Rheological studies of the fucose-rich exopolysaccharide FucoPol

International Journal of Biological Macromolecules 79 (2015) 611–617

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Rheological studies of the fucose-rich exopolysaccharide FucoPol Cristiana A.V. Torres a , Ana R.V. Ferreira b , Filomena Freitas a , Maria A.M. Reis a , Isabel Coelhoso b , Isabel Sousa c , Vítor D. Alves c,∗ a b c

UCIBIO-REQUIMTE, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal LAQV-REQUIMTE, Chemistry Department, FCT/Universidade Nova de Lisboa, 2829-516 Caparica, Portugal LEAF – Linking Environment, Agriculture and Food, Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017, Portugal

a r t i c l e

i n f o

Article history: Received 17 April 2015 Received in revised form 14 May 2015 Accepted 18 May 2015 Available online 23 May 2015 Keywords: Fucose-rich polysaccharide Solution properties, Rheology

a b s t r a c t In this work, the solution properties of the bacterial fucose-rich polysaccharide, FucoPol, were studied. The effect of pH (3.5–10.0) and ionic strength (0.02–1.0 M NaCl) on the intrinsic viscosity and steady shear flow were evaluated using a central composite rotatable design of experiments and surface response methodology. FucoPol’s intrinsic and apparent viscosities presented a quite low variation under a wide range of pH (3.5–8.0) and ionic strength (0.05–0.50 M NaCl) values. FucoPol produced viscous solutions with shear-thinning behavior at different polymer concentrations (0.2–1.2 wt.%). Flow curves were successfully described by the Cross model. The viscosity of the first Newtonian plateau varied from 0.01 to 2.47 Pa s for polymer concentrations from 0.2 to 1.2 wt.%, and the dependence of the estimated relaxation time with polymer concentration suggests a large degree of interaction between FucoPol molecules. Given the results obtained, FucoPol is proposed as thickening agent for applications in which stability of the apparent viscosity under pH and ionic strength variations is required. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Water soluble polysaccharides are industrially important materials due to their rheological properties in aqueous systems. Understanding polysaccharides’ properties in aqueous solutions is critical to forecast their potential industrial applications in specific areas, such as food products, cosmetics, pharmaceuticals, oil drilling fluids and paints, being important to the manufacture, distribution, storage and consumption of many products. These properties can be affected by several parameters, such as pH, salt concentration, temperature, polymer average molecular weight and shear rate [1]. For some applications, it is important to have viscous solutions at low concentrations, and stable under a wide range of temperatures, pH and ionic strengths, namely in emulsions in the food industry [2,3] and in oil drilling fluids [4,5]. Bacterial polysaccharides frequently present distinctive solution properties that are not demonstrated by traditional polymers derived from other natural sources (plants, algae and animals) [6,7]. Examples of commercial bacterial polysaccharides include xanthan gum, gellan gum, fucogel, hyaluronic acid and welan gum. Others, with either none or low commercial expression have been reported,

∗ Corresponding author. Tel.: +351 21 3653546; fax: +351 21 3653200. E-mail address: [email protected] (V.D. Alves). http://dx.doi.org/10.1016/j.ijbiomac.2015.05.029 0141-8130/© 2015 Elsevier B.V. All rights reserved.

for example colanic acid [8], clavan [9] and rhamsan [10]. Beyond the solution properties, they may also present interesting biological activities, namely those composed of l-fucose, l-rhamnose or uronic acids residues. Such functional properties are the driving force for the search of new bacterial polysaccharides with potential to be used on specific applications [6,11]. FucoPol is a fucose-rich polysaccharide synthesized by the bacterium Enterobacter A47. It presents a high molecular weight (5 × 106 ) and a low polydispersity index (1.3), being composed of fucose (32–36 mol%), galactose (25–26 mol%), glucose (28–37 mol%), glucuronic acid (9–10 mol%) and acyl groups, namely succinyl (2–3 wt.%), pyruvyl (13–14 wt.%) and acetyl (3–5 wt.%). The purified polymer samples present a protein content below 5 wt.% and traces of inorganic salts [12–14]. The presence of glucuronic acid, as well as pyruvyl and succinyl, confers FucoPol a polyelectrolyte character. Previous works have demonstrated that FucoPol solutions, with a concentration around 1 wt.%, have a shear-thinning behavior and mechanical spectra revealing viscous solutions with entangled polymer molecules, in the range of temperatures within 15–65 ◦ C [12,13]. Moreover, the apparent viscosity and the viscoelastic properties, measured at 25 ◦ C, were maintained after consecutive heating and cooling cycles, indicating a good thermal stability under temperature fluctuations [15]. However, additional studies are necessary in order to improve the knowledge of FucoPol aqueous solutions properties, namely

