observed at high concentrations of calcium (Ca2þ > 8 mM), may be responsible for the decrease in the gel strength. 3.3. Microstructure of gellan systems Confocal laser scanning microscopy is an optical tool that is useful for the visualization of the 2D and 3D structure of biopolymers on the micrometer scale (van de Velde, Weinbreck, Edelman, van der Linden, & Tromp, 2003). This microscopy technique allowed the visualization of the extent of gellan associations in the systems. Fig. 9 shows the micrographs of some gellan systems. The clear zones correspond to the fluorescence of labeled gellan and the dark ones to the solvent or regions devoid of gellan. Gellan without added calcium formed homogeneous systems (Fig. 9a and b). The presence of a thin and continuous structure was detected in the 0.07% gellan (Fig. 9b). This structure appeared slightly reinforced in the presence of 0.7 mM CaCl2 as the weak interconnections were more noticeable but still tenuous (Fig. 9d). The abundant and thin structure correlated well with the rheological characteristics of a ‘pre-gel’ with a wide linear viscoelastic zone. It was likely that the

Fig. 8. Effect of the R ratio (a) and the total ionic concentration CT (b) on G0 for 0.02 (circles), 0.05 (squares) and 0.07% (triangles) gellan. Data were obtained from mechanical spectra at 25
C and 6.28 rad/s after a cure test at 25
C. Error bars represent the standard deviation.

ions added, 0.7 mM CaCl2, which are not enough to screen the carboxyl groups of the gellan backbone (R ¼ 1.29), promoted the formation of a weakly associated structure, but elastic. It can be described as structured liquid from their physical appearance. At high gellan concentrations, systems formed more compact structures with progressively smaller voids throughout the volume of the gel. The compactness of the network structure was controlled by gellan, while added calcium reinforced the strands of that network. This effect was noticeable when the microstructures with the same gellan concentrations were compared. For example, Fig. 9e shows a network with thicker strands compared to those

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

299

Fig. 9. Confocal laser scanning micrographs of gellan systems at 25
C with different concentrations of calcium ions. Images were obtained at a depth z z 15 mm on the xy plane with a 60 lens. Micrograph in g) is a 100 magnification of the sample in e). Clear zones correspond to gellan networks. Image size from a) to f) is 212  212 mm.

shown in Fig. 9c; the increase in concentration of the external counter-ions from 1.2 to 6 mM in 0.02% gellan resulted in more robust network structures, as can be seen in the amplification of the image shown in Fig. 9g. The rheology of gellan systems was consistent with the structures observed. The elastic character was enhanced as calcium ion concentration increased. 0.02% gellan was high enough to produce a continuous and well distributed network; further increments in gellan concentration resulted in a more evident solid-like behavior. Moreover, when the concentration of calcium ions was increased up to a critical value, double helices might have been reinforced by ionic bonding and the solid-like character was enhanced as a consequence. This correlated well with the presence of more robust networks observed by CLSM. Conversely, attenuation of the solid-like behavior at high calcium concentrations might have been the result of an oversaturation of the anionic sites on the gellan backbone because of its low charge density compared to other anionic polysaccharides as carrageenans and pectins. 4. Conclusions At the low gellan concentrations used in this study, the results reveal that the R ratio did not have strong effect on the solid character of gellan systems. On the other hand, the total ionic concentration was found to be the triggering and critical factor for the gelation and elasticity of gellan systems. Knowledge of the solegel transition of gellan as a function of total ionic concentration will allow gellan to be better used as a food ingredient. Unlike most of food hydrocolloids, gellan was functional at very low concentrations. It is possible to create a wide spectrum of rheological behaviors from structured liquids to strong gels through the accurate selection of gellan and cation concentrations, even at very low levels of gellan. This work has shown that more interconnected structures are formed as gellan concentration increases; while the progressive increase in calcium ion concentration does reinforce the gellan network. Acknowledgments Financial support from “Cátedra Coca-Cola para Jóvenes Investigadores, CONACyT-Fundación Coca-Cola, México-2005” and

