Influence of temperature, mono- and divalent cations on dilute solution properties of sage seed gum

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Influence of temperature, mono- and divalent cations on dilute solution properties of sage seed gum

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A.R. Yousefi a , Seyed M.A. Razavi a,∗ , S.H. Khodabakhsh Aghdam b a Food Hydrocolloids Research Center, Department of Food Science and Technology, Ferdowsi University of Mashhad (FUM), PO Box 91775-1163, Mashhad, Iran b Department of Chemical Engineering, Ferdowsi University of Mashhad (FUM), PO Box 91775-1111, Mashhad, Iran

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a r t i c l e

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a b s t r a c t

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Article history: Received 20 January 2014 Received in revised form 25 February 2014 Accepted 17 March 2014 Available online xxx

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Keywords: Hydrocolloid Chain flexibility Chain stiffness Intrinsic viscosity Salt temperature

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1. Introduction

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The functional properties of food hydrocolloids are remarkably affected by the quality of solvent/cosolutes and temperature in a food system. In this paper, dilute solution properties of sage seed gum (SSG) as a function of salt type (NaCl, KCl, MgCl2 and CaCl2 ), salt concentration (10, 50, 100 and 200 mM) and temperature (25, 45 and 65 ◦ C) were investigated. Among various models, Higiro model showed a higher performance to determine intrinsic viscosity of SSG at all temperatures and cosolutes. From 25 to 65 ◦ C for every 20 ◦ C rise in temperature, intrinsic viscosity decreased about 18.99 and 63.86%, respectively. The divalent cations had more reduction effect on intrinsic viscosity than monovalet cations. More flexibility of SSG in monovalent salts solutions compared with divalent ones was observed. A high value for activation energy (2.53 × 107 J/kg mol) and chain flexibility (3046.45) of SSG was obtained, which was higher than many hydrocolloids. The shape factor of SSG macromolecules at 25–65 ◦ C was an oblate or prolate and for all used cosolutes, the shape was roughly found to be ellipsoidal. © 2014 Published by Elsevier B.V.

Hydrocolloids are widely used in food systems to enhance their quality by affecting the physical and organoleptic attributes as thickening and gelling agents, stabilizers and texture modifiers [1]. Nowadays, the demand for hydrocolloids from plants (e.g. plant cell walls, tree exudates, seeds, seaweeds) is greater than those from animals (hyaluronan, chitin, chondroitin sulphate) because of more benefits and friendly image toward consumers [2]. There are some Iranian endemic plants that their seeds can be used as a new hydrocolloid sources for food and pharmaceutical systems [3]. Salvia contains about 700–900 species of herbaceous and woody plants of the mint family, Lamiaceae. About 200 species of this genus grow in some provinces of Iran [4]. Wild sage (Salvia verbenaca) is one these endemic plants and its seed (Salvia macrosiphon) mucilage has a potential alternative to some commercial gums [5]. The extraction conditions for sage seed mucilage was optimized using response surface methodology [6]. The steady shear flow behavior of sage seed gum (SSG) demonstrated that it has strong shear thinning characteristics at different temperature

∗ Corresponding author. Tel.: +98 511 8795618/+98 511 8795620; fax: +98 511 8787430. E-mail addresses: [email protected], sma [email protected] (S.M.A. Razavi).

and concentrations which is comparable to xanthan [3]. Structural characteristics of SSG revealed that mannose (61.50%) and galactose (33.15%) are the main carbohydrate fractions, but glucose (2.78%), arabinose (1.41%) and rhamnose (1.17%) are insignificant ones [4]. It was concluded that SSG polysaccharide is a galactomannan with a 1.78–1.93:1 mannose/galactose ratio. It was recently found that SSG enhanced the stability of oil-in-water emulsion containing whey protein concentrate (5–15% w/v) by modification of the flow behavior and induction of intermolecular interactions [7]. The dynamic rheological properties of SSG at all concentration within the range of 0.5–2% (w/w) have shown weak gel behavior like xanthan and psyllium gums that was more elastic compared with other galactomannans [2]. The viscosity behavior of macromolecular substances in a solution is one of the most frequently used approaches to determine its specification. In dilute solution, it is assumed that macromolecule chains are separated without intermolecular interactions [8,9]. Investigation of molecular properties such as macromolecule-solvent interaction, molecular weight, molecular shape and conformation seems to be useful to understand and control the behavior of a hydrocolloid in dilute solution at different conditions. Intrinsic viscosity [] is a measure of the capability of a polymer in solution to increase the viscosity of the solution. Much information for fundamental properties of a solute and its interaction with