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regarding the effect of different pH, ionic strength and polymer concentration. In this work, the solution properties of the fucose-rich exopolysaccharide FucoPol were evaluated. The assessment of the polymer’s behavior in aqueous solutions involved the study of (i) the impact of pH and ionic strength on the intrinsic viscosity; (ii) the steady shear behavior, with focus on the effect of polymer concentration, pH and ionic strength on the apparent viscosity; and (iii) the concentration regimes. These studies are essential to envisage FucoPol’s potential applications, since polysaccharides are mainly used in aqueous formulations, which are the base of many systems, such as drilling fluids, food products, and some cosmetics and pharmaceuticals.

Table 1 Central composite rotatable design (CCRD) with two independents variables X1 (Ionic Strength, IS) and X2 (pH), and the observed responses studied Y1 (intrinsic viscosity []) and Y2 (zero-shear rate viscosity 0 ) for 1 wt.% FucoPol solution. Run number

pH X2

[] (dL/g) Y1

0 (Pa s) Y2

Factorial design

1 2 3 4

0.15 0.65 0.15 0.65

4.50 4.50 9.50 9.50

8.17 7.37 7.38 7.21

0.94 0.88 0.41 0.43

Central point

5 6 7

0.40 0.40 0.40

7.00 7.00 7.00

8.30 8.28 8.23

1.20 1.20 1.20

8 9 10 11

0.05 0.75 0.40 0.40

7.00 7.00 3.47 10.54

8.54 5.65 8.07 5.51

0.64 0.61 1.07 0.37

Axial points

2. Materials and methods

IS (M NaCl) X1

2.1. FucoPol production and extraction FucoPol was obtained by cultivation of the bacterium Enterobacter A47 (DSM 23139) on mineral medium supplemented with glycerol byproduct from the biodiesel industry, as previously reported [12,14]. The culture broth recovered from the bioreactor at the end of the cultivation was diluted with deionized water (1:2, v/v) for viscosity reduction and centrifuged (13 000 × g, 1 h) in order to remove cells. The cell-free supernatant was subjected to protein thermal denaturation (70 ◦ C, 1 h), to avoid enzymatic polymer degradation during the subsequent purification steps, followed by their separation by centrifugation (13 000 × g, 1 h). FucoPol-rich supernatant was dialysed with a 10 000 MWCO membrane (Snake SkinTM Pleated Dialysis Tubing, Thermo Scientific) against deionized water, for 48 h at 4 ◦ C. Finally, the purified supernatant was freeze-dried. 2.2. FucoPol solutions FucoPol, purified as described in Section 2.1, was added to NaCl solutions or deionized water, depending on the ionic strength value used, and stirred overnight at room temperature. FucoPol concentration ranged from 0.02 to 0.06 wt.% for intrinsic viscosity measurements, and from 0.2 to 1.2 wt.% for apparent viscosity measurements. The solutions’ ionic strength ranged from roughly 0.05 to 0.075 M NaCl. The pH of the solutions was adjusted to the desired values by addition of small drops of HCl (15 wt.%) and/or NaOH (10 wt.%) aqueous solutions.

