rheological properties of xanthan-sodium caseinate mixtures

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Rheological Properties and Phase Separation of Xanthan-Sodium CaseinateMixtures Analyzed by a Response Surface MethodAbdelkader HadjSadok a; Nadji Moulai-Mostefa b; Mounia Rebiha a

a Institut de Chimie Industrielle, Université de Blida, Route de Soumaa, Blida, Algeria b LPTRR,Université Yahia Fares de Medea, Ain D'Heb, Medea, Algeria

Online publication date: 03 March 2010

To cite this Article HadjSadok, Abdelkader, Moulai-Mostefa, Nadji and Rebiha, Mounia(2010) 'Rheological Properties andPhase Separation of Xanthan-Sodium Caseinate Mixtures Analyzed by a Response Surface Method', InternationalJournal of Food Properties, 13: 2, 369 — 380To link to this Article: DOI: 10.1080/10942910802532531URL: http://dx.doi.org/10.1080/10942910802532531

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International Journal of Food Properties, 13: 369–380, 2010Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print / 1532-2386 onlineDOI: 10.1080/10942910802532531

369

RHEOLOGICAL PROPERTIES AND PHASE SEPARATION OF XANTHAN-SODIUM CASEINATE MIXTURES ANALYZED BY A RESPONSE SURFACE METHOD

Abdelkader HadjSadok1, Nadji Moulai-Mostefa2, and Mounia Rebiha1

1Institut de Chimie Industrielle, Université de Blida, Route de Soumaa, Blida, Algeria2LPTRR, Université Yahia Fares de Medea, Ain D’Heb, Medea, Algeria

Effects of xanthan and sodium caseinate concentrations on the rheological properties oftheir mixture in an aqueous medium were investigated at neutral pH. It was deduced fromthe use of an experimental design methodology, the existence of a critical concentration ofsodium caseinate, which depends on the xanthan quantity, and beyond which repulsive seg-regation interactions occur, generating a weakening of the elastic modulus and an embit-terment of the colloidal system structure. In addition, it was observed that when the Cassonviscosity value of the aqueous solution was above 0.1 Pa.s, phase separation of the systemwas observed. The phase diagram of the solution was established using a polynomial fit.

Keywords: Xanthan, Sodium caseinate, Rheology, Interactions, Response SurfaceMethod.

INTRODUCTION

Proteins and polysaccharides are commonly used in many food industries becauseof their potentiality to improve texture, stability and dietetic aspect of the products withcomplex structure, in particular the food emulsions where both biopolymers are present.[1,2]

The proteins are used for their surface properties, to stabilize the oily droplets, whilepolysaccharides are usually added to increase the viscosity of the continuous phase. In anaqueous medium, phase separation due to the thermodynamic incompatibility results inmutual repulsive interactions of segregation of the two biopolymer macromolecules. Inthis case, the polysaccharide-polysaccharide or protein-protein interactions are suffi-ciently high in comparison to the biopolymer-solvent interactions.[3] Conditions of incom-patibility depend on several factors, in particularly, temperature, pH, ionic force,concentration and conformation of biopolymers.[4,5] On the other hand, in the case of floc-culation by depletion, the non-adsorbed polymer is excluded from the internal space of thetwo colloidal particles, as their surface of separation becomes smaller than the polymerchain size.[6] An osmotic pressure difference is then created and consequently flocculation

Received 30 March 2008; accepted 7 October 2008.Address correspondence to Nadji Moulai-Mostefa, LPTRR, Université Yahia Fares de Medea, Ain D’Heb

26001 Medea, Algeria. E-mail: [email protected]

Downloaded By: [Mostefa, Moulai] At: 09:01 4 March 2010

370 HADJSADOK, MOULAI-MOSTEFA AND REBIHA

of the two particles is observed. In the case where the inter-colloids attraction interactionis significant, the system can record a phase separation.[7]