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a specific solvent can be obtained by determination of intrinsic viscosity [10,11]. There are some linear (Huggins and Kraemer) and non-linear (Tanglertpaibul & Rao and Higiro) equations to determine intrinsic viscosity [12–15]. Razavi et al. [9] found that the Tanglertpaibul and Rao and Higiro models were the best ones for intrinsic viscosity determination of SSG at different temperatures (20, 30 and 40 ◦ C) and salts concentrations (0.5, 20 and 50 mM), respectively. Behrouzian et al. [16] also showed that the Tanglertpaibul and Rao model was the best one to determine intrinsic viscosity of cress seed gum dilute solutions. Since intrinsic viscosity affect by changes in solvent properties like ionic strength, then study of its change could be a good instrument to track the changes in molecular attributes [1,9,11,16]. Literature shows that higher ionic strength and temperature lead to decrease in solution viscosity [9,11,15,17–20]. For instance, Behrouzian et al. [16] found that increase in concentration of NaCl (25–100 mM), CaCl2 (5–15 mM), sucrose (up to 30%) and lactose (up to 5%) caused a reduction of intrinsic viscosity. Razavi et al. [9] also showed that CaCl2 had a more pronounced effect on intrinsic viscosity of SSG than NaCl. The similar results were reported by Mohammad Amini and Razavi [11] for Balangu (Lallemantia royleana) seed gum. Furthermore, they reported that the effect of temperature on intrinsic viscosity of Balangu seed gun was significant, so that each 10 ◦ C temperature increase from 20 ◦ C to 50 ◦ C caused a decrease in intrinsic viscosity approximately 15.12%, 24.10%, and 30.84%, respectively. In salt-free situation or low salt concentration, the expansion of polymer chain occurs because of interachain electrostatic repulsion. At higher salt concentration, screening of the charge occurs and the electrostatic interactions diminish and the chain conformation becomes more compact. The aim of the present study was to develop our previous works about SSG. For this purpose, the influence of different mono- and divalent ions (NaCl, KCl, MgCl2 and CaCl2 ) and temperatures (25, 45 and 65 ◦ C) on intrinsic viscosity of SSG was investigated. In addition, some molecular parameters of SSG including conformation, relative stiffness, persistence length, chain flexibility, voluminosity, shape factor, coil radius and volume were determined and their relation with the intrinsic viscosity of SSG were discussed.

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2. Materials and methods

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2.1. Preparation of materials

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The sage seeds were purchased from a local market in Mashhad, Iran. Several cleaning steps manually were used to ensure removing all of undesirable stuffs. Extraction of the sage seed gum was performed using the method described by Bostan et al. [6]. Finally, the SSG was subjected to a force convection oven (Model 4567, Kimya Pars Com., Iran) overnight at 70 ◦ C prior to be milled and sieved using a mesh 18 sifter. The SSG powder contained, on averagely, 6.72% moisture, 0.85% lipid, 8.17% ash, 2.84% protein, 1.67% crude fiber, and 79.75% carbohydrate. 2.2. Preparation of solutions Stock SSG solutions were prepared at concentration of 0.25% by mixing 0.1 g (d.b.%) of SSG powder in 40 ml of de-ionized water and a range of NaCl, KCl, MgCl2 and CaCl2 concentrations (10, 50, 100 and 200 mM) at room temperature. For this reason, the prepared gum powder was gradually added into the vortex formed due to whirl of magnetic stirrer. After that, the attained deionized water, NaCl, KCl, MgCl2 and CaCl2 -SSG suspensions were mixed using a roller mixer (Hematology Cell Mixer; Pars, Iran) for 15 min without heating and was retained 24 h for complete hydration. In order to discard any insoluble residues, all of the prepared SSG solutions

were centrifuged at 10,000 × g. Eventually, the supernatant parts of the centrifuged samples were filtered via a methyl-cellulose membrane with a pore size of 0.45 ␮m. It should be noted that the dilute solutions of SSG were obtained by diluting the filtrated part (0.25%, d.b.). 2.3. Density measurements

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Density of solvent (0 ) and solution () were obtained by means of a standard 25 ml pycnometer. The temperature for density measurements was similar to that for viscosity measurements. The partial specific volume (¯v) of SSG solution in deionized water was determined through density increment () vs. concentration curve at 25 ◦ C. 2.4. Viscosity measurement

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Dilute SSG solutions were made by adding distinct extent of the solvents (de-ionized water, NaCl, KCl, MgCl2 and CaCl2 solutions) to the stock solutions. The viscosity of SSG solutions was measured using a Cannon-Ubbelohde viscometer (Cannon Instruments, USA; viscometer constant, k = 0.007690 mm2 /s2 ) immersed in a paraffin bath to maintain at 25 ◦ C for 15 min. The kinematic viscosity was measured by allowing the solutions to flow due to their gravity through the capillary part of the viscometer. All the measurements were done as triplicates and the average values are reported. Intrinsic viscosity [] can be determined by measuring the viscosity of very low concentration solutions through the calculation of the following viscosities:

