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Food Hydrocolloids 28 (2012) 248e257

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Food Hydrocolloids

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Viscoelastic behavior and microstructure of aqueous mixtures of cross-linkedwaxy maize starch, whey protein isolate and k-carrageenan

Alejandra Espinosa-Dzib a, Mariana Ramírez-Gilly b, Alberto Tecante b,*

a Programa de Maestría y Doctorado en Ingeniería, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, D.F., 04510, MexicobDepartamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México, Cd. Universitaria, D.F., 04510, Mexico

a r t i c l e i n f o

Article history:Received 14 May 2011Received in revised form30 November 2011Accepted 20 December 2011

Keywords:StarchWhey proteinsCarrageenanMicroscopyRheologyGels

* Corresponding author.E-mail address: tecante@unam.mx (A. Tecante).

0268-005X/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.foodhyd.2011.12.011

a b s t r a c t

The viscoelasticity and microstructure of mixtures of cross-linked waxy maize starch (CH10), wheyprotein isolate (WPI) and k-carrageenan (kC) at pH 7.0 with 100 mM NaCl were investigated by oscil-latory rheometry and confocal laser scanning microscopy (CLSM). Mixtures were heated to 90 �C (1.5 �C/min), held for 10 min at this temperature and cooled. Within the range of concentrations studied, CH10swollen granules reinforced WPI and kC networks. The mechanical behavior of the three-componentmixtures was modified by different WPI concentrations, but kC governed the overall response due toits gelling ability. CLSM images of three-component mixtures showed particulate systems in whichswollen starch granules are surrounded by kC and WPI. CH10 granules were immersed in a single phaseand a separate phase of kC and WPI, for low and high concentrations of these components, respectively.Therefore, it is possible to obtain two- and three-phase mixtures.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Starch is widely used in prepared foods as a thickening andgelling agent. Cross-linked waxy maize starch (XLWMS) is essen-tially amylopectin (<1% amylose), and chemical modificationmakes it highly resistant to thermal and mechanical treatments(Loisel, Fayolle, Cantoni, & Doublier, 1997). The cooked pastes ofXLWMS are two-phase suspensions of swollen granules dispersedin an aqueous phase, which show remarkable shear-thinning andsolid-like viscoelastic behaviors influenced by the volume fractionand deformability of the swollen granules (Abdulmola, Hember,Richardson, & Morris, 1996; Nayouf, Loisel, & Doublier, 2003;Steeneken, 1989; Tecante & Doublier, 1999). Cross-linked starchesare used in pie fillings, breads, puddings, baby foods, soups, sauces,gravies, salad dressings, retorted foods and aseptically processedfoods (Hirsch & Kokini, 2002).

Whey protein isolate (WPI) is also an important ingredient inmany food applications, such as processed meats, bakery products,ice creams, confections, infant foods, pastas, spreads, dips andbeverages, because of their nutritional value and functional prop-erties as emulsifying, foaming, water-holding, thickening andgelling agent (Fitzsimons, Mulvihill, & Morris, 2008; Vu Dang,Loisel, Desrumeaux, & Doublier, 2009). WPI is a high protein

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material (>95%) that contains mainly b-lactoglobulin (more than70% of total protein) and a-lactalbumin (about 15 to e20% totalprotein); minor amounts of bovine serum albumin, immunoglob-ulins, lactose, minerals and fat are also present (Fitzsimons et al.,2008; Vu Dang et al., 2009). The thermal gelation of WPI globularproteins involves a denaturation-unfolding step followed byprotein aggregation, which leads to three-dimensional networkformation through hydrophobic and hydrogen bond interactionsand disulphide bridge formation (Vu Dang et al., 2009). Aggrega-tion depends on pH, type and concentration of ions present in theaqueous phase and WPI concentration. At pH 7, heat-denatured b-lactoglobulin, the major component of WPI, can form aggregateswhich have a broad size distribution. At low ionic strength,formation of larger aggregates is impeded by electrostatic repulsion(Le Bon, Nicolai, & Durand, 1999).