2.4. Apparent viscosity measurements The apparent viscosity of FucoPol’s aqueous solutions was measured by loading directly the solutions on a cone and plate geometry (diameter 35 mm, angle 2◦ ) of a controlled stress rheometer (Rheostress RS 75, Haake, Germany). The shearing geometry was covered with paraffin oil in order to minimize sample dehydration. The samples were equilibrated at 25.0 ± 0.1 ◦ C, for 10 min, after which the flow curves were obtained using a steady-state flow ramp (torque was imposed using a logarithmic ramp) in the shear rate range from 1 to 700 s−1 . 2.5. Experimental design Response surface methodology (RSM) was applied to evaluate the combined effect of the ionic strength (NaCl concentration) and the pH, both on the observed intrinsic viscosity [] (Y1 ), and on the zero shear rate viscosity (first Newtonian plateau) (Y2 ) of solutions with a FucoPol concentration of 1 wt.%. A central composite rotatable design (CCRD), with two independent variables, where X1 is the ionic strength, IS (M, NaCl concentration), and X2 is the pH (Table 1), was used. The conditions of the central point of the design (NaCl 0.40 M, at pH 7.0) were tested three times, to allow estimating the experimental error. All experiments were carried out on a randomized order to prevent the effect of unexplained variability due to exogenous factors. The system’s behavior was evaluated by fitting the experimental data to the following second order model:

2.3. Intrinsic viscosity measurements Capillary viscosity measurements were performed using a glass viscometer (Schott Micro-Ubbelohde Viscometer Ic) immersed in a water bath at constant temperature (25 ± 0.5 ◦ C). The reduced and inherent viscosities were measured from capillary flow times corrected for density perturbations, for at least seven FucoPol concentration values (three replicas for each concentration). The intrinsic viscosity was calculated by extrapolation to zero concentration of Huggins (Eq. (1)) and Kraemer (Eq. (2)) equations: sp = [] + KH []2 c C

(1)

ln(rel ) = [] + KK []2 C C

(2)

Yp = b +

3  i=1

ai Xi +

3 3   i=1 j=1,j = / i

aij Xi Xj +

3 

aii Xi2

(3)

j=1

where Yp is the predicted response, Xi is the coded value of the independent variable i; b is the intercept and ai , aij , aii are the linear, interaction and quadratic coefficients, respectively [16]. In order to identify an appropriate reduced quadratic model, the significance of each source of variation was obtained from statistical analysis (ANOVA). The statistical analysis was carried out using an appropriate software. 3. Results

where sp (dL/g), [] (dL/g) and rel are the specific, intrinsic and relative viscosities, respectively. KH and KK are the Huggins and Kraemer constants, and c (g/dL) is the polymer concentration. All experiments were carried out in the range 1.2 < rel < 2.0 in order to ensure a good accuracy in the extrapolations to zero concentration.

3.1. Solution properties in dilute regime 3.1.1. Polyelectrolyte behavior in deionized water and in 0.1 M NaCl Fig. 1a presents the variation of the reduced viscosity as a function of FucoPol concentration in a salt free aqueous

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Table 2 Analysis of variance of the second order model for parameter [] (intrinsic viscosity). Source of variation

IS pH IS.IS pH.pH IS.pH Lack-of-fit Pure error Total (corr.) R2

Fig. 1. Reduced viscosity as a function of FucoPol concentration in salt-free solution (a) and determination of the intrinsic viscosity in 0.1 M NaCl using the Huggins (䊐) and Kraemer (䊏) equations (b).

solution, showing a non-linear dependence of these two parameters. As the polymer concentration decreased, there was a decline of the reduced viscosity until a polymer concentration of 0.02 g/L, followed by a steep increase for concentration values below 0.02 g/L. This behavior is usually perceived at low ionic strength aqueous solutions of polyelectrolytes [17], being attributed to repulsive forces occurring between intra-chain groups with the same charge, which leads to an increase of the molecules hydrodynamic volume, and consequently, to an increase of the intrinsic viscosity. As the biopolymer concentration increases, inter-chain interactions prevail over intra-chain repulsive forces, becoming dominant at higher concentration values, leading to the reduced viscosity-concentration dependence normally observed for uncharged polymers [18]. The addition of ions to FucoPol solution leads to polysaccharide’s negative charges shielding and disabling of intra-chain repulsions. As a consequence, when the measurements were performed in the presence of salt (0.1 M NaCl), at 25 ◦ C and pH 5.6 ± 0.05 (pH after FucoPol dissolution, without any pH adjustment), linear plots for Huggins and Kraemer extrapolations were obtained (Fig. 1b), enabling the determination of the intrinsic viscosity. The value obtained was 8.86 ± 0.09 dL/g, which is in accordance with the previously reported value, 8.9 dL/g [13], and also within the values referred in the literature for commercial polysaccharides, such as xanthan, guar gum and Fucogel (5–50 dL/g) [19,20]. The Huggins constant (KH ) determined for FucoPol was 0.58 ± 0.04, which is quite similar to that measured for Fucogel, a commercial fucose-containing bacterial polysaccharide (KH = 0.55 [20]). According to Morris et al. [21], KH should lie between 0.3 and 0.8, while values of KH higher than 1.0 are indicative of molecular aggregation. 3.1.2. Effect of pH and ionic strength on intrinsic viscosity To get a better knowledge of FucoPol’s intrinsic viscosity, the surface response methodology with a central composite rotatable design with two independent variables was performed. The