141 142 143 144 145 146 147 148 149 150 151 152

 = s

(1)

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sp = rel − 1

(2)

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(3)

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rel

[] = limc→0

sp C

where  is the solution viscosity, s is the solvent viscosity, rel is relative viscosity and sp is the specific viscosity. There are several developed equations to determine the intrinsic viscosity. According to Huggins model [12] (Eq. (4)), the intrinsic viscosity [] obtained by extrapolating sp /C data to zero concentration simply through a linear regression: sp = [] + k []2 C C

(4)

where k is the Huggins constant. Kraemer [13] reported that the intrinsic viscosity [] could be obtained by extrapolation of ln rel /C values to zero concentration (Eq. (5)): ln rel = [] + k []2 C C

(5)

where k is the Kraemer constant. It is demonstrated that the methods in which the intrinsic viscosity is calculated based on the slopes of plots had higher correlation coefficient and lower standard errors in comparison with those are calculated through intercepts of plots [9,21]. Based on this finding, three equations are shown as follow to determine the intrinsic viscosity of the solutions based on the slope of plots: Tanglertpaibul and Rao [14]: rel = 1 + []C

(6)

Higiro et al. [15]: rel = e rel =

[]C

1 1 − []C

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(7)

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(8)

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2.5. Estimation of molecular conformation

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The following relationship was used for the estimation of b component from the slope of a double logarithmic plot of sp vs. concentration. The conformation of a polysaccharide can be estimated through this parameter [22].

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sp = aC b

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(9)

2.6. Determination of relative stiffness parameter (B) and persistence length (q) The stiffness parameter (S) was calculated using the following equation from the intrinsic viscosity’s slope at various ionic strengths against the inverse square root of ionic strength (I−0.5 ) plot [18]. [] = []∞ + SI −0.5

(10)

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where []∞ is the intrinsic viscosity at infinite ionic strength. Although the constant S is a criterion of polymer stiffness, but it is powerfully molecular weight dependent. For this reason, Smidsrod and Huga [23] introduced B as an independent stiffness parameter which was calculated accordingly:

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S = B([]0.1 )

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(11)

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where the  parameter was found to be at range of 1.2–1.4 which the average value of 1.3 is broadly used as a constant number. The value of []0.1 is the intrinsic viscosity at an ionic strength of 0.1 M [24]. Smidsrod and Christiansen [25] introduced another character entitled “persistence length” (q) which is a criterion of the length is related to B parameter. Indeed, q is a measure of the length over which the chain persists’ in the direction of the first bond of the chain. The mathematical relation between B and q parameters is shown in follow equation:

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q=

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0.26 B

(12)

2.7. Determination of the chain flexibility parameter

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 = AeEa /RT

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2.8. Voluminosity and shape factor of the polymer coil VE parameter is a measure of volume of a solvated polymer molecule which could be obtained through intercept of plotting Y value (as shown in Eq. (14)) vs. C [9,27]

  c

In brief, VE exhibited the conformation of a polymer in different solvent condition [9]. The fallowing equation revealed the relationship between VE and intrinsic viscosity via another parameter which is known as shape factor (). [] = VE

(15)

The shape of polymer’s particles in a solution could be estimated through  factor. 2.9. Coil radius and volume

 3[] × M 1/3 w

10 × NAV

(16)

where, Mw and NAV are the polymer weight average molecular weight and Avogadro’s number (6.022 × 1023 mol−1 ), respectively. Coil volume, Vcoil , can be determined through the following equation, by the assumption that shape of the coil be sphere like [11]: 4 3 R 3 coil

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Antoniou et al. [27] proposed the following equation to calculate the hydrodynamic coil radius, Rcoil . Rcoil =

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(17)

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3. Results and discussion

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3.1. Density

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(13)

where  is the dynamic viscosity (Pa s), A is a constant number, Ea is the activation energy of the flow process (kJ/kg mol), R is the universal gas constant (8.314 kJ/kg mol K) and T is the absolute temperature (K). Mohammad Amini and Razavi [11] reported that the chain flexibility of a polymer can be calculated from the Ea value, which the chain flexibility decreases with increasing temperature. By replacement of the intrinsic viscosity with the dynamic viscosity, the calculated slope for natural logarithmic intrinsic viscosity vs. the inverse of absolute temperature (1/T) can be used to calculate the chain flexibility of a polymer due to its relation to the Ea .

Y=

Fig. 1. Dependence of density increment () on concentration. The slope of this dependence is the partial specific volume (¯v) (the error bars are standard deviation).