Kappa carrageenan (kC) is a sulfated polysaccharide extractedfrom red seaweeds used in many food products as a gelling agent.The high sensitivity of kC to potassium ions leads to a variety ofsystems, solutions and gels, with a wide spectrum of rheologicalbehaviors (Núñez-Santiago, Tecante, Garnier, & Doublier, 2011).Gelation of kC in the presence of potassium ions is thermo-reversible and involves a two-step solegel transition mechanismin which there is a coilehelix conformational change followed byaggregation of helical structures when hot kC solutions are cooled(Rochas & Rinaudo, 1984). kC can also form thermo-reversible gelswith sodium ions, although gels with sodium are weaker than

A. Espinosa-Dzib et al. / Food Hydrocolloids 28 (2012) 248e257 249

those with potassium for the same level of both ions. kC may notform gels at sodium ion concentrations for which it does withpotassium ions (e.g., 10 mM). The mechanism of gel formation inthe presence of sodium ions is different from potassium ions; kCaggregation occurs without previous conformational transition.Gelation would be the result of phase separation into zones rich inkC concentration and zones with kC concentration below theaverage. Aggregation of kC leading to gel formation would occur inthe rich zones. The potassium to sodium ion concentration ratioalso has an effect on gelation of kC and on the strength of its gels(Mangione et al., 2005).

Proteins and polysaccharides are present in many food systems,and both types of macromolecules contribute to the structure,texture and stability of foods (Doublier, Garnier, Renard, & Sanchez,2000). Consequently, investigation of their possible interactions isimportant to understand the behavior of complex multi-component systems. Studies involving starch, WPI and kC arerestricted to two-component systems made up of combinations ofany two of these ingredients. For example, mixtures of kCeWPIhave garnered considerable attention (de Jong, Klok, & van deBelde, 2009; de la Fuente, Hemar, & Singh, 2004; Flett & Corredig,2009; Gaaloul, Corredig, & Turgeon, 2009; Gaaloul, Turgeon, &Corredig, 2009; Leng & Turgeon, 2007; Mleko, Li-Chan, & Pikus,1997; Turgeon & Beaulieu, 2001). The same is true for starchekCand starcheWPI mixtures (Shim & Mulvaney, 2001), but thosespecifically involving XLWMS are considerably less abundant(Fitzsimons et al., 2008; Ravindra, Chan, Lee, & Upender, 2007;Ravindra, Chan, & Upender, 2007; Ravindra, Genovese, Foegeding, &Rao, 2004; Tecante & Doublier,1999; Vu Dang et al., 2009). A reviewcovering different aspects of interactions and rheological propertiesof starcheWPI mixtures was published several years ago (Ravindra,Chan, Lee, et al., 2007; Ravindra, Chan, & Upender, 2007). Althoughstudies with mixtures containing tapioca starch, carrageenan andwhey protein concentrate added to low-fat pork sausages (Lyons,Kerry, Morrissey, & Buckley, 1999) and with skimmed milkproteins, k-carrageenan, adipate-substituted waxy maize starch,sucrose and water present in sterilized dairy desserts (Verbeken,Bael, Thas, & Dewettink, 2006) have been reported, the rheolog-ical and microstructural properties of mixtures ofXLWMSeWPIekC have not been investigated.

The aim of this research was to study the viscoelastic propertiesand microstructure of aqueous mixtures of XLWMS, WPI and kC,using oscillatory shear and confocal laser scanning microscopy(CLSM), respectively, to understand the way these macromolecularcomponents interact in the presence of external sodium ions. Theimportance of this study lies in the wide use of these macromole-cules as ingredients normally added to meet desired functions orproperties in prepared foods.

