Journal of Food Engineering 100 (2010) 435–445

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Journal of Food Engineering

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Linear viscoelasticity of carob protein isolate/locust bean gum blends

Lidia S. Zárate-Ramírez, Carlos Bengoechea, Felipe Cordobés, Antonio Guerrero *

Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, C/Profesor García González, 1, 41012 Sevilla, Spain

a r t i c l e i n f o

Article history:Received 23 December 2009Received in revised form 19 April 2010Accepted 20 April 2010Available online 23 May 2010

Keywords:Carob protein isolateLBGLinear viscoelasticitySegregative separationCLSM

0260-8774/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2010.04.028

* Corresponding author. Tel.: +34 954 557179; fax:E-mail address: aguerrero@us.es (A. Guerrero).

a b s t r a c t

Linear viscoelastic properties of locust bean gum (LBG), carob protein isolate (CPI) and LBG–CPI aque-ous dispersions were studied at different concentration and pH values. LBG and CPI dispersions showmarked different linear viscoelasticity behaviour. The former system exhibits a fluid-like behaviour forall the concentrations considered (2–4 wt.%), whereas the later shows gel-like mechanical spectra. Anincrease in linear viscoelastic functions, G0 and G00, takes place as protein concentration becomeshigher, as a consequence of a strengthening of the gel network. CPI dispersions were affected bythe pH value, in agreement with the CPI solubility profile. The behaviour observed for CPI–LBG dis-persions shows an apparent evolution with time, suggesting occurrence of thermodynamic incompat-ibility between both biopolymers that finally leads to a segregative separation of LBG-rich and CPI-rich phases. Both microstructure and viscoelastic properties of the final LBG–CPI systems are highlydependent on pH value.

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1. Introduction

Proteins and polysaccharides are among the most widely natu-ral biopolymers used in foodstuffs. Food industry typically usesone of them or their blends in order to control structure, textureand stability of a wide variety of processed foods, many of whichexhibit multiphase nature (Neirynck et al., 2004; Tolstoguzov,1991; Vega et al., 2005; Benichou et al., 2007). It was recognizedthat polysaccharides could interact at the interfaces with otherpolymers, as well as with groups residing there (protein–water,oil–water, or air–water) leading to the formation of aqueous struc-tured materials with useful viscoelastic properties under condi-tions of low shear strain (Dickinson and Pawlowsky, 1997;Galazka et al., 2000; Benichou et al., 2007). Thus, rheological prop-erties resulting from these polymer–polymer interactions consti-tute an important subject of study of hydrocolloids, as has beenrecently shown by different authors (i.e. Musampa et al., 2007;Zhu et al., 2008).

From a thermodynamic point of view, protein and polysaccha-rides may be compatible or incompatible in an aqueous solution.According to Tolstoguzov (1991), protein–polysaccharide mixturesmay lead to three different interaction patterns: co-solubility,which is limited to very dilute solutions; association or complexcoacervation; and incompatibility. In complex coacervation, asso-ciative phase separation of both biopolymers from a phase mainlycontaining solvent occurs. Coacervation phenomena are due to

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electrostatic attraction between oppositely charged biopolymers(Turgeon et al., 2003; Nayebzadeh et al., 2007). However, the mostusual pattern corresponds to thermodynamic incompatibility be-tween protein and polysaccharide molecules. Thermodynamicincompatibility occurs when the interaction between different bio-polymers is energetically less favourable than the average interac-tion between similar biopolymers. This incompatibility results inthe formation of two separated phases, each being rich in one ofthe biopolymers (Abassi and Dickinson, 2004; Dickinson and Hong,1995; Dickinson and Pawlowsky, 1997; Syrbe et al., 1998; Nayeb-zadeh et al., 2007). The incompatibility behaviour depends onthose factors that affect the behaviour of either of the two biopoly-mers in solution (i.e. ionic strength, pH, temperature, charge den-sity and biopolymer concentration) (Tolstoguzov, 1991).

Caroubin, the water-insoluble protein isolated from carob beanembryo, is a mixture composed of a large number of polymerizedproteins of different size (Wang et al., 2001). It is obtained fromthe germ of the seeds in the fruit pod of the carob tree (Ceratoniasiliqua L.), found in Mediterranean regions. This protein systemhas similar rheological properties than gluten, though caroubinpossesses a more ordered structure, with minor changes in second-ary structure when hydrated. The composition and microstructureof caroubin has been studied in a previous work (Bengoechea et al.,2008).