Vcoil =

Goycoolea et al. [26] reported that under C* concentration of a polymer solution (Newtonian region), increasing temperature causes to decrease in viscosity which follows Arrhenius law:

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0.5 −1 rel 1.350.5 − 0.1 rel



(14)

A partial specific volume (¯v) of 0.48 ml/g was obtained via calculating the slop of ( − 0 ) vs. concentration (Fig. 1). This value was similar to the value 0.47 ml/g obtained for carboxymethylchitins [28], but lower than citrus pectin (0.57 ml/g) and xanthan (0.60 ml/g) and konjac glucomannan (0.63 ml/g) and Balangu seed gum (0.61 ml/g) reported by Harding et al. [29], Dhami et al. [30], Kok et al. [31] and Mohammad Amini and Razavi, [11], respectively. The buoyancy of particles in food systems is an important factor that affects sedimentation phenomena. The buoyancy of a specific particle increases with the increase in partial specific volume, therefore the more partial specific volume for a polymer, the less sedimentation [30]. Durchschlag [32] reported that the partial specific volume of native conjugated proteins in aqueous solution vary in the range of 0.59–1.05 ml/g. 3.2. Solution viscosity Fig. 2a depicts changes in relative viscosity by increasing SSG solutions concentration at temperatures of 25, 45 and 65 ◦ C. To investigate the cosolutes impact, the effect of MgCl2 solutions concentration on the relative viscosity is shown in Fig. 2b. It can be seen

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0.0098 0.0096 0.0078 0.0023 0.9983 0.9994 0.9966 0.9995 4.31 2.18 2.41 2.18 0.0392 0.0017 0.0168 0.0097 0.9976 0.9999 0.9925 0.9993 5.56 3.50 2.79 2.48 0.0734 0.0062 0.0259 0.0174 0.9900 0.9994 0.9882 0.9974 7.18 2.87 3.24 2.81 0.0251 0.0564 0.0531 0.0162 0.9744 0.9153 0.9161 0.9540 4.04 2.58 2.25 2.08

0.0152 0.0134 0.0104 0.0092 0.9998 0.9988 0.9993 0.9983 4.41 2.29 2.24 1.87 0.0346 0.0043 0.0017 0.0033 0.9937 0.9999 0.9999 0.9998 5.74 2.64 2.58 2.09 0.0708 0.0043 0.0064 0.0021 0.9810 0.9997 0.9995 0.9998 7.48 3.05 2.97 2.33 0.0863 0.0117 0.0073 0.0045 0.8692 0.9214 0.9402 0.9705 4.72 2.78 2.70 2.19

0.0371 0.0165 0.0093 0.0202 0.9908 0.9980 0.9972 0.9946 4.91 4.53 4.31 3.80 0.0077 0.0205 0.0261 0.0295 0.9993 0.9992 0.9987 0.9999 7.11 5.79 5.37 4.86 0.0171 0.0481 0.0494 0.0632 0.9991 0.9963 0.9971 0.9981 8.12 7.46 6.71 6.18 0.0180 0.0403 0.0658 0.0784 0.9333 0.9532 0.921 0.9072 5.15 4.81 4.16 3.81

0.0219 0.0261 0.0261 0.0199 0.9957 0.9981 0.9962 0.9969 5.48 3.96 3.57 3.14 0.0156 0.0268 0.0129 0.0413 0.9997 0.9983 0.9999 0.9973 7.00 5.21 4.55 3.87 0.0419 0.0649 0.0430 0.0587 0.9993 0.9940 0.9993 0.9950 8.99 6.90 5.81 5.41

0.0089 0.0236 0.0082 0.9985 0.9952 0.9995 13.59 10.75 3.46 0.0540 0.1008 0.0046

0.0457 0.0208 0.0344 0.1608

0.0306 0.0197 0.0016 0.9990 0.9997 0.9999 10.11 8.19 2.96

R [] RMSE R [] RMSE

Tanglertpaibul and Rao

2

0.9788 0.8672 0.8488

0.0131 0.0142 0.0436 0.0139

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0.9920 0.9881 0.9500 0.9902

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0.1830 0.0081 0.0081 0.0050

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0.9512 0.9793 0.9921 0.9582

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0.0232 0.0500 0.0829 0.0684

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0.9924 0.9934 0.9833 0.9912

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0.0492 0.0839 0.0325 0.1717

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0.9940 0.9803 0.9950 0.9715

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12.18 9.28 3.02

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0.0785 0.1463 0.0059

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0.7449 0.8329 0.9948

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Temperature ( C) 25 12.85 45 9.41 2.99 65 NaCl (mM) 5.25 10 3.99 50 3.74 100 2.65 200 KCl (mM) 5.12 10 4.12 50 100 3.44 200 3.21 MgCl2 (mM) 10 3.96 2.84 50 2.63 100 2.23 200 CaCl2 (mM) 3.18 10 2.57 50 2.11 100 2.01 200