[] Sum of squares

Df

Mean squared

F-value

p-value

5.706 1.999 5.013 3.097 0.666 0.055 0.002 10.476 0.994

1 1 1 1 1 2 2 10

5.706 1.999 5.013 3.097 0.666 0.027 0.001 1.164

595.128 208.478 522.783 323.038 69.510 20.033

<0.001 <0.001 <0.001 <0.001 0.001 0.047

values of the independent variables, IS (M, NaCl) and pH, and of the dependent variable, [], are listed in Table 1. For the central point runs (0.40 M NaCl and pH 7.0), the [] achieved was within 8.23–8.30 dL/g, a value similar to the one obtained without pH adjustment (8.86 ± 0.09 dL/g, at 0.1 M NaCl and pH ≈ 5.6). Furthermore, results have also shown that within the wide range of ionic strengths and pH tested, [] remained within 7.21 and 8.54 dL/g. Only for the highest ionic strength (0.75 M NaCl; pH = 7) and highest pH (0.4 M NaCl; pH = 10.5) values tested, under intensive charge shielding, the intrinsic viscosity demonstrated a pronounced decrease to 5.65 and 5.51 dL/g, respectively. Statistical analysis was used to evaluate the significance of ionic strength (M NaCl) and pH effect and their interactions on the quadratic model used for describing []. An appropriate analysis of variance (ANOVA) of the second order model showed a good fit (R2 = 0.99) and a sum of squares (SS) of 10.476, with 10 degrees of freedom (Table 2) [22]. Despite that, there was evidence of lackof fit (p < 0.05), which means that the error predicted by the model was above the error of the replicates. Such result could be explained by the pure error (calculated by the replicas of the central point), which was close to zero, giving an artificial sense of model with lack of fit. The linear (IS; pH), quadratic (IS.IS; pH.pH) and the interaction (IS.pH) effects of ionic strength (M NaCL) (X1 ) and pH (X2 )) on [] is described in Table 2. The intrinsic viscosity was influenced by all the effects of ionic strength and pH (linear, quadratic and interaction), for a significance level of 5% (p < 0.05). Such correlation between response and independent variables can be graphically illustrated by the 3D response surface plots (Fig. 2). It can be observed that the intrinsic viscosity was kept practically unchanged for a wide range of ionic strength and pH values (0.05–0.50 M NaCl and 3.0–8.0, respectively). Withal, it decreased for combinations of ionic strength and pH above 0.50 M NaCl and 8.0, respectively. Results from Fig. 2 seems to indicate that for ionic strengths above 0.75 M NaCl, the intrinsic viscosity decreases for any value of pH tested between 3.5 and 10. The reduction of [] values with the increase of ionic strength is common to other polysaccharides solutions with a polyelectrolyte behavior, such as colanic acid, for which the intrinsic viscosity decreases from 47.5 to 22.7 dL/g when the ionic strength increases from 0.002 to 0.2 M NaCl [23], and also for the exopolysaccharide produced by Pseudomonas oleovorans, for which the intrinsic viscosity decreases from 14.0 to 4.9 dL/g when the ionic strength increases from 0.01 to 0.5 M NaCl [24]. With a higher ionic strength there’s a strong polymer charge shielding which decreases the intramolecular repulsion and leads to a lower hydrodynamic volume of the polymer molecule [24,25]. The difference is that, for FucoPol, a substantial decrease of the intrinsic viscosity takes place only at higher values of ionic strength (>0.5 M NaCl).