The carob gum (locust bean gum, LBG) is obtained by crushingthe endosperm of the seeds from the carob fruit pod. LBG was thefirst galactomannan used both industrially (paper, textile, pharma-ceutical, cosmetic and other industries) and in food products (icecream and other preparations) (Dakia et al., 2008). It is used as a

436 L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445

thickening agent in aqueous solutions. LBG chemical structure isbased on a 1,4-linked b-D-mannan backbone which is solubilizedby 1,6-linked a-D-galactose side chain residues.

Since LBG biopolymer is uncharged, the lack of attractive inter-actions between both polymers, irrespectively of the pH value, re-stricts complex coacervation between them. On the contrary,segregative separation would be expected (McClements, 2006). Inspite of this limitation, there is a strong interest in the evaluationof the potentials of protein/polysaccharide blends to improve pro-cessing, such as emulsification, and properties of the final productsuch as emulsion rheology and stability. This evaluation may becarried out by analyzing the rheological behaviour and microstruc-ture of protein/polysaccharide blends in aqueous solution.

The objective of this work was to select optimal conditions forLBG/CPI blends in aqueous systems, at different relative contentsand pH values, on the basis of the analysis of their rheological

Table 1Critical stress (sc) and strain (cc) values for stress sweep tests at 6.28 rad/s for LBG/water systems at pH 6 as a function of LBG concentration.

LBG (wt.%) sc (Pa) cc

2 2.04 ± 0.09 0.36 ± 0.032.5 17.04 ± 0.13 1.03 ± 0.043 22.79 ± 0.99 0.53 ± 0.034 75.48 ± 3.69 0.43 ± 0.02

2

0

10

20

30

40

50

60

G´ 1,G

´´ 1 / (P

a)

LBG Concentr

B

10-2 10-110-4

10-3

10-2

10-1

100

101

102

103

A

G´,G

´´/(P

a)

ω /( rad

Fig. 1. Linear viscoelastic properties for LBG–water systems at 20 �C and pH 6 as a functio(B) SAOS parameters at 1 rad/s (G01, G001 and tan d1).

properties and microstructure, which should contribute to the de-sign of continuous phases with suitable potentials to produce con-centrated food emulsions either at lab or pilot plant scale. Aprevious step would be to select the protocol of preparation forbinary and ternary systems. Therefore, both biopolymers were firststudied separately in dispersions at different concentrations andpH, comparing the results to those obtained for LBG/CPI systemsin order to identify occurrence of synergistic effects.

2. Experimental

2.1. Sample preparation

Carob flour (46% protein content) was obtained from Alimcarat(Caratina, Mallorca, Spain). A carob protein isolate (CPI, min. 96%protein content) was isolated from the carob flour applying theprocedure of alkaline solubilization followed by isoelectric precip-itation described in a previous paper (Bengoechea et al., 2008). LBGwas purchased from Sigma (Sigma–Aldrich, St. Louis, MO). Theaverage molecular weight of LBG supplied by Sigma is 310 kDa.

Samples were prepared by adding the correspondent amount ofbiopolymer to water, adjusting afterwards the pH by NaOH/HCl(2 M) addition. A mechanical stirrer (Heidolph RZR1, Germany)provided with an anchor stirrer was used at constant temperaturefor 30 min in order to obtain a homogeneous system.

2

4

6

8

10

4

G′1

G′′1

ation (%wt)

tan

δ 1

tan δ1

100 101

/s)

n of LBG content: (A) storage, G0 , and loss, G00 , moduli as a function of frequency and

L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445 437

2.2. Rheological characterization

Small Amplitude Oscillatory Shear (SAOS) measurements wereperformed in a controlled-stress rheometer (RS-100) from Haake(Germany) in the linear viscoelastic region, using plate-and-plategeometry (60 mm) with a rough surface and a gap between platesof 1 mm. Shear stress sweep tests (from 0.01 to 1000 Pa) were pre-viously performed for each sample at 6.28 rad/s in order to find thelinear viscoelastic range. Frequency sweep tests (ranging from 10to 0.02 rad/s) were carried out at a constant stress well withinthe linear viscoelastic range. At least two replicates were made.All the systems studied had the same thermorheological history.All the tests were performed at 20 �C.

2.3. Microstructural characterization

The microstructure of some selected samples was analyzed bymeans of a Confocal Laser Scanning Microscopy, CLSM (Leica

Table 2Critical stress (sc) and strain (cc) values for stress sweep tests at 6.28 rad/s for CPI/water systems at pH 6 as a function of CPI concentration.