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R

278

[]

277

RMSE

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R

275

◦

274

[]

273

0.9234 0.9815 0.9082 0.8995

0.9922 0.9964 0.9984 7.59 6.22 2.54

R2 [] RMSE

Higiro et al. (Eq. (8)) 272

2

Higiro et al. (Eq. (7)) 271

2

270

Kraemer

269

2

268

that increasing the salt concentration and temperature decreases the relative viscosity value. Intrinsic viscosity [] is a criterion of the hydrodynamic volume occupied by a macromolecule, which is closely related to the size and conformation of the macromolecule chains in a particular solvent [9,10,15]. In this paper, the intrinsic viscosity value was determined by two methods, which in first method, [] obtained from the intercept of plots (Eqs. (4) and (5)), while in second method, it was calculated from the slope of plots (Eqs. (6)–(8)) [9,21]. The intrinsic viscosity values calculated by five models for SSG dilute solutions at different temperatures (25, 45 and 65 ◦ C) and salt concentrations of the mono (Na+ and K+ ) and divalent (Mg2+ and Ca2+ ) cations are shown in Table 1, respectively. It is clear that in both investigations (influence of temperature and salt), the obtained intrinsic viscosity form the models in which this valve calculated through the slopes of plots had higher determination coefficient (R2 ) and lower root mean square error (RMSE) than the intrinsic viscosity obtained via intercepts of plots. Similar results have been reported by McMillan [21], Razavi et al. [9], Behrouzian et al. [16]. In both investigations, Higiro model (Eq. (7)) was the most suitable model for determination of intrinsic viscosity which had the highest R2 in range of 0.9925–0.9999 and the lowest RMSE in the range of 0.0017–0.0413. Razavi et al. [9] reported that Tanglertpaibul and Rao model was the best model to estimate the intrinsic viscosity of SSG at 20, 30 and 40 ◦ C, but in agreement with the results of this research, Higiro model (Eq. (7)) was the best model to determine the intrinsic viscosity value of SSG at different concentration (0.5, 20, 50 mM) of NaCl and CaCl2 . Amongst the models, Tanglertpaibul and Rao and Huggins had the highest and the lowest estimation of intrinsic viscosity. According to the results, by increasing the temperature from 25 to 65 ◦ C, the intrinsic viscosity decreased from 10.11 to 2.96 dl/g. This amount of decrease demonstrates significant effect of temperature

Huggins

267

Treatment

266

Table 1 Intrinsic viscosity values determined by five models (Eqs. (4)–(8)) for SSG at selected temperatures and cosolutes (25 ◦ C).

Fig. 2. Relative viscosity of SSG as a function of gum concentration at (a) different temperatures (25–65 ◦ C, 61.06–192.31 s−1 ) and (b) different MgCl2 concentrations (10–200 mM, 59.89–99.39 s−1 ) (the error bars are standard deviation).

6.05 4.43 4.08 3.96

0.0818 0.0709 0.0135

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RMSE

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on intrinsic viscosity, as each 20 ◦ C increase in temperature results in a decrease in intrinsic viscosity of SSG about 18.99 and 63.86%, respectively, possibly due to the increased kinetic energy and phase separation. These results were in agreement with previous studies [1,11,18–20]. As Table 1 illustrates, regardless of the ion type, the intrinsic viscosity drastically decreases even at 10 mM concentration of the mono and divalent salts compared with SSG solutions in deionized water at 25 ◦ C (10.11 dl/g). It can be seen that divalent cations (Mg2+ and Ca2+ ) had more effective reduction in the intrinsic viscosity value compared with the monovalent cations (Na+ and K+ ) when the salts concentration rose to 200 mM. This reduction for NaCl and KCl solutions at 200 mM were 61.72% and 51.93%, while for MgCl2 and CaCl2 were 79.33% and 75.47%, respectively. This must be due to higher capacity of Mg2+ and Ca2+ than Na+ and K+ to create intermolecular junction between SSG macromolecules, which resulted in a greater extent of contraction. It is obvious that when the salt concentration increases the macromolecule contract due to the increased screening of interamolecular electrostatic repulsion. Therefore, more contract in the polymer conformation, less value of intrinsic viscosity. Similar results have been reported for hsiantsao leaf gum [18], xanthan [15], sodium alginate [10], Balangu seed gum [11] and cress seed gum [16]. The results of statistical t-test demonstrated that there was no significant difference between intrinsic values obtained in NaCl and KCl as well as MgCl2 and CaCl2 solutions (p > 0.05), but the difference between each pair consist of a mono and a divalent solution was significant (p < 0.05). Investigation of the influence of the salt concentration and temperature on the intrinsic viscosity of SSG clearly revealed that increase in the salt concentration regardless of its type from 10 to 200 mM had greater reduction effect than increase in temperature from 25 to 65 ◦ C. It was in agreement with the results of Mohammad Amini and Razavi [11]. They reported that increase in NaCl and CaCl2 concentration from 5 to 50 mM had a more pronounced effect on decrease of intrinsic viscosity than increase in temperature from 20 to 50 ◦ C.