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C.A.V. Torres et al. / International Journal of Biological Macromolecules 79 (2015) 611–617 Table 3 Cross model parameters estimated for different FucoPol concentrations. FucoPol (wt.%)

Cross model 0 (Pa s)

0.20 0.35 0.45 0.50 0.60 0.80 0.90 1.00 1.20 RE =

0.011 0.054 0.074 0.096 0.134 0.439 0.759 1.095 2.465

n i=1

± ± ± ± ± ± ± ± ±

 (s) 0.000 0.006 0.002 0.004 0.008 0.038 0.024 0.036 0.066

0.005 0.028 0.016 0.022 0.033 0.098 0.159 0.282 0.553

m ± ± ± ± ± ± ± ± ±

0.000 0.012 0.002 0.004 0.007 0.018 0.018 0.036 0.052

0.825 0.613 0.634 0.693 0.735 0.677 0.707 0.645 0.666

± ± ± ± ± ± ± ± ±

0.092 0.058 0.028 0.046 0.066 0.036 0.030 0.024 0.016

(|xexp,i − xcalc,i |/xexp )/n is between 0.002 and 0.070.

Newtonian plateau at low shear rates, followed by a shear thinning region [23,28]: 1  − ∞ = o − ∞ 1 + ( ) ˙ m

Fig. 2. Response surface plot of intrinsic viscosity [] as a function of pH and ionic strength.

3.2. Steady shear measurements 3.2.1. Effect of polymer concentration on apparent viscosity The effect of polymer concentration (0.2–1.2 wt.%) on the steady shear behavior of FucoPol aqueous solutions was evaluated. The apparent viscosity increased with increasing biopolymer concentration and, for all the concentration values studied, flow curves showed a shear-thinning behavior, approaching a Newtonian plateau at lower shear rates (Fig. 3). This behavior is typical of solutions composed of entangled macromolecules [21,23,26,27] and it had already been observed in a previous work focused on the effect of temperature on the rheological properties of 1.0 wt.% FucoPol solutions [15]. In addition, for all concentrations studied, the apparent viscosity did not change when reducing the applied shear rate, right after shearing the sample up to a shear rate of 700 s−1 . The flow curves were fitted to the Cross model (Eq. (4)) using the software package ScientistTM from MicroMath® , assuming  » ∞ and 0 » ∞ , often used to describe flow curves presenting a

Fig. 3. Shear rate dependence of viscosity for different concentrations of FucoPol. () 0.20 wt.%; () 0.35 wt%; (䊐) 0.45 wt.%; (*) 0.50 wt.%; (♦) 0.60 wt.%; (䊉) 0.80 wt.%; (×) 0.90 wt.%; (䊏) 1.0 wt.%; () 1.2 wt.%. Lines represented the fitted Cross equation.

(4)

where ˙ is the shear rate (s−1 ),  is the apparent viscosity (Pa s), 0 is the viscosity of the first Newtonian plateau (Pa s), 0 is the viscosity of the second Newtonian plateau (Pa s),  is a relaxation time (s) and m is a dimensionless constant, which may be related to the exponent of the power law (n) by m = 1 − n. Eq. (4) was fitted to the experimental data (Fig. 3) and the estimated parameter values are summarized in Table 3. The time constant  increases as FucoPol concentration increases. This fact means that the relaxation time of the sample is higher due to the higher overall number of entanglements established as more biopolymer molecules are present in solution. Hence, the critical shear rate value, i.e. the value corresponding to the transition from Newtonian to shear-thinning behavior, which corresponds to the reciprocal of , decreases as the polymer concentration increases. Flow curves depicted in Fig. 3 were overlaid by scaling vertically, dividing by the respective viscosity of the first Newtonian plateau (0 ), and horizontally, multiplying by the relaxation time (), which generated a master curve (Fig. 4). Such approach enables to understand FucoPol’s behavior for a wider range of shear rates. It was employed successfully for diverse polysaccharide [21,24,29]. With the application of the generalized equation: /0 =

1 1 + ( ) ˙ m

(5)

Fig. 4. Master curve obtained shifting vertically dividing by 0 , and horizontally multiplying by relaxation time (). Inset:  as function of FucoPol concentration.