The proteins of sodium caseinate are a mixture of four caseins. They are amphiphilicand anionic macromolecules (above isoelectric point) and can form aggregates in an aque-ous medium by hydrophobic interactions, in the form of sub-micelles.[8] It was shown, bymeans of the multiangle laser light scattering that sodium caseinate solutions not purified(with its lipidic matter) had a radius gyration (Rg) values ranged from ∼50 to 120 nm.[9]

Recently, by dynamic light scattering, we have found that sub-micelles of sodium casein-ate (purified and in presence of 100mM of NaCl), have a dynamic radius of about11 nm.[10]

The xanthan gum is an anionic natural biopolysaccharide, produced by the Bacte-rium of Xanthomonas Campestris and made up of various monosaccharides, mannose,glucose, and glucurinic acids. The macromolecules of xanthan can have the conformationof a simple, double or triple helicoidally flexible chains,[11] which are able to interactbeyond a critical concentration of 0.005 % approximately and to form aggregates startingfrom 0.07 %.[12] Thus, this biopolymer has the capacity to increase the viscosity of thesolution.[13]

Knowledge of rheological behavior of food preparations based on soluble biopoly-mer content is quite important in quality control, storage and processing. The rheologicaland structural properties of protein–polysaccharide mixtures depend on biopolymer inter-actions that can be influenced by the concentration and molecular structure of biopoly-mers.[14,15] Knowledge of the types of interactions that occur in polysaccharide–proteinsystems is important for providing optimum food quality and for the design of new prod-ucts with attractive structures and textures. Recent studies on the aqueous mixture contain-ing sodium caseinate (NaCN) and xanthan, at neutral pH, were carried out by usingapparent viscosity, confocal microscopy[16] and dynamic light scattering,[17] in order toinvestigate the phase behavior and the mixture microstructure. It was found that bothbiopolymers produce a phase separation, when the xanthan concentration is about 0.5%(in wt.%) with 5% of NaCN. However, the authors did not determine the phase diagram ofthe system.

In conventional multifactor design, experiments are usually carried out by varyinga single factor while keeping all other factors fixed at a specific set of conditions. It isnot only time-consuming, but also usually incapable of reaching the true optimum dueto ignoring the interactions among variables. Thus, it is desirable to develop an accept-able process in shortest possible time using minimum number of men, hours, and rawmaterials. The technique of the experimental design is an efficient method of indicatingthe relative significance of a number of variables and their interactions.[18] For thispurpose, response surface method (RSM) was proposed to determine the influences ofindividual factors and their interactive influences. RSM is a statistical technique fordesigning experiments, building models, evaluating the effects of several factors andtheir interactions.[19]

In this work, the interactions between the ingredients in sodium caseinate-xanthanmixtures by determining the rheological properties were investigated. RSM was used todetermine the effects of the two biopolymer concentrations on the rheological behavior oftheir aqueous mixture at neutral pH. In particular, investigations were focused on the neg-ative inter-biopolymer interactions, which can take place, and are generally responsiblefor phase separation. The phase diagram of the colloidal system was determined using apolynomial fit.


RHEOLOGICAL PROPERTIES OF XANTHAN-SODIUM CASEINATE MIXTURES 371

MATERIALS AND METHODS

Materials

Sodium caseinate (NaCN) was obtained from Armor Proteins (France), containing0.09 wt% calcium, 1.45 wt% sodium, 2.67 wt% phosphate, and 5.8 wt% moisture. Xanthangum was purchased from Rhodia (Algeria). All the other used chemicals were of analyticalgrade and were obtained from Sigma Chemical Co (Switzerland).

Preparation of Xanthan/Protein Mixtures

The mixtures concentrations of sodium caseinate and xanthan, in the solution, werevaried from 1–6% and from 0–0.5% (in wt.) respectively. The quantities of powder of thetwo ingredients were dissolved simultaneously under stirring in distilled water, during 20hours at room temperature. A quantity of 0.01% (in wt.) of sodium azide was added inorder to protect the mixture from microbial contamination. The pH of all samples wasadjusted to 7 by slow addition of 0.01M NaOH under continuous stirring.