337

3.3. Coil overlap parameter

301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335

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The conformation of many macromolecules in a solution is random coil in which the shape fluctuates consecutively under Brownian motion [33]. In a dilute solution, the individual polymer coils move independently because they are far enough apart that have negligible influence on each other [9,10,22]. During transition from dilute solutions to concentrated solutions an abrupt change in concentration occurs, which is dependent to the solution viscosity [34,35]. The corresponding polymer concentration is called critical or coil overlap concentration (C*). To determine this parameter for SSG, master curve which is the plot of logarithm of sp vs. logarithm of C[] was used. Morris et al. [34] reported that this slope in a dilute solution domain is close to 1.4, while in a concentrated domain the slope increase to 3.3. It was found that SSG solutions at all selected temperatures and salts concentrations were in dilute regime in which no molecular entanglements occur (Fig. 3).The slope values obtained for SSG at selected temperatures were in the range of 1.04 to 1.13, whereas they were ranged from 1.32 to 1.71 for NaCl, from 1.13 to 1.47 for KCl, from 1.03 to 1.40 for MgCl2 and from 1.08 to 1.51 for CaCl2 . When the Berry number (C[]) becomes more than one, the molecular entanglement begins in concentrated regime [36]. It was found that this value for SSG was within the range of 0.12–0.78 at all selected temperatures and salts concentrations, indicating no coil overlap and molecular entanglements occurred (Table 2). The slope of power-law model (Eq. (9)) or exponent b value obtained at different solution conditions is shown in Table 2. Morris et al. [34] declared that in dilute regime, b values greater than unity

5

Fig. 3. Master curve for SSG determined at (a) selected temperatures, (b) different concentration of monovalent salts and (c) different concentration of divalent salts.

are related to random coil conformation, whereas less values are associated with rod-like conformation. The obtained b values for SSG at selected temperatures were within the range of 1.04–1.13, indicating that the molecular conformation of SSG is between random coil and rod like [3,16]. Behrouzian et al. [16] reported similar results for cress seed gum solution (b = 1.08) in deionized water at 25 ◦ C. It can be seen that for both monovalent salts (NaCl and KCl), b value increased with the increase of salt concentration, but on the Table 2 The values of Berry number (C[]) and exponent b (slope of log sp vs. log C) of SSG at selected temperatures and salts concentrations. Temperature (◦ C)

25

45

65

B C[] Concentration (mM) NaCl b C[] KCl b C[] MgCl2 b C[] CaCl2 b C[]

1.04 ± 0.01 0.37–0.68 10

1.10 ± 0.00 0.29–0.66 50

1.13 ± 0.01 0.21–0.37 100

200

1.40 ± 0.03 0.35–0.58

1.44 ± 0.01 0.36–0.65

1.32 ± 0.02 0.28–0.57

1.71 ± 0.04 0.16–0.27

1.13 ± 0.00 0.26–0.78

1.42 ± 0.04 0.32–0.58

1.47 ± 0.03 0.30–0.54

1.45 ± 0.02 0.27–0.60

1.40 ± 0.05 0.28–0.64

1.05 ± 0.01 0.13–0.33

1.08 ± 0.01 0.12–0.32

1.03 ± 0.00 0.10–0.26

1.51 ± 0.06 0.27–0.62

1.08 ± 0.02 0.17–0.44

1.28 ± 0.04 0.13–0.34

1.24 ± 0.02 0.12–0.31

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6

Fig. 5. An Arrhenius-type plot drawn for SSG in deionized water (the error bars are standard deviation). Fig. 4. Intrinsic viscosity of SSG as a function of the inverse square root of ionic strength (25 ◦ C) (the error bars are standard deviation).

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contrary increase in divalent salts (MgCl2 and CaCl2 ) concentration resulted in the decrease in this value. Similar results were reported by Higiro et al. [15] for locust been gum. They found that b value decreased by increasing CaCl2 concentration from 0 to 50 mM. tTest results showed that there was a significant difference between obtained b values in mono and divalent ionic solutions (p < 0.05). In agreement with the obtained results in this study, Higiro et al. [15] reported that locust bean gum had greater b value in NaCl and KCl solutions in comparison with CaCl2 solution. Lai and Chiang [18] found that b value for hsian-tsao leaf gum in the dilute domain was from 0.78 to 0.8 and concluded that the molecular conformation of hsian-tsao gum was more rod-like than random coil. 3.4. Relative stiffness parameter (B) and persistence length (q) (R2