scholarship to S. J. Pérez-Campos from PIFI P/CA-3 2006-14-04 “Consolidación del Cuerpo Académico de Biotecnología Agroalimentaria” are gratefully acknowledged. The authors express their gratitude to Gabriel Orozco and Araceli Patrón for their technical support on confocal microscopy. References Axelos, M. A. V., & Kolb, M. (1990). Crosslinked biopolymers: experimental evidence for scalar percolation theory. Physical Review Letters, 64, 1457e1460. Coviello, T., Matricardi, P., Marianecci, C., & Alhaique, F. (2007). Polysaccharide hydrogels for modified release formulations. Journal of Controlled Release, 119, 5e24. Crescenzi, V., & Dentini, M. (1987). The influence of side-chains on the dilutesolution properties of three structurally related, bacterial anionic polysaccharides. Carbohydrate Research, 160, 283e302. Cuvelier, G., Peigney-Noury, C., & Launay, B. (1990). Viscoelastic properties of physical gels: critical behaviour at the gel point. InPhillips, G. O., Wedlock, D. J., & Williams, P. A. (Eds.). (1990). Gums and stabilisers for the food industry, Vol. 5 (pp. 549e552). Oxford, England: IRL Press. Dai, L., Liu, X., Liu, Y., & Tong, Z. (2008). Concentration dependence of critical exponents for gelation in gellan gum aqueous solutions upon cooling. European Polymer Journal, 44, 4012e4019. Dai, L., Liu, X., & Tong, Z. (2010). Critical behavior at solegel transition in gellan gum aqueous solutions with KCl and CaCl2 of different concentrations. Carbohydrate Polymers, 81, 207e221. De Belder, A. N., & Wik, K. O. (1975). Preparation and properties of fluoresceinlabelled hyaluronate. Carbohydrate Research, 44, 251e257. Doublier, J. L., & Cuvelier, G. (1996). Gums and hydrocolloids: functional aspects. In A. C. Eliasson (Ed.), Carbohydrates in food (pp. 312e313). New York: Marcel Dekker. Ferry, J. D. (1980). Viscoelastic properties of polymers (3rd ed.). New York: John Wiley and Sons. Fujimori, S., Nakajima, H., Wada, Y., & Doi, M. (1975). Computer simulation of brownian motion of a polyelectrolyte molecule (charged bead-spring model). Journal of Polymer Science, 13, 2135e2153. Garnier, C., Axelos, M. A. V., & Thibault, J. F. (1993). Phase-diagrams of pectincalcium systems e influence of pH, ionic strength and temperature on the gelation of pectins with different degrees of methylation. Carbohydrate Research, 240, 219e232. Goycoolea, F. M., Milas, M., & Rinaudo, M. (2001). Associative phenomena in galactomannan-deacetylated xanthan systems. International Journal of Biological Macromolecules, 29, 181e192. Jampen, S., Britt, I. J., & Tung, M. A. (2000). Gellan polymer solution properties: dilute and concentrated regimes. Food Research International, 33, 579e586. Jansson, P., Lindberg, B., & Sandford, P. A. (1983). Structural studies of gellan gum, an extracellular polysaccharide elaborated by Pseudomonas elodea. Carbohydrate Research, 124, 135e139. Kasapis, S., Giannouli, P., Hember, M. W. N., Evageliou, V., Poulard, C., TortBourgeois, B., et al. (1999). Structural aspects and phase behavior in deacylated and high acyl gellan systems. Carbohydrate Polymers, 38, 145e154.

300

S.J. Pérez-Campos et al. / Food Hydrocolloids 28 (2012) 291e300

Kiani, H., Mousavi, M. E., Razavi, H., & Morris, E. R. (2010). Effect of gellan, alone and in combination with high-methoxy pectin, on the structure and stability of doogh, a yogurt-based Iranian drink. Food Hydrocolloids, 24, 744e754. Launay, B., Doublier, J. L., & Cuvelier, G. (1986). Flow properties of aqueous solutions and dispersions of polysaccharides. In J. R. Mitchell, & D. A. Ledward (Eds.), Functional properties of food macromolecules (pp. 1e77). New York: Elsevier Applied Science. León, P. G., Lamanna, M. E., Gerschenson, L. N., & Rojas, A. M. (2008). Influence of composition of edible films based on gellan polymers on L-(þ)-ascorbic acid stability. Food Research International, 41, 667e675. Lopes da Silva, J. A. L., & Gonçalves, M. P. (1994). Rheological study into the ageing process of high methoxyl pectin/sucrose aqueous gels. Carbohydrate Polymers, 24, 235e245. Lopes da Silva, J. A., & Rao, M. A. (2007). Rheology behavior of food gels. In M. A. Rao (Ed.), Rheology of fluid and semisolid foods: Principles and applications. Food engineering series, (pp. 355). New York: Springer. Lu, L., Liu, X., Dai, L., & Tong, Z. (2005). Difference in concentration dependence of relaxation critical exponent n for alginate solutions at sol-gel transition induced by calcium cations. Biomacromolecules, 6, 2150e2156. Mao, R., Tang, J., & Swanson, B. G. (2001). Water holding capacity and microstructure of gellan gels. Carbohydrate Polymers, 46, 365e371. Michon, C., Cuvelier, G., & Launay, B. (1993). Concentration dependence of the critical viscoelastic properties of gelatin at the gel point. Rheologica Acta, 32, 94e103. Milas, M., & Rinaudo, M. (1996). The gellan sol-gel transition. Carbohydrate Polymers, 30, 177e184. Milas, M., Shi, X., & Rinaudo, M. (1990). On the physicochemical properties of gellan gum. Biopolymers, 30, 451e464. Miyoshi, E., & Nishinari, K. (1999). Rheological and thermal properties near the solgel transition of gellan gum aqueous solutions. Progress in Colloid and Polymer Science, 114, 68e82. Mohammed, Z. H., Hember, M. W. N., Richardson, R. K., & Morris, E. R. (1998). Kinetic and equilibrium processes in the formation and melting of agarose gels. Carbohydrate Polymers, 36, 15e26. Núñez-Santiago, M. C., & Tecante, A. (2007). Rheological and calorimetric study of the solegel transition of k-carrageenan. Carbohydrate Polymers, 69, 763e773. Nickerson, M. T., & Paulson, A. T. (2004). Rheological properties of gellan, k-carrageenan and alginate polysaccharides: effect of potassium and calcium ions on macrostructure assemblages. Carbohydrate Polymers, 58, 15e24.