2. Materials and methods

2.1. Materials

Cross-linked adipate and stabilized acetate waxy maize starch(Clearam CH10�) was supplied by Roquette Frères (Lestrem, France);its moisture content was 9.20%. Whey protein isolate (WPI) waspurchased from Nutrical (Mexico), and its moisture and proteincontents were 5.50% and 95.0% as determined by oven drying at100 �C and macro Kjeldahl analysis (N x 6.38), respectively. The ioncontents (mg/kg) of WPI from atomic absorption analysis wereNaþ¼ 749.2, Kþ¼ 3868, Mg2þ¼ 474.1 and Ca2þ¼ 2328. Food gradek-carrageenan (kC) was kindly provided by Cargill Texturizing Solu-tions (Baupte, France); its moisture content was 5.20%. It contained(mg/kg) Naþ¼ 15807 and Kþ¼ 64423. Fluorochromes (Sigma-Aldrich,Mexico) and other chemicals for CLSM included 8-anilino-1-

naphtalene sulfonic acid (ANS) for WPI and fluorescein iso-thiocyanate (FITC) for CH10 and rhodamine B isothiocyanate (RITC)for kC, as well as dimethyl sulfoxide (DMSO, Sigma-Aldrich, Mexico),dibutiltin-dilaurate (DBTDL, Sigma-Aldrich, Mexico), absoluteethanol (J.T. Baker, México) and acetone (J.T. Baker, Mexico). Labelingwas carried out following established protocols (Núñez-Santiagoet al., 2011; Vu Dang et al., 2009). Other materials included bluedextran (Mol. wt. 2�106, Sigma-Aldrich, Mexico), reagent gradesodium chloride (Sigma-Aldrich, Mexico) and sodium azide (Sigma-Aldrich, México). Deionized water was used throughout.

2.2. Preparation of samples

The concentrations (wt%) were: 1.0, 2.0, 3.0 and 4.0 for CH10;0.5, 1.0, 2.0, 3.0, 3.5 and 3.9 for WPI; and 0.25, 0.50, 0.75 and 1.0 forkC. WPI solutions were prepared by dispersing the powder inwaterunder strong mechanical stirring. Once dispersed, milder stirringwas continued for 1 h. Sodium chloride was added to a concentra-tion of 100 mM, and the pH was adjusted to 7.0 with 0.1 M NaOH.The liquid was left overnight at 4 �C to completely hydrate anddissolve the WPI. In the two-component mixtures containing WPI,the other componentwas either slurried (CH10) or dispersed (kC) inthe WPI solution. In CH10ekC mixtures, the starch was slurried inthe kC dispersion. In the three-component mixtures, kC wasdispersed completely in the WPI solution and then CH10 wasslurried in the WPIekC mixture. All of the aqueous systems wereprepared at 25 �C under mechanical stirring with the same NaClconcentration and pH of individual WPI solutions. The thermaltreatment included heating from 25 to 90 �C at 1.5 �C/min, holdingat 90 �C for 10 min and cooling to 25 �C at the same rate. First, thesample was heated from 25 to 65 �C under magnetic stirring ina jacketed stainless steel vessel fitted to a programmable circulatorbath (Polystat, Cole-Parmer, USA), and once at 65 �C magneticstirring was stopped and the sample was transferred to cap-screwed aluminum molds (25 mm radius, 10.5 cm long). Thisprocedure avoided sedimentation of starch granules. Then, themolds were heated from 65 to 90 �C in the circulator bath and thethermal treatment continued as described. At the end of thethermal treatment, the aluminum molds were kept at 4 �C over-night until further testing. This procedurewas used to ensure starchgelatinization, protein denaturation and carrageenan dissolution.

2.3. Solubility, swelling, particle size and particle size distribution ofstarch granules

The swelling capacity (g/g) and solubility (%)were determined inquadruplicate. A known mass of the hot starch paste was dilutedwith solvent at 60 �C to a final concentration of 0.5 wt%. Aftercooling, 8 mL of the dilutionwas centrifuged at 2200 rpm for 10 min(Leach, McCowen, & Schoch, 1959), and another 8 mL portion wasdried overnight at 100 �C to obtain the drymass of starch. Solubility,S, was referred to the mass of dry starch in the total volume ofsupernatant (Doublier, 1987), which was quantified by thephenolesulfuric method (Dubois, Gilles, Hamilton, Rebers, & Smith,1956). As this method quantifies total sugars, S was corrected whenrequired by a blank with deionized water. Swelling of starch gran-ules, G, was calculated as reported by Nayouf et al. (2003). A 0.01%blue dextran solution was prepared in 100 mM NaCl. Seventy-fivegrams of cooked CH10 was mixed with 25 g of blue dextran solu-tion in a flask and stirred. Ten grams of thismixturewas centrifugedfor 15 min at 250 rpm (Hermle Z200A, Labnet, Germany) to sepa-rate starch granules and the concentration of blue dextran in thesupernatant was determined by reading the maximum absorbanceat 620 nm in a spectrophotometer (Genesys 10 UV, USA). Thevolume fraction of swollen granules, 4, was calculated from