The linear dependence > 0.975) of intrinsic viscosity on the reciprocal of the square root of ionic strength (I−0.5 ) for selected mono and divalent salts is shown in Fig. 4. From the Eq. (10), the stiffness parameter (S) and intrinsic viscosity at infinite ionic strength ([]∞ ) can be estimated in presence of selected mono and divalent salts (Table 3). As expected, the monovalent salts had greater []∞ compared with divalent ones so that the greatest value of []∞ obtained in KCl solution (4.42 dl/g). The relative stiffness parameter (B), which is independent of Mw , calculated from the Eq. (11). These two parameters (S and B) obtained for SSG in NaCl and KCl solutions were lower than those in MgCl2 and CaCl2 solutions, indicating more flexibility of SSG in monovalent salts solutions compared with divalent ones. It is due to greater extent contraction in SSG chains in divalent salts than monovalent ones that proved by the results of intrinsic viscosity. The obtained stiffness parameter (S) for SSG in NaCl solution (0.381) was close to that reported for Balangu seed gum (0.346) by Mohammad Amini and Razavi [11] and lower than reported for tragacantin (0.6) by Mohammadifar et al. [24]. In addition, this parameter in presence of CaCl2 for SSG (0.821) was greater than that for Balangu seed gum (0.507) [11]. In brief, it can be concluded that SSG in monovalent solutions has a rather flexible conformation, whereas in divalent solutions has a semi-flexible/stiff one. By using value of 1.3 for  in Eq. (12), the persistence length parameter (q) was calculated for SSG in selected ionic solutions (Table 3). It can be seen that q values of monovalent Table 3 The molecular parameters determined for SSG by using Eqs. (10)–(12). Solvent

[∞ ] (dl/g)

S

B

q (nm)

NaCl KCl MgCl2 CaCl2

3.26 4.42 0.91 1.61

0.381 0.273 0.688 0.821

0.053 0.038 0.096 0.114

4.892 6.827 2.709 2.270

salts (4.892 and 6.827 nm) were more than those for divalent ones (2.709 and 2.270 nm), confirming more flexibility of SSG molecular chains in NaCl and KCl than MgCl2 and CaCl2 solutions. The obtained q values in selected solution for SSG were lower reported ones for tragacantin [24] and xanthan [37]. 3.5. Chain flexibility parameters The plot of ln[] against the inverse of absolute temperature (1/T) with the slope of dln[]/d(1/T) for SSG is shown in Fig. 5. The obtained slope for the plot is a measure of macromolecular chain flexibility. The calculated chain flexibility parameter (Ea /RT) and activation energy (Ea ) for SSG at selected temperatures were 3046.45 and 2.53 × 107 J/kg mol, respectively. The chain flexibility value of SSG was much higher than those reported for chitosan (666–1334) by Chen and Tsaih [38], xanthan (900–1100) by Milas and Rinaudo [39] and Balangu seed gum (1156.23) by Mohammad Amini and Razavi [11].The calculated Ea for SSG was very close to the value (2.5 × 107 J/kmol) reported for chitosan with 91% DD [40], while it was much higher than the value (1.5 × 107 J/kmol) determined for chitosan with 75% DD [40] and for Balangu seed gum (1 × 107 J/kmol) [11], indicating that SSG macromolecules entangled more easier within themselves which resulted in higher Ea value. 3.6. Coil radius and volume Razavi et al. [9] reported that the weight-average molecular weight of SSG is about 1.5 × 106 Da at 25 ◦ C. Using their results, Rcoil and Vcoil of SSG in selected ionic solutions at 25 ◦ C were estimated and tabulated in Table 4. It was found that both salt concentration and salt type (mono or divalent) had a pronounced influence on Rcoil and Vcoil values of SSG macromolecules. Therefore, in divalent solutions and higher concentrations regardless of the salt type, these values reduced to a greater extent. These results were in accordance with the results of intrinsic viscosity (Table 1), which demonstrates that Rcoil and Vcoil of SSG macromolecules can be strongly affected by intrinsic viscosity. The obtained Rcoil value for SSG in 50 mM NaCl solution (4.96 nm) was similar to that for Balangu seed gum (4.60 nm), while was different at the same concentration in CaCl2 solution (4.37 nm for SSG and 3.77 nm for Balangu seed gum) [11]. Antoniou et al. [27] found that Rcoil value for dextran T500 in single and binary good + bad solvents at 20 ◦ C was between 13.7 and 19.7 nm, which was much higher than the obtained Rcoil value for SSG in this research. 3.7. Voluminosity and shape factor of the polymer coil Voluminosity and shape factor results of SSG at selected temperatures and cosolutes are shown in Table 4. It can be seen that for every 20 ◦ C rise in temperature, E decreased 22.08 and 66.66%,