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615

Table 4 Analysis of variance of the second order model for parameter 0 (viscosity of the first Newtonian plateau estimated by the Cross model). Source of variation

IS pH IS.IS pH.pH IS.pH Lack-of-fit Pure error Total (corr.) R2 Fig. 5. Effect of ionic strength (M NaCl) and pH on FucoPol solutions’ viscosity dependency on shear rate (1 wt.% FucoPol). () 0.15 M NaCl – pH 4.5; (䊏) 0.65 M NaCl – pH 4.5; () 0.15 M NaCl – pH 9.5; (×) 0.65 M NaCl – pH 9.5; (*) 0.40 M NaCl – pH 7.0; (䊉) 0.40 M NaCl – pH 7.0; (+)0.40 M NaCl – pH 7.0; (䊏) 0.05 M NaCl – pH 7.00; (䊏) 0.75 M NaCl – pH 7.00; (♦) 0.40 M NaCl – pH 3.47; (䊐) 0.40 M NaCl – pH 10.54. Lines represent the fitted Cross equation.

a value of m = 0.681 ± 0.008 was obtained. This value is in accordance with the one presented by Morris, m = 0.76, for polysaccharides presenting strong interactions between polymer chains, such as hydrogen bonds [30]. The concentration dependence of  is illustrated in the inset of Fig. 3. The solid line suggests  ∼ c4.12 for concentrations above 0.6 g/dL, with an exponent near 4, which is referred in the literature for polysaccharides exhibiting a strong inter-chain association [21,31]. By the contrary, polysaccharides with linear chains revealing no interchain association presented exponent values of 2.08 [32]. Therefore, we may infer that, for the concentration range studied (0.2–1.2 wt.%), FucoPol presents a rheological behavior of a macromolecular entangled solution with high inter-chain association. 3.2.2. Effect of ionic strength and pH on the apparent viscosity The effect of pH and ionic strength on the steady shear behavior of FucoPol solutions was also studied using the response surface methodology coupled with a central composite rotatable design, according to Table 1. The study was carried out with a constant FucoPol concentration of 1.0 wt.%. Fig. 5 presents the flow curves of FucoPol aqueous solutions with different ionic strength and pH values. The results show that all solutions presented a shear thinning behavior, as in the studies where the effect of concentration was evaluated. The flow curves were fitted by Eq. 4, which enabled the estimation of 0 values, presented in Table 1. For the central point runs (0.40 M NaCl and pH 7.0), 0 achieved the highest value (1.20 Pa s), which was similar to the one obtained for the 1.0 wt.% FucoPol aqueous solution without pH adjustment (1.10 Pa s, at 0.1 M NaCl and pH ∼5.6). The lowest values were obtained for the solutions with 0.40 M NaCl – pH 10.54, 0.15 M NaCl – pH 9.5 and 0.65 M NaCl – pH 9.5, with zero-shear viscosities (0 ) of 0.37, 0.45 and 0.43 Pa s, respectively. Statistical analysis was also used to evaluate the impact of ionic strength NaCl (M) and pH on the quadratic model for describing 0 . ANOVA of the second order model showed a satisfactory fit (R2 = 0.85) (according to Lundstedt et al. [16]), a sum of squares (SS) of 1.109, with 10 degrees of freedom, and a non-significant lack-of fit (p = 0.120) (Table 4). Table 4 shows the linear (X1 ; X2 ), quadratic (X1 X1 ; X2 X2 ) and the interaction (X1 X2 ) coefficients of ionic strength (M NaCl) (X1 ) and pH (X2 ) on 0 . Linear pH and quadratic ionic strength are the factors which had a significant effect on 0 (p < 0.05). The 3D response surface plot (Fig. 6) evidenced the correlation between 0 and the independent variables pH and ionic strength.