Table 1Average median diameter (D[v, 0.5]), swelling power (SP), and size distributionrange (SDR) for CH10 pastes.

CH10 (wt%) D[v, 0.5] (mm) SP SDR (mm)

1.0 43.3� 0.151 23.3� 0.177 4.37e1052.0 40.3� 0.176 18.8� 0.184 5.75e1203.0 40.2� 0.035 18.6� 0.007 5.01e91.24.0 40.4� 0.098 18.9� 0.056 5.01e91.2

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4¼ [1� (S/100)]$C$G (Doublier, 1987), where S is solubility, C is theinitial starch concentration and G is granule swelling.

Particle size and the particle size distribution were determinedat least in triplicate at room temperature in aMalvernMastersizer S2000 laser diffraction analyzer with a size range of 0.02 to 200 mm(Malvern Instruments, Ltd. UK.), including the Small VolumeSample Dispersion Unit (SVSDU) (Hydro 200 MU). At the end ofpasting, a given volumewas dispersed in the SVSDU to carry out themeasurements. This technique is based on Mie’s theory, andparticle sizes are given by reference to equivalent spheres (MalvernInstrument Ltd., 1990). We chose the median volume-baseddiameter, D[v, 0.5], to allow comparison with reported data(Ziegler, Thompson, & Casasnovas, 1993). This diameter representsthe size at which 50% of particles by volume are smaller and 50% arelarger. The refractive index of starch and solvent was 1.5295 and1.3300, respectively, with an adsorption of 0.1 for the former(Tecante & Doublier, 1999). Data are presented as swelling power(SP) and size distribution range (SDR) for 1.0e4.0% CH10 pastes.SP¼ (Dmax/Di)3, i.e. the cube of the ratio of the maximum diameterto the initial diameter (Ziegler et al., 1993). The values of SP werecalculated using D[v, 0.5] and the initial diameter of the uncookedpastes (15.2 mm) (Núñez-Santiago & Tecante, 2007). The SDR is theminimum and maximum values of granule diameter.

2.4. Rheological measurements

The viscoelastic behavior was characterized using small-amplitude oscillatory shear tests. Measurements were made in anARES-RFS III rheometer (TA Instruments, USA) using the serrated-surface parallel plates fixture. Gels were cut into disks (25 mmdiameter, 2.5 mm thickness) and placed onto the bottom plate. Thegap between plates was 2.5 mm, and any excess sample wastrimmed. The fixture rimwas covered with mineral oil to minimizemoisture loss during the test. Strain sweeps at 25 �C and 6.28 rad/swere run to determine the region of linear viscoelasticity.Frequency sweeps at 25 �C were run over an angular frequencyrange of 0.1e100 rad/s for different strains depending on samples,but within the linear region. Temperature sweeps were run at anangular frequency of 6.28 rad/s as follows: (1) temperature increaseat a rate of 1.5 �C/min from 25 to 90 �C at 50% strain; (2) 10 min at90 �C at 30% strain; (3) cooling at the same rate of heating from90 �C to 5 �C at 20% strain; (4) 10 min at 5 �C at 5% strain. Differentstrains were used to work within the zone of linear viscoelasticity.