Rheological Analysis

Rheological measurements were performed using a controlled stress rheometer(Paar Physica MCR 300, equipped with the US software 200), in a steady and oscillatoryshear with cone–plate geometry. The flow properties were measured by applying asequence of constant stress values to the samples and measuring the corresponding shearrate. Oscillatory stress sweep tests were always performed at a fixed frequency in order toknow the linear viscoelasticity range. For this test, the colloidal solutions were subjectedto a ramp of a small oscillatory deformation at a frequency of 10Hz and at a temperatureof 20°C. All the frequency sweep measurements were performed well within the linearrange.

Phase Separation

The phase separation of the aqueous mixture was obtained under normal conditions.The samples were put in transparent test tubes at room temperature and the phase separation(an upper phase rich in xanthan and a lower phase rich in NaCN) was detected for a periodof six months. However, in spite of the presence of sodium azide in the solutions, a beginningof mixture degradation was observed after three months. Under these work conditions, itwas not possible to measure the heights of the two phases, because they vary slowly in thetime, and thus it was not possible to know the time of their stabilization. From this analysis,a characteristic response of the system state stability (St) was obtained, St = 1, the mixtureis stable and St = 0, there is a macroscopic phase separation.

Experimental Design

The influence of varying concentrations of xanthan and NaCN was studied using inparticular a D-optimal design. The D-optimal method is relatively a new technique,related to response surface methodology, used for carrying out the design of experiments,the analysis of variance, and the empirical modeling.[20] Table 1 presents the levels of pre-dictor variables tested following D-optimal design of experiments. The design consisted



of 15 experiments including four repetitions for the calculation of the pure error. Themathematical model suggested, binds the NaCN and xanthan concentrations with therheological parameters, noted Y, and is of polynomial quadratic type. It takes into accountthe effects of both biopolymers and their interactions:

The quality of this model and its power of prediction, are related to the variance coeffi-cient, R2 and to prediction coefficient, Q2 respectively. The responses fixed as objectivesin this formulation are the rheological parameters of the Casson model (hc (Pa.s) and s0(Pa)); the elastic modulus G’0 (Pa), and the parameter of the state of stability St. Themodel of surface response corresponding to the D-optimal experimental design takes intoaccount all the principal retained factors and their interactions.

RESULTS AND DISCUSSION

Rheological Behaviour of Xanthan/NaCN Solutions

The initial part of the experimental plan was devoted to the analysis of the flowbehavior of xanthan solutions and its dependence on the protein concentration. Themechanical behavior of the solutions studied is presented in Fig. 1. The shear stress wasvaried from 1 to 1000 Pa at 20°C. A clearly non-Newtonian behavior of a shear-thinning(pseudoplastic) fluid was observed. Similar curves were obtained for all preparations. Theshear rate–shear stress data at all biopolymer concentrations were well described by thepower law model with yield stress values.

Some rheological models were tested, but did not adequately fit the flow curves,such as Oswald ( ), Herschel Bulkley ( ), Sisko( ) and generalized Casson ( ). is the shear rate and

Table 1 Experimental Matrix (Factor levels and responses).

Test NaCN (%) Xanthan (%)hc

(Pa.s)t0

(Pa) G’o (Pa) St

01 1 0 0.005 0.00 0.70 102 6 0.50 0.151 0.41 1.57 003 6 0 0.015 0.00 0.70 104 1 0.50 0.112 2.29 12.60 105 1 0.33 0.041 1.59 6.57 106 1 0.16 0.026 0.66 2.27 107 6 0.16 0.104 0.36 1.50 008 6 0.33 0.133 0.29 1.66 009 4.33 0.50 0.099 2,04 15.00 110 2.66 0.50 0.079 2.25 14.30 111 4.33 0 0.011 0.00 0.70 112 3.50 0.25 0.079 1,15 4.80 113 3.50 0.25 0.081 1.24 5.00 114 3.50 0.25 0.080 1.16 5.12 115 3.50 0.25 0.077 1.28 4.50 1