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433

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A.R. Yousefi et al. / International Journal of Biological Macromolecules xxx (2014) xxx–xxx Table 4 Some molecular parameters of SSG determined by Eqs. (14)–(17) at selected temperatures and cosolutes. Solvent Deionized water 25 ◦ C 45 ◦ C 65 ◦ C NaCl (mM) 10 50 100 200 KCl (mM) 10 50 100 200 MgCl2 (mM) 10 50 100 200 CaCl2 (mM) 10 50 100 200

Rcoil (nm)

Vcoil (nm)3

E (dl/g)



6.23 – –

1012.35 – –

4.62 3.60 1.20

2.19 2.27 2.46

5.51 4.96 4.77 4.52

700.36 510.87 454.38 386.61

1.99 1.83 1.76 1.68

3.52 2.84 2.58 2.30

5.54 5.17 5.04 4.88

711.86 578.54 539.99 486.55

2.03 1.97 1.73 1.57

3.50 2.94 3.10 3.09

5.16 3.98 3.95 3.68

575.19 263.94 258.02 208.64

1.94 1.12 1.07 0.88

2.96 2.36 2.41 2.37

5.10 4.37 4.05 3.89

555.36 349.39 278.12 246.44

1.7 1.02 1.06 0.88

3.27 3.43 2.63 2.82

7

best one to estimate [] of SSG using highest R2 (0.9925–0.9999) and lowest RMSE (0.0016–0.0413) values obtained in all selected conditions. It was observed that divalent cations have more effective reduction in the intrinsic viscosity value compared with the monovalent cations when the salts concentration rises to 200 mM due to providing a greater extent of contraction. It was found that at all selected temperatures and ionic concentrations, the Berry number (C[]) was within the range of 0.12–0.78, indicating no coil overlap and molecular entanglements occurred. The stiffness (S) and relative stiffness (B) values obtained for SSG in NaCl and KCl solutions were lower than those obtained in MgCl2 and CaCl2 solutions, indicating more flexibility of SSG in monovalent salts solutions compared with divalent ones. The high values of activation energy (2.53 × 107 J/kg mol) and chain flexibility (3046.45) obtained for SSG indicate that SSG macromolecules entangled more easily within themselves. It was generally observed that the voluminosity and shape factor of SSG coils reduced to a greater extent in divalent solutions and higher concentrations regardless of the salt type. From the results, the shape factor of SSG macromolecules at 25, 45 and 65 ◦ C was an oblate or prolate, whereas in all selected cosolutes the shape was roughly found to be ellipsoidal. In general, it can be concluded that the solvent quality significantly decreased with increase in salt concentration and temperature.

References

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respectively. It is indicates that the coil dimension of SSG macromolecules or the solvent power decreased [9]. It can be seen that the divalent ions (Mg2+ and Ca2+ ) had more pronounced reduction effect on E values than the monovalent ions (Na+ and K+ ). Therefore, it seems that divalent ions provided more molecular contraction in comparison with monovalent ones. The obtained results were in accordance with the results of intrinsic viscosity. Similar results were observed during investigation of concentration influence regardless of the ion type on voluminosity and shape factor (Table 4). Based on experimental observations, the physical meaning of  can be summarized as follows: (a) a value of 2.5 for  indicates spherical shape, (b) higher value is associated with ellipsoidal shape, (c) different values of  suggest oblate or prolate shape for a polymer coil [27]. Accordingly, it can be seen that the shape factor of SSG macromolecule at all selected temperatures was an oblate or prolate, whereas in all selected cosolutes the shape was roughly found to be ellipsoidal. In a poor solvent, the monomers of individual polymer effectively attract each other to minimize their contacts with the solvent molecules, so a roughly spherical or ellipsoidal shape forms which have lesser flexibility [41]. Therefore, from the results of shape factor it can be concluded that deionized water solvent at selected temperatures (especially at 25 and 45 ◦ C) has had better quality than mono- and divalent cosolutes. It was observed that with increasing temperature from 25 to 65 ◦ C, the shape factor value increased from 2.19 to 2.46, indicating lesser flexibility of SSG macromolecules at higher temperatures. These results were in agreement with the results of intrinsic viscosity (Table 1). Similar results in case of temperature effect was reported by Razavi et al. [9]. Mohammad Amini and Razavi [11] showed that  value of Balangu seed gum more affected by Na+ than Ca2+ . It was also found that SSG macromolecule is more sensitive to increase in temperature than Balangu seed gum.

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4. Conclusion

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In this work, the influence of monovalent (Na+ and K+ ) and divalent ions (Mg2+ and Ca2+ ) as well as different temperature consisting 25, 45 and 65 ◦ C on dilute solution properties of SSG have been studied. It was found that Higiro model (Eq. (7)) was the

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Q4

576