2.5. Confocal microscopy

Covalent labeling of kC with RITC was carried out using theprocedure described by Núñez-Santiago et al. (2011). One gram ofkC was dissolved in 50 mL of DMSO with magnetic stirring at roomtemperature for 15 min. Then 1.5 mL of pyridine, 0.06 g of RITC and50 mL of DBTDL were added, and the mixture was heated withcontinuous stirring for 2 h at 40 �C. After this time, the labeled kCwas precipitated with ethanol (95%), filtered through a porous(40e60 mm) membrane (Millipore, USA), dehydrated with absoluteethanol and acetone to remove the free dye and finally dried ina vacuum oven (Barnstead, Lab-line, USA) at 30 �C for 24 h. Thedried kC-RITC was dissolved in water at 80 �C for 15 min, dialyzed(Spectra/Por 28.6 mm, Spectrum Medical Industries, Inc. USA)against deionized water with 0.02% sodium azide to preventmicrobial growth, lyophilized and stored at 4 �C. The fluorochromewas excited at 568 nm and emission recorded at 580e600 nm.

WPI was non-covalently labeled as follows; 0.02 g of ANS wasdissolved in 100 mL water and stirred for 5 min. 50 mL of thismixture was added to 100 mL of WPI solution. ANS was excitedusing an UV laser at 364 nm and its emission recorded at 450 nm.

CH10 was non-covalently labeled with FITC; 0.02 g of the fluoro-chrome was dissolved in 10 mL of a 50/50 (DMSO/H2O) solution.50 mL of this solution was added to the starch suspension, and thestarch was pasted. FITC was excited at 488 nm, and its emissionwasrecorded at 565 nm. When the mixtures reached 65 �C, a samplewas placed on the slide, and the cover slip was sealed with nailenamel and brought to 4 �C overnight. Observations were carriedout on an Olympus FV1000 confocal microscope (Olympus, Japan)using an argon/krypton laser and an oil-immersed 40x objective.

2.6. Phase separation

Mixtures of WPI and kC were centrifuged to detect phaseseparation. Stock solutions of both components were mixed atdifferent ratios at pH 7.0 and thoroughly stirred at room tempera-ture in cap-screwed glass test tubes. The tubes were subjected tothe thermal treatment described previously and held at 90 �C for10 min without stirring. Then the mixtures were transferred whilehot to centrifuge tubes and centrifuged (Hermle Z200A, Labnet,Germany) at 5000 rpm for 30 min.

2.7. Statistical analysis

Significant differences between experimental data were deter-mined by a three-way ANOVA followed by residual analysis by theDurbineWatson test using the software Stat Graphics Centurion XVfor Windows.

3. Results and discussion

3.1. Solubility, swelling, particle size and particle size distribution ofstarch granules

Starch solubility ranges from 2.16 to 2.60%, and granule swellingis 22.8� 4.07, 20.2� 3.89, 22.6� 0.410 and 20.5� 0.026 g/g, for 1.0,2.0, 3.0 and 4.0% CH10, respectively. The corresponding volumefractions are 0.23, 0.37, 0.55 and 0.74. Therefore, 4.0% starch pastesare concentrated particle dispersions and 1.0 and 2.0% starch pastesare diluted dispersions. The particle size distributions of swollenstarch granules aremonomodal (not shown) and broad as shown bythe SDRs in Table 1. This table shows also the median diameter, D[v,0.5], and swelling power (SP) for all the CH10 pastes. The mediandiameters for 2.0, 3.0 and 4.0% CH10 are not significantly different(p> 0.05); however, they are significantly different (p< 0.05) withrespect to 1.0% CH10. This difference of about 6% inD[v, 0.5] between1.0% CH10 and the other starch concentrations could be due to thefact that when starch concentration is low and the volume fractionis small, there is more water available for granule swelling.

3.2. Viscoelastic behavior and microstructure of two-componentmixtures

Fig. 1 shows the variation of G0 at 6.28 rad/s with WPI concen-tration for different CH10 concentrations. Three main conditionsare observed. First, in the absence of CH10, gels are not formed

Fig. 1. Variation of G0 at 6.28 rad/s with WPI concentration for CH10 concentrations of:0 (circles), 1.0 (squares), 2.0 (triangles), 3.0 (inverted triangles) and 4.0% (diamonds).Lines are included as a guide.