CPI (wt.%) sc (Pa) cc

10 1.48 ± 0.03 0.15 ± 0.0211 4.26 ± 0.32 0.12 ± 0.0112.5 39.2 ± 1.60 0.47 ± 0.0315 375 ± 7.76 0.51 ± 0.01

10 12

0

200

400

600

800

G1

G1

11

G',

G''/(

Pa)

CPI concentrat

Btan

10-2 10-1100

101

102

103

104

A G' G'' % wt 10 11 12.5

G',

G''/(

Pa)

ω/(rad/

Fig. 2. Linear viscoelastic properties for CPI–water systems at 20 �C and pH 6 as a functioand (B) SAOS parameters at 1 rad/s (G01,G001 and tan d1).

Mycrosystems, Heidelberg, Germany). The CLSM was used in thefluorescent mode, and a wavelength of 488 nm from the laserwas used. Wavelengths above 500 nm were analyzed. A 100�objective was used. It was not necessary to stain the aqueous phasedue to the autofluorescent properties shown by the protein used.

3. Results and discussion

3.1. LBG/water systems

As is well known, LBG shows poor solubility in water at roomtemperature. Therefore, heat treatment is usually required to fullyhydrate LBG in water and achieve the best water binding capacity(Gainsford et al., 1986; Hui and Neukom, 1964; Maier et al., 1993).To point out the importance of heating to solubilize LBG, linear dy-namic viscoelastic properties (G00 and G0) of LBG/water systemsusing different protocols were studied (data not shown). The LBGcontent was kept constant at 2 wt.%. The lowest viscoelastic prop-erties correspond to the system prepared at room temperature, atwhich LBG is poorly hydrated. In contrast, when LBG is solved inwater at 80 �C, G0 and G00 are much higher (i.e. the viscous modulusis about 25 times higher). According to these results it is generallyaccepted that heat processing is essential for optimal performanceof LBG as a thickener agent. However, due to this difference in vis-cosity, for certain applications it may be advisable to apply heattreatment in a later stage of the process. For instance, the emulsi-fication process may be greatly benefited from the use of a low vis-

0.20

0.22

0.24

0.26

0.28

0.30

14 16

' ''

ion (%wt)

δ1

tan

δ 1

100 101

15

s)

n of CPI concentration (A) storage, G0 , and loss, G00 , moduli as a function of frequency

438 L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445

cosity continuous phase, since the energy input required could besubstantially reduced and the efficiency of the process could besignificantly improved (McClements, 2004). Once the emulsion isprepared, heat treatment may be applied to enhance emulsionthickness. A further benefit may be achieved by following this pro-cedure, since heat treatment may also contribute to reinforceemulsion microstructure by means of heat-induced protein dena-turation and aggregation. In fact, this procedure has been previ-ously used to enhance protein-based emulsion stability(Dickinson and Hong, 1995; Moros et al., 2003; Romero et al.,2009). Both linear viscoelastic functions may reach the level thatcorresponds to the fully hydrated LBG system either by preparing

Table 3Critical stress (sc) and strain (cc) values for stress sweep tests at 6.28 rad/s for 10 wt.%CPI/water systems as a function of pH.

pH sc (Pa) cc

2 33.6 ± 1.52 0.34 ± 0.012.7 2.50 ± 0.10 0.20 ± 0.016 2.01 ± 0.03 0.27 ± 0.027.2 53.3 ± 1.61 0.57 ± 0.038 205 ± 1.87 1.35 ± 0.2910 217 ± 15.94 2.15 ± 0.17

1 2 3 4 5 6

20

40

60

80

100

120

140

160

180

pH

10-2 10-1100

101

102

103

G' G'' pH 2 2.7 6 7.2 8

G',

G''/(

Pa)

ω /(rad/s

A

B

11

G',

G''/(

Pa)

Fig. 3. Linear viscoelastic properties of 10 wt.% CPI/water systems at 20 �C and different pparameters at 1 rad/s (G01, G001 and tan d1).

the mixture at high temperature or by applying heat treatmentafter mixing at room temperature (data not shown). Thus, these re-sults indicate that the order of the heat treatment stage may beconveniently changed in any sequential process without producingany further modification in LBG functionality. Accordingly, a low-temperature protocol for preparation of LBG/water dispersionshas been selected in this work.