0 Sum of squares

Df

Mean squared

F-value

p-value

0.001 0.384 0.480 0.265 0.001 0.163 0.001 1.109 0.850

1 1 1 1 1 2 2 10

0.001 0.384 0.480 0.265 0.001 0.054 0.001 0.123

0.018 9.344 11.687 6.446 0.035 37.209

0.899 0.038 0.027 0.064 0.860 0.120

The quadratic effect demonstrated that for the higher and lower ionic strength and pH values studied (axial points) the zero-shear viscosity decreased. It presented higher values (0.88 < 0 < 1.20) for ionic strength and pH within 0.20–0.60 M NaCl and 3.0–8.0, respectively, corresponding to the ranges for which a higher intrinsic viscosity was observed (Fig. 2), i.e. where no significant shielding from the ions in solution is expected. 3.3. Concentration regimes The concentration regimes are generally obtained from the concentration dependence of zero shear-rate specific viscosity (sp,0 ) with biopolymer concentration, an approach used by several authors [18,20,33–35]. In this study, for concentration values below 0.1 wt.%, sp,0 values were determined from the capillary viscometry data used to calculate the intrinsic viscosity in section 3.1.1. For concentration values above 0.1 wt.%, sp,0 was calculated using the 0 values estimated in section 3.2.1 and presented in Table 3. Fig. 7 displays the concentration dependence of zero shear-rate specific viscosity (sp,0 ) with FucoPol concentration. The curve has three distinct linear zones, characterized by different slopes, and separated by two critical concentrations (c* and c**). This type of correlation is analogous to the one exhibited by other high molecular weight microbial polysaccharides, such as xanthan

Fig. 6. Response surface of zero-shear viscosity (0 ) as a function of pH and ionic strength.

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References

Fig. 7. Concentration dependence of zero-shear specific viscosity for FucoPol samples (0.1 M NaCl; pH = 5.6).

[36], the galactose-rich polysaccharide produced by P. oleovorans [24] and the exopolysaccharide produced by P. acidi-propionici [29]. The dilute regime is characterized by isolated polymer molecules with a free mobility and a negligible influence on each other. c* sets the dilute regime boundary, marking the onset of significant molecules overlapping [29]. The overlap parameter (C∗ []), or space occupancy, was found to be approximately 0.8. With increasing concentration, polymer molecules start being directly affected by each other [19]. This leads to a change from one stage to another, thus resulting in a significant increase of the slope. The intermediate concentration region has a slope of 2.43, with space occupancy between 0.8 and 5.34. c** corresponds to the beginning of the entangled regime, where molecules interact intensively with each other [37]. The estimated values (c* ∼ 0.09 g/dL, c** ∼ 0.6 g/dL) are higher than the ones obtained for xanthan at the same ionic strength (c* ∼ 0.024 g/dL, c** ∼ 0.092 g/dL) [36]. This fact may be related to a smaller hydrodynamic volume of FucoPol molecules, expressed by its lower intrinsic viscosity ([] = 8.86 ± 0.09 dL/g), when compared to that of xanthan ([] = 47.5 dL/g). The slope above c** (3.78) is in the range (3.3–4.0) presented for several random coil polymers in the literature (alginate, xanthan, colanic acid, galactomannans) [21,23,38,39]. 4. Conclusions This work showed that FucoPol produces viscous solutions with shear-thinning behavior at different polymer concentrations. Being a polyelectrolyte, its solution properties are dependent on pH and ionic strength. Though, it presents intrinsic and apparent viscosity values with little variation within a wide range of pH and ionic strength values. From the results obtained, FucoPol shows a great potential to be used as thickening agent in diverse aqueous formulations prepared with a wide range of pH and ionic strength values, to be used in applications such as oil drilling fluids, paints, pharmaceuticals, cosmetics and food products. Acknowledgements This work was supported by Fundac¸ão para a Ciência e a Tecnologia (FC&T, Portugal) through projects UID/Multi/04378/2013, and PTDC/AGR-ALI/114706/2009. PEst-OE/AGR/UI0245/2014 Cristiana A.V. Torres, Filomena Freitas and Ana R.V. Ferreira acknowledge FC&T for fellowships SFRH/BPD/87774/2012, SFRH/BPD/72280/2010 and SFRH/BD/79101/2011, respectively.

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