Y a a NaCN a Xant

a NaCN a Xant a NaC

1 2

112

222

12

= + +

+ + +0

NN X ant.(1)

s s g= +0 K n� , s g h g¥= +K n� � ,s g h g¥= +K n� � , s s h g g¥= + +0 � �K �g



s is the shear stress. From Fig. 1, it was noticed that the experimental data can be fitted tothe Casson model, with a satisfactory correlation (R close to 1) for all the concentrationsconsidered. The Casson model is given by the equation:

The characteristic parameters of this model are the Casson viscosity, hc obtained whenthe shear rate is infinite ( ) and the yield stress s0 obtained at .Xanthan solutions were also examined under oscillatory shear conditions (Fig. 2), inorder to define the upper limit of the linear viscoelastic range and determine themechanical spectrum for each concentration level. The experimental data obtained fromstress sweep tests performed at 10 Hz are shown in Fig. 2. A typical curve of the varia-tion of the elastic modulus, G’, according to g, is presented. G’0 represents the plateauof the curve in the range of linear viscoelasticity.

Statistical Analysis

Table 1 summarizes the characteristic parameters values of the selected testsobtained from the realization and characterization of the different mixtures. The examina-tion of these experimental values shows that the values of yield stress, so and elastic mod-ulus G’0 are strongly correlated: s0 = 0.190G’0 – 0,154; R = 0.94. Consequently, these twocharacteristics are dependent and are controlled by the same phenomena. So it is judiciousto keep only one of them (it was proposed to keep only the response G’0). The parametersof the polynomial model associated to the two characteristics (Log(G’0), hc are calculated

Figure 1 Typical rheogram and its adjustment by the Casson model, at T = 20°C[10].

0 100 200 300 400 500 600 700

σ (P

a)

0

2

4

6

8

10

12Experimental Casson

γ (s–1)•

s s h g1 201 2 1 2/ / /( )= + c � (2)

h s g g ¥c = →( / )( )� � �g = 0



by the multilinear regression method, by using the experimental values of G’0 and hc cor-responding to the concentrations of NaCN and xanthan:

Statistical testing of the model was performed with the Fisher’s statistical test for analysisof variance (ANOVA). The ANOVA of these responses demonstrated that the model ishighly significant as is evident from the value of Fstatistic (the ratio of mean square due toregression to mean square to real error), (Fmodel (a) = 29,9228 and Fmodel (b) = 13,3230) anda very low probability value (P = 0,001). Obviously, the quality of these three equations issatisfactory. This leads us to determine, by simulation, the concentration effects of eachcomponent, on the characteristics of the aqueous mixture, in the domain of study.

Influence of Xanthan and NaCN Concentrations on the Elastic Modulus

In Figs. 3 and 4, are shown the simulated curves of the effects of xanthan and NaCNrespectively on the elastic modulus (G’0). The two figures are equivalent, but allow us toappreciate the intrinsic effects of each of both substances. Figure 3 shows the effect ofxanthan at various levels of NaCN (from 1% to 6%) and experimental reference curve ofxanthan solution (CNaCN = 0%). Obviously, the effect is positive, but tends to decreasewith the high NaCN concentrations. In this case, polysaccharide loses its aptitudes of sta-bilization, and if it is used with a relatively high concentration, the aqueous mixture whichresults (tests 2, 7, and 8) develops a phase separation (St = 0). The upper phase is rich inNaCN and the lower phase is rich in xanthan. This phase separation could be caused by

Figure 2 Typical curve of variation of the elastic modulus, G’, according to g.[10]

γ0.1 1 10

G' (

Pa)

1

10

G'0

Log G’ 4 26 NaCN 4 23Xant 4NaCN

33 Na

20 0 0 0 0 0

0

( ) = − + +. . . .

.

−

− CCN Xant 2 45Xant2· − .(3)

nc

2

7 3NaCN 5Xant

1NaCN 1NaCN

= + +

+ +

0 0 0 0 0 0

0 0 0 00

. . .