Table 1 shows the critical stress and strain values for the onsetof non-linear viscoelasticity obtained from stress sweep tests as afunction of LBG concentration. Systems containing LBG at concen-trations below 2 wt.% undergo phase separation and consequentlywere not evaluated by rheological measurements. A higher stress isrequired to disrupt the structure as concentration is increased,whereas the critical strain undergoes a slight decrease.

Mechanical spectra obtained from frequency sweep tests forgum dispersions at different LBG concentrations (2, 2.5, 3 and4 wt.%), pH 6 and prepared at room temperature, are shown inFig. 1A. The loss modulus, G00, is always higher than the elasticmodulus, G0, in the whole frequency range considered. In this fig-ure, the viscous component shows a lower slope (close to 1) thanthe elastic component at the low frequency region. This behaviouris typical of low-concentrated macromolecular solutions showingan apparent fluid character (i.e. relaxation time values lower than0.1) with a tendency to a crossover between G0 and G00 in the high

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

7 8 9 10

G1' G1''

tan

δ 1

tan δ1

100 101

10

)

H values: (A) storage, G0 , and loss, G00 , moduli as a function of frequency and (B) SAOS

L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445 439

frequency regime. Thus, this crossover is observed in Fig. 1A for thesystem containing 4% LBG. Similar results were obtained by Oaken-full et al. (1997).

Fig. 1B shows the values of G0, G00 and tan d at 1 rad/s (G01, G001 andtan d1) obtained at different LBG concentrations. The values of G01and G001 follow an exponential growth with LBG concentrationwhereas tan d1 continuously decreases. This evolution indicatesthat the elastic response tends to overcome the predominantly vis-cous character of LBG dispersions as the concentration is raised,which subsequently leads to an enhancement in the stabilityagainst phase separation.

3.2. CPI/water systems

3.2.1. CPI concentration effect studyCPI/water systems were prepared at room temperature to avoid

heat-induced denaturation. Preliminary studies for CPI/water dis-persions at pH 6 evidenced an apparent phase separation at CPIconcentrations below 10 wt.%. These studies also showed that CPIdispersions were difficult to prepare and homogenize at CPI con-tent higher than 15 wt.%. The CPI concentration range consideredin the present study is therefore 10–15 wt.%.

Fig. 4. Microscope visible (LM) images of CPI/water systems (10 wt.% CPI) atdifferent pH values: (A) pH 2, (B) pH 6, and (C) pH 8.

According to Table 2, stress and strain critical values tend to in-crease with CPI concentration, resulting in a wider linear viscoelas-tic region. These results suggest an increase in structural resistanceof the system with protein concentration.

Fig. 2A shows the results obtained from frequency sweep testsfor different CPI dispersions at pH 6. The mechanical spectra ob-tained for all the CPI dispersions prepared describe a gel-likebehaviour where G0 shows higher values than G00 in the experimen-tal frequency window.

Fig. 2B shows the evolution of G0, G00 and tan d values at 1 rad/sfor different CPI concentrations. Parameters G01 and G001 undergo aremarkable increase with CPI concentration. A decrease in tan d1

values is also induced by a rise in the CPI content. Therefore, theevolution of linear viscoelastic properties with CPI content revealsa reinforcement of the gel network formed among proteinsegments.

3.2.2. Influence of pHTable 3 shows the values of the critical stress and strain that de-

limit the linear viscoelasticity range for aqueous dispersions con-taining 10 wt.% CPI at different pH values (from 2 to 10). Anincrease in pH leads to a U-shaped evolution for either of thetwo critical parameters, being very similar to the CPI solubility pro-file reported in a previous paper (Bengoechea et al., 2008). In fact,near the isoelectric point, at which the minimum in CPI solubilitywas previously obtained (pH �4), CPI/water dispersions undergophase separation and therefore the viscoelastic properties of thesystem could not be measured.

Fig. 3A represents the mechanical spectrum of 10 wt.% CPI dis-persions at different pH values. The storage modulus G0 is alwayshigher than the loss modulus G00 within the experimental fre-quency range, both linear viscoelastic functions showing a moder-ate dependence on frequency as corresponds to a gel-likebehaviour. Fig. 3B shows the evolution of parameters G01, G001 andtan d1 obtained from the mechanical spectrum at 1 rad/s, as afunction of pH. An increase in the pH value, from pH 2, leads toa dramatic reduction of both parameters G01, G001, as well as an in-crease in the loss tangent, which eventually gives rise to phaseseparation in the proximity of the isoelectric point. A further in-crease in pH induces a recovery in the stability of CPI/water sys-tem, which is apparent at pH 6, as well as in the linearviscoelastic properties. Thus, either of the two parameters G01and G001 passes through a peak value around pH 8, while the losstangent reaches a minimum value, which corresponds to a max-imum elastic character for CPI/water dispersions. At pH 10 anew reduction in G01, G001, and a further increase in tan d1 takesplace. Again, this behaviour is quite similar to that one foundfor the CPI solubility curve.