Nickerson, M. T., Paulson, A. T., & Hallet, F. R. (2008). Pre-gel solution properties of gellan polysaccharides: effect of potassium and calcium ions on chain associations. Food Research International, 41, 462e471. Nickerson, M. T., Paulson, A. T., & Speers, R. A. (2003). Rheological properties of gellan solutions: effect of calcium ions and temperature on pre-gel formation. Food Hydrocolloids, 17, 577e583. Nitta, Y., Kim, B. S., & Nishinari, K. (2003). Synergistic gel formation of xyloglucan/ gellan mixtures as studied by rheology, DSC and circular dichroism. Biomacromolecules, 4, 1654e1660. Nitta, Y., Takahashi, R., & Nishinari, K. (2010). Viscoelasticity and phase separation of aqueous Na-type gellan solution. Biomacromolecules, 11, 187e191. Noda, S., Funami, T., Nakauma, M., Asai, I., Takahashi, R., Al-Assaf, S., et al. (2008). Molecular structures of gellan gum imaged with atomic force microscopy in relation to the rheological behaviour in aqueous systems. 1. Gellan gum with various acyl contents in the presence and absence of potassium. Food Hydrocolloids, 22, 1148e1159. Rodríguez-Hernández, A. I., Durand, S., Garnier, C., Tecante, A., & Doublier, J. L. (2003). Rheology-structure properties of gellan systems: evidence of network formation at low gellan concentrations. Food Hydrocolloids, 17, 621e628. Rojas-Graü, M. A., Tapia, M. S., Rodríguez, F. J., Carmona, A. J., & Martin-Belloso, O. (2007). Alginate and gellan-based edible coatings as carriers of antibrowning agents applied on fresh-cut Fuji apples. Food Hydrocolloids, 21, 118e127. Sakurai, M., Tanaka, Y., & Nakamura, K. (1995). Viscosities, densities and sound velocities of dilute aqueous gellan solutions. Food Hydrocolloids, 9, 189e194. Sosa-Herrera, M. G., Berli, C. L. A., & Martínez-Padilla, L. P. (2008). Physicochemical and rheological properties of oil-in-water emulsions prepared with sodium caseinate/gellan gum mixtures. Food Hydrocolloids, 22, 934e942. Sunil, A. A., Sheetal, S. J., & Tejraj, M. A. (2006). Controlled release of cephalexin through gellan gum beads: effect of formulation parameters on entrapment efficiency, size & drug release. European Journal of Pharmaceutics and Biopharmaceutics, 63, 249e261. Sworn, G., Sanderson, G. R., & Gibson, W. (1995). Gellan gum fluid gels. Food Hydrocolloids, 9, 265e271. Tang, J., Tung, M. A., & Zeng, Y. (1996). Compression strength and deformation of gellan gels formed with mono and divalent cations. Carbohydrate Polymers, 29, 11e16. van de Velde, F., Weinbreck, F., Edelman, M. W., van der Linden, E., & Tromp, H. (2003). Visualisation of biopolymers mixtures using confocal scanning laser microscopy (CLSM) and covalent labeling techniques. Colloids and Surfaces B: Biointerfaces, 31, 159e168. Winter, H. H., & Chambon, F. (1986). Analysis of linear viscoelasticity of a crosslinking polymer at the gel point. Journal of Rheology, 30, 367e382.