. . •XXant 2Xant2− 0 0.(4)



depletion interaction, as that proposed in the case of casein-guar,[21] casein-carrageenan,[22]

casein-pectin,[23] or casein-xanthan systems.[16] However, attraction by depletion of theprotein particles generally generates a rise in the viscosity of the system.[24] In our case,phase separation was accompanied by a drastic loss of the storage modulus parametervalue (G’0). Thus, rare are the authors who worked with the NaCN-xanthan systems in anaqueous medium. Among them, Nash et al.[17] showed, by using confocal microscopy, that

Figure 3 Na-CN’s curves influence on the storage modulus (G’o) at various xanthan concentrations.[10]

NaCN (%)

1 2 3 4 5 6

G' 0

(Pa)

0

2

4

6

8

10

12

14

16

18Xanthan = 0%0.125% 0.25% 0.375 % 0.5%

Figure 4 Xanthan’s curves influence on the storage modulus (G’o) at various Na-CN concentrations.[10]

Xanthan (%)0.0 0.1 0.2 0.3 0.4 0.5 0.6

G' 0

(Pa)

0

2

4

6

8

10

12

14 NaCN = 0% 1% 2% 3% 4% 5% 6%



the NaCN-xanthan system is not controlled by the same mechanism of interaction as thatof the casein-xanthan system (depletion flocculation). According this author, in the case ofNaCN-xanthan system, the mixture appears not homogeneous and the aggregate proteinsof NaCN are not visible on the microscope. Indeed, we have showed, in a recent study[10],by using dynamic and static light scattering that the increase of the concentration ofcaseinate, does not produce an increase of the apparent hydrodynamic radius of sub-micelles and their apparent molar mass. Therefore, there is no aggregation of NaCN mole-cule due to the increase of the concentration of caseinate. It was possible to deduce that thethermodynamic incompatibility mechanism supported by the electrostatic repulsive inter-actions, is responsible for the phase separation because the two biopolymers have thesame charges.[3]

Figure 4 shows the effect of NaCN on G 0́, with various xanthan concentrations.Except for the curve associated with 0% of xanthan, all the curves are no monotonous,with an increasing section of G 0́ and another decreasing section of G 0́. Obviously, thereis a critical concentration of NaCN, from which the structure rigidity, with its storagemodulus G 0́ decreases. In this last case, the segregation interactions of xanthan-NaCNcan be occurred. The critical NaCN concentration, denoted NaCN*, corresponds to thebeginning of reduction in G 0́, and depends on the xanthan concentration. However, itshould be noticed that this concentration is not synonymous of a macroscopic phase sepa-ration of the mixture. The critical concentration, NaCN*, is associated with the optimalvalue of the polynomial expression given by the Eq. 3. Therefore:

A simple calculation shows that it is expressed as follows:

The maximum critical value of NaCN* (3.39%), associated to 0% of xanthan, correspondsto the value, to which, the least quantity of the added xanthan would give rise to negativeinteractions. Moreover, the minimal critical concentration of NaCN* (1.27%) correspondsto the quantity which can potentially develop segregative interactions with the maximumof quantity of xanthane (0.5%). Thus, For CNaCN ≤ 1.27% and 0% ≤ Cxanthan ≤ 0.5%, thereis no segregative interactions between the two biopolymers, and thus would be co-soluble.

Influence of Xanthan and NaCN Concentrations on the Casson

Viscosity

Figure 5 shows the response surfaces for Casson viscosity as a function of the xan-than and caseinate concentrations. As can be observed the Casson viscosity increased asbiopolymers concentrations increased. ANOVA applied to the results indicated thatbiopolymer concentration significantly influenced the viscosity. Tavares et al.[25] reportedthe same behavior for frozen concentrated orange juice. They found that the index consis-tency is influenced by fruit concentrate related to pectin content, fibers, and pulp content.

The limiting Casson viscosity (hc) is a parameter which explain the structural stateof the matter, when this one is in extreme flow associated to an infinite shear deformation.