This behaviour may be explained in terms of the balance offorces among protein molecules which in turn depends on thepH of the aqueous media. At pH near the isoelectric point, thenet charge is approximately zero; leading to a lack of electrostaticinteractions that favours protein aggregation and gives rise to a de-crease in effective solubility and viscoelastic properties. When thepH value departs from the isoelectric point, the subsequent elec-trostatically-driven increase in protein solubility leads to an in-crease in viscoelastic properties. As may be observed in Fig. 4,which shows LM images for 10 wt.% CPI/water systems, anenhancement in the gel-like microstructure of the system takesalso place, either by reducing pH (Fig. 4A) or by increasing pH(Fig. 4C). In both cases, a fairly homogeneous microstructure isapparent, in contrast to the rather heterogeneous microstructurefound near the pI (Fig. 4B).

Therefore, the above mentioned results and discussion puts for-ward a direct relationship between protein solubility and visco-elastic properties. However, viscoelastic properties do not depend

10-2 10-1 100 101 10210-1

100

101

102

103 C

G' G''

G' G'' t(h) 0 3 6 24 96168264

G´,G

´´ / (

Pa)

ω / (rad/s)

10-2 10-1 100 101 10210-2

10-1

100

101

102

103

B

LBG

CPI

G' G''

G´ G´´ t(h) 0 3 6 24 96 168 264

G´,

G´´

/ (Pa

)

ω / (rad/s)

10-2 10-1 100 101 10210-1

100

101

102

103

G' G'' LBG

CPI

A G' G'' t(h)

0 3 6 24 96264

G´,

G´´/

(Pa)

ω / (rad/s)

Fig. 5. Mechanical spectra of CPI/LBG blends (10 wt.% CPI, pH 6) at different LBG concentrations. (A) 2 wt.%, (B) 3 wt.% and (C) 4 wt.%. Spectra of the correspondent CPI/waterand LBG/water systems are also included.

Table 4Sinergistic parameter (S0(x)) and Grunberg–Nissan interaction parameter, GLBG–CPI,values for LBG/CPI blends containing 10 wt.% CPI as a function of frequency (x) andLBG content.

LBG (wt.%) S0(x) GLBG–CPI

Frequency, x (rad/s) Frequency, x (rad/s)

0.1 (%) 1 (%) 10 (%) 1

2 1099 1159 1002 20.73 392 310 56 9.24 308 53 �63 6.6

440 L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445

only on protein solubility since the peak in both viscous and elasticparameters takes place at pH 8, lower than that one correspondingto the maximum protein solubility (pH 10). In addition, it shouldbe taken into account that an increase in electrostatic interactions(i.e. caused by similarly charged protein surfaces found at high pH)may leads to inhibition of hydrophobic interactions. This effectmay play also an important role since it might restraint proteinaggregation to a high extent, leading to a weakening of the gel-likenetwork. This fact may explain the behaviour found at pH higherthan 8.

3.3. CPI/LBG/water systems

3.3.1. Influence of LBG contentAgeing time did not produce any significant change on linear

viscoelastic properties of CPI or LBG aqueous dispersions. On theother hand, CPI/LBG blends exhibit a remarkable evolution alongtime in aqueous media. This effect may be deduced from Fig. 5 thatplots the mechanical spectra obtained for three different LBG con-centrations (pH 6) as a function of ageing time. The mechanicalspectra obtained for their corresponding CPI/water and LBG/watersystems are also included in Fig. 5 as a reference. G0 values are gen-erally higher than G00 for any of the three LBG concentrations stud-ied. Both linear viscoelastic functions undergo a remarkabledecrease with time showing an asymptotic tendency towards anequilibrium value at long time. However, the kinetics of this evolu-tion slows down with an increase in LBG content. These resultssuggest that the influence of protein on the linear viscoelasticbehaviour of the system prevails. This predominant effect rein-forces with time as deduced from the decrease of the slopes ofeither G0 and G00-frequency curves. The shape of the initial spectrasuggests an incompatibility between both biopolymers, which isrelatively frequent in the literature of polymer blends (Fitzsimons

102 103 104 105 106 107

101

102

103

103 104 105 106

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1B 2% LBG

3% LBG 4% LBG

tan

δ 1

t / (s)

A G' G'' % LBG 2 3 4

t / (s)

11

G',

G''/(

Pa)

Fig. 6. Evolution of SAOS parameters with time for ternary (CPI/LBG/water) systems with different LBG contents: (A) parameters G01, and G001 and (B) parameter tan d1.