¶¶

G o

NaCN

’

( )*= =0 for NaCN NaCN (5)

NaCN 4 24 Xant 3 39* . .= − + (6)



The macromolecules in suspension, under the effect of this maximum shearing, record atotal disintegration accompanied by an alignment of the chains according to the directionof shearing. The attractive or repulsive interactions under these conditions are negligiblecompared to repulsion forces of shearing. Consequently, the effect of the addition of xan-than or NaCN can be only positive on hc because the increase in the number of chains orsub-micelles and thus of obstacles in the solution, generates more resistance to the flow.However, Hemar et al.[16] noticed that the apparent viscosity at high shearing rate( >1000 S-1) of the mixture containing xanthan and NaCN is qualitatively constant,according to the addition of NaCN.

Phase Separation of the Polymeric Mixtures

In Fig. 6 is shown the dependence of the state of stability of the polymeric mixturein solution to hc. It was observed clearly that the macroscopic phase separation, one rich inxanthan and the other in NaCN, cannot take place unless the limiting Casson viscosity isbeyond a critical value, hc = 0.1 Pa.s. This critical Casson viscosity corresponds in fact tothe state where the system contains an important number of sub-micelles of NaCN andchain of xanthan. So segregative interactions become intense, and they will lead the aque-ous system to a phase separation. Thus, while replacing, in Eq. (4), the Casson viscosityhcby the critical value, noted hc* = 0.1 Pa.s, the implicit equation of the binodal line isobtained (Eq. (7)), which separates the domain on two areas: a macroscopic phase corre-sponding to a homogeneous phase and a biphasic system.

Figure 5 Na-CN’s curves influence on the Casson viscosity at various xanthan concentrations.[10]

NaCN (%)

0 1 2 3 4 5 6 7

η c (

Pa.s

)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Xanthan 0.1% 0.3%0.5%

�g

00 0 0 0 0 0 0 0

0 00.

. . . .

. •1

7 3 NaCN 5 Xant 1NaCN

1NaCN X

2

−+ + +

+ aant 2Xant

2− 0 00

.

⎛

⎝⎜

⎞

⎠⎟ = (7)



In Fig. 7 is shown the phase diagram, obtained by simulation, in which the twodomains are separated by the binodal line. It appears that the critical Casson viscosity varia-tion depends strongly on the xanthan concentration. When the xanthan quantity is relativelyhigh, the viscosity decreases and becomes sensitive to NaCN concentration. Unfortunately,there is no literature that gives the phase diagram of xanthan-NaCN systems to comparethese results.

Figure 6 State of macroscopic stability of the aqueous mixture according to the variation of the viscosity ofCasson’s model.[10]

ηc (Pa.s)

0.00 0.05 0.10 0.15 0.20

Stab

ility

0

1

Figure 7 Phase diagram of the solution mixture as a function of the simultaneous variation of Na-CN andxanthan concentrations.[10]

Xanthan %

NaC

N %

1

2

3

4

5

6

0.0 0.1 0.2 0.3 0.4 0.5

homogensystems

biphasic systemsbinodal line

0.01 Pa.s 0.04 Pa.s 0.07 Pa.s

0.1 Pa.s



CONCLUSION

In this work, effects of sodium caseinate and xanthan concentrations and their inter-actions on the rheological properties of their aqueous mixture were studied. For thispurpose, series of tests were prepared and characterized, and the results were modeled bypolynomial functions, with the assumption to consider that these effects are nonlinear andthat the interactions of order one can exist. It arises from this effect study on the rheologi-cal properties, that beyond a certain NaCN concentration which depends on the xanthanquantity used (pH 7 and 3mM of sodium azide), repulsive interactions occur and conse-quently a decrease in the storage modulus G’o is obtained. This phenomenon does notgenerate systematically a macroscopic phase separation. However, in the case where theCasson viscosity exceeds a critical value (0.1 Pa.s), segregative interactions between xan-than and NaCN inevitably will lead the system to a phase separation, one rich in NaCNand the other in xanthan. Thus, only the repulsive interactions, which are responsible forthe brittleness of the polymeric network are of thermodynamic nature, and are probablycaused by the differences on the chemical structure of the hydrocolloidal and polymericsurfactant.

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