L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445 441

et al., 2008; Quiroga and Bergenstahl, 2008). However, LBG/CPI/water systems eventually reach a well defined gel-like behaviour,characteristic of an elastic network, which is very similar to thatone shown by CPI/water systems.

In order to analyze the synergistic effects taking place at longtime, a synergistic index may be defined for the elastic componentas follows:

S0ðxÞ ¼ G0MðxÞ � ðG0CPIðxÞ þ G0LBGðxÞÞ

ðG0CPIðxÞ þ G0LBGðxÞÞð1Þ

where G0MðxÞ is the storage modulus obtained at long time for LBG/CPI blends. The values obtained for this synergistic parameter at dif-ferent frequencies are shown in Table 4.

A remarkable synergistic effect takes generally place. This effectis more relevant at the lowest LBG concentration, where no signif-icant influence of frequency takes place and the viscoelastic re-sponse is clearly dominated by CPI. A further increase in LBGcontent produces a reduction in the synergistic parameters andthe effect of frequency becomes more pronounced, leading to areduction in S0. Actually, the highest values for LBG concentrationand frequency lead to an apparent inversion in the synergistic ef-fect. This negative value for S0 may be caused by the fact that G0

for 10 wt.% CPI/water system is much lower than the value ob-tained for 4 wt.% LBG/water system, that in fact corresponds tothe crossover point of a Maxwellian behaviour. However, themechanical spectrum of the LBG/CPI ternary system corresponds

to a gel-like behaviour which indicates that the predominant effectof the protein component still remains.

In order to contrast the synergistic index defined, the interac-tion parameter, Gij, from the Grunberg–Nissan equation (Grunbergand Nissan, 1949) was also estimated from the following equation:

lngm ¼Xn

i¼1

xi ln gi þ12

Xn

i¼1

Xn

j¼1;j–i

xixjGij ð2Þ

where m is the liquid mixture viscosity (mPa s), i is the viscosity ofthe ith component (mPa s), xi and xj are the molar fractions of theith and jth components, Gij is the interaction parameter (mPa s),and n is the number of pure components in the mixture. The valuesobtained for this interaction parameter, GLBG–CPI, (calculated at 1 Hzfor an average molecular weight for the protein) are also shown inTable 4. A similar evolution in S0() and GLBG–CPI can be observed.

Fig. 6 shows the evolution of parameters G01, G001 and tan d1 withageing time for ternary systems at three different LBG concentra-tions. Fig. 6A confirms the above mentioned asymptotic evolutionfor the viscoelastic moduli, represented by parameters G01 and G001,as well as the decrease in kinetics taking place as LBG concentra-tion is raised. Thus the asymptotic value is reached at much shortertime for the lowest LBG content. It should be noticed that this is thesystem that also exhibits a remarkable synergistic effect with thehighest final values for G01, G001 and the lowest tan d1, as may be ob-served in Fig. 6B. In fact, the final values for the loss tangent areeven lower than those obtained for the corresponding CPI binary

10-2 10-1 100 101 10210-2

10-1

100

101

102

103

C

LBG

CPI

G´ G´´

G´ G´´ t(h) 0 1 24 72

G´,G

´´/(P

a)

ω/(rad/s)

10-2 10-1 100 101 10210-2

10-1

100

101

102

103

B

LBG

CPI

G´ G´´

G' G'' t(h) 0 3 6 24 96 264G

´, G

´´/ (P

a)

ω / (rad/s)

10-2 10-1 100 101 10210-2

10-1

100

101

102

103 A

LBG

CPI

G´ G´´

G´ G´´ t(h) 0.1 1.3 2.9 24 48

G´,G

´´/(P

a)

ω/(rad/s)

Fig. 7. Mechanical spectra of CPI/LBG blends (10 wt.% CPI, 2% LBG) at different pH values: (A) pH 2, (B) pH 6 and (C) pH 8. Spectra of the correspondent CPI/water and LBG/water dispersions are also included.

Table 5Sinergistic parameter (S0(x)) and Grunberg–Nissan interaction parameter, GLBG–CPI,values for LBG/CPI blends containing 10 wt.% CPI and 2 wt.% LBG as a function offrequency (x) and pH.

pH S0(x) GLBG–CPI

Frequency, x (rad/s) Frequency, x (rad/s)

0.1 (%) 1 (%) 10 (%) 1

2 398 485 735 18.46 1099 1159 1002 20.78 32 42 49 6.1

442 L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445

system, within the whole frequency range. Addition of LBG to theCPI/water dispersion always produces the same evolution ontan d1. This evolution consists of an initial increase up to a maxi-mum value followed by a decrease with a tendency towards anequilibrium value, at least at the lowest LBG content. The increasein LBG content leads to a displacement of the maximum value to-wards longer times. This evolution may be attributed to two differ-ent effects. The first one is the increase in viscous character

contributed by LBG, which is more important at the highest LBGcontent. On the other hand, a segregative separation of a CPI-richphase from another LBG-rich phase depleted in protein seems totake place at longer times, being more evident at higher LBG con-tent. Segregative separation is typically produced as a consequenceof a relatively strong repulsion between protein and polysaccha-ride, usually due to a steric exclusion mechanism, that may occurwhen one of the biopolymers is uncharged, as it is the case forLBG (McClements, 2006).

3.3.2. Influence of pHFig. 7 shows the evolution of the mechanical spectra over stor-

age time obtained for 10 wt.% CPI, 2 wt.% LBG aqueous dispersionsat three different pH values. The mechanical spectra for the disper-sions containing either of the two components, at the same condi-tions (pH and concentration), are also shown as a reference. As maybe observed, the evolution after the protein–hydrocolloid mixingstage shows a quite different behaviour depending on the pH va-lue. At low pH, not significant evolution of the mechanical spec-trum can be noticed within 48 h, showing a gel-like behavioursimilar to that found for the CPI dispersion. However, an apparent

L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445 443

decrease in G0 and G00 takes place along time at pH 6. At this pH,which is not far from the isoelectric point, the contribution of elec-trostatic interactions can be neglected and the system exhibits thelowest values of linear viscoelastic functions. The evolution of theprotein–polysaccharide mixed dispersion at pH 8 is different, sincean enhancement of G0 (and a decrease in G00) takes place over stor-age time leading to an extension of the plateau region.

All the CPI/LBG dispersions show a positive synergism for bothviscoelastic moduli, as may be observed in Table 5. The synergisticeffect is less pronounced at pH 8 at which the gel-like viscoelasticbehaviour is more similar to that found for the corresponding CPI/water system. The most remarkable synergistic effect for the elas-tic component corresponds to the closest value to the isoelectricpoint (pH 6).

The evolution of some linear viscoelastic parameters is shownin Fig. 8 as a function of time, for the three pH values studied.Fig. 8A displays the results obtained for G01 and G001. The higher val-ues of both parameters correspond always to pH 2, and the lowestto the pH close to the pI. The CPI/LBG blend prepared at pH 8 showsan intermediate behaviour (although closer to the one found at pH6). Fig. 8B shows the values of the loss tangent and the slope for theelastic modulus (n0). It may be pointed out that the evolution of theloss tangent and parameter n0 are also dependent on the pH value.Thus, an increase in both parameters with time is shown in Fig. 8Bfor the CPI/LBG blend at the lowest pH value, whereas a clear de-

103 1040.1

0.2

0.3

0.4

0.5

0.6B

tan

δ, n

'

t

102 103 1101

102

103

A

G´ G´´ pH 2 6 8

G1´,G

1´´/(P

a)

t

1

Fig. 8. Evolution of SAOS parameters with time at 1 rad/s for ternary systems (10 wt.% CPand n0 (slope of G0 vs. frequency).

crease takes place at pH 6 and 8. Moreover, these two systemseventually develop lower values for the loss tangent and for theslope n0, which is indicative of an enhancement of gel-like networkmicrostructure. The above mentioned extension of the plateau re-gion described for the blend prepared at pH 2 may be clearly ob-served in this figure.

In addition, all the dispersions show slopes for G0 vs. frequencythat are intermediate between the protein-gel (showing values be-tween 0.1 and 0.14, depending on pH) and the polysaccharide sys-tem (with a value of 1.3), being clearly closer to the former,particularly at pH 6 and 8. These results suggest that CPI contrib-utes to the gel-like network structure to a higher extent thanLBG. However LBG molecules produce a certain degree of perturba-tion depending on the pH value.

Fig. 9 shows CLSM and LM images obtained for CPI/LBGaqueous systems for the three pH values studied at long storagetime. At pH 6, which is relatively close to the isoelectric point,the micrographs shown in Fig. 9B reveal a high degree of pro-tein aggregation related to a limited presence of charges at pro-tein surfaces. As a result, the system resembles a dispersion oflarge random aggregates in a continuous LBG phase. Fig. 9Csuggests that an increase up to pH 8 produces an inversion ofthe system, giving rise to a completely different scenario wheresome LBG-rich regions are randomly distributed within the CPImatrix.

105 106

tan δ n' pH 2 6 8

/(s)

04 105 106

/(s)

I, 2% LBG) at different pH values: (A) parameters G01, and G001 and (B) parameters tan d1

Fig. 9. CLSM and LM images of CPI/LBG//water systems (10 wt.% CPI, 2% LBG) at different pH values: (A) pH 2, (B) pH 6 and (C) pH 8.

444 L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445

At pH 2 (Fig. 9A), the hydrophobically-driven tendency of pro-tein molecules to aggregate is limited by electrostatic interactionsamong the charged protein surfaces. As a result, protein aggregatesare much smaller than those formed at pH 6. Moreover, the abovementioned segregative separation due to steric exclusion is re-duced, leading to a stronger mixed gel with higher viscoelasticfunctions.

4. Concluding remarks

With the experimental protocol used in this study, LBG/watersystems exhibit a fluid-like rheological behaviour typical of a dilute

polymer solution, whereas CPI/water dispersions show a gel-likebehaviour that is related to the development of a protein network.Linear viscoelasticity functions of CPI/LBG/water blends also dis-play a gel-like behaviour showing a gel strength that depends onLBG content and pH. An apparent evolution of both moduli withtime takes place at pH close to the isoelectric point, showing a ten-dency towards equilibrium values. These results suggest that ther-modynamic incompatibility between both biopolymers occurs,leading to a segregative separation of a CPI-rich phase from an-other LBG-rich phase depleted in protein. The kinetics of this evo-lution slows down with an increase in LBG content. A remarkableresult is the occurrence of a maximum in tan d1 at intermediate

L.S. Zárate-Ramírez et al. / Journal of Food Engineering 100 (2010) 435–445 445

ageing time, that even leads to a critical gel behaviour at the high-est LBG content (where tan d is close to unity at the whole fre-quency window). An important technological consequence maybe extracted from this result, as the time for this maximum maybe considered optimal for carrying out some processing that in-volve a mixing stage such as emulsification. Current studies carriedout by our research group are focused on this possibility.

An apparent decrease in viscoelasticity with time takes place atpH 6 and 8. At pH 6, at which the lowest values of G01 and G001 arefound, the system resembles a dispersion of random protein aggre-gates in a continuous LBG phase. An increase up to pH 8 producesan inversion of the system, giving rise to a dispersion of LBG intothe CPI matrix (the development of the gel network after the inver-sion process takes certain time to be completed). At pH 2, at whichno significant evolution of viscoelastic parameters can be noticed,electrostatic interactions leads to formation of smaller aggregates.The above mentioned segregative separation due to steric exclu-sion is reduced, leading to a stronger mixed gel with higher visco-elastic functions.

In summary, for the experimental conditions selected in thiswork (i.e. low-temperature protocol) the protein dominates thebehaviour of the final ternary systems that show mechanical spec-tra similar to those found for CPI/water dispersions and LBG onlymodulate this behaviour depending on LBG/CPI ratio and pH.Although the role of LBG as a thickener agent does not seem tobe very remarkable when a low-temperature protocol for LBG/water systems is used, it is reasonable to expect that this factmay change if a heat treatment of the emulsion is performed afteremulsification. This potential improvement in emulsion rheology,microstructure and stability should be explored in the near future,as well as the use of a high-temperature protocol for LBG/watersystems to allow for complete polysaccharide hydration.

Acknowledgements

This study was supported by MEC/FEDER (research projectAGL2007-65709) and Junta de Andalucía (excellence project P08-AGR-03974). The financial support is gratefully acknowledged.

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