Table 2One phase (þ) and two-phase (þþ) WPI-kC mixtures.

WPI (%)

kC(%) 0.50 1.0 1.5 2.0 2.5 3.0 3.5 3.90.15 þ þ þ þ þ þ þ þ0.25 þ þ þ þþ þþ þþ þþ þþ0.50 þ þ þþ þþ þþ þþ þþ þþ0.75 þ þþ þþ þþ þþ þþ þþ þþ1.0 þ þþ þþ þþ þþ þþ þþ þþ

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below 2.0% WPI, whereas above this concentration self-standinggels are obtained. Second, swollen starch granules act as reinforc-ing particles but the effect is different depending on CH10 and WPIconcentrations; for 1.0 and 2.0% starch, below 2.0%WPI, rheologicalsynergism (Abdulmola et al., 1996; Dickinson & Chen, 1999; Morris,1998) occurs leading to increases in G0 of about two to four loga-rithmic decades with respect to individual components. Above 2.0%WPI, G0 increases just one to two decades with respect to individualcomponents, but no significant difference exists between G0 ofmixtures with 1.0 and 2.0% CH10 in the range of 3.0e3.9% WPI.Finally, for 3.0 and 4.0% CH10, the reinforcing effect of starchgranules is still present, but G0 increases moderately with WPIconcentration. This can be attributed to the high volume fraction ofstarch. Starch granules are surrounded by protein aggregates, butabove 2.0% WPI the rigidity of the gels is mainly provided by theprotein matrix because an increase in CH10 from 3.0 to 4.0% resultsin G’ values of the same order of magnitude. Fig. 2 shows the CLSM

Fig. 2. CLSM image of a 1.0% CH10þ 2.0% WPI mixture. Dark zones correspond tountagged CH10 granules and blue zones correspond to WPI non-covalently taggedwith ANS. (For interpretation of the references to color in this figure legend, the readeris referred to the web version of this article.)

image of a 2.0%WPIþ 1.0% CH10mixture. Dark zones correspond tountagged starch granules, and blue zones indicate WPI non-covalently tagged with ANS. The surface of swollen granules isnot smooth but convoluted so that cavities are formed. Thesecavities are occupied by WPI, which explains why blue proteinzones are surrounded by dark zones, corresponding to non-fluorescent starch granules.

Table 2 shows the concentrations ofWPI and kC for which phaseseparation is observed. kC and WPI concentrations of 0.15% and1.5%, respectively, are included only to obtain a more completephase separation scheme. For 0.15% kC, one phase solutions areobtained irrespective of WPI concentrations. As kC concentrationincreases, two-phase systems are formed at lower WPI concen-trations. Thus, phase separation occurs in mixtures having 1.0% ofboth components. Fig. 3 shows the variation of G0 at 6.28 rad/s withWPI concentration for different kC concentrations. Referring toTable 2, it is possible to identify the concentrations for whichsingle- and two-phase gels are obtained. The presence of kC resultsin gel formation irrespective of WPI concentration. In single phasegels, e.g. 0.25% kC with less than 2.0% WPI, noticeable increases inG0 with respect to individual WPI occur. Such increases are lower intwo-phase gels, e.g. 0.25% kC with 2.0e3.9% WPI. Subsequentincreases in kC concentration results in additional increases in G0

with respect to individual WPI and mixtures with 0.25% kC.However, for 0.5e1.0% kC, G0 increases moderately within the entirerange of WPI concentration. A greater amount of kC, weakens thecontribution of WPI to the rigidity, G0, of the mixture. This behaviorresults from the ability of kC to form gels, even at 0.25%. kC gelationresults from its interaction with external sodium counterions andcations already present in the commercial preparation. The rigidity

Fig. 3. Variation of G0 at 6.28 rad/s with WPI concentration for kC concentrations of:0 (circles), 0.25 (squares), 0.5 (triangles), 0.75 (inverted triangles) and 1.0% (diamonds).Lines are included as a guide.

Fig. 4. Variation of G0 (Pa) during heating (continuous line, temperature profile) for3.9% WPI (a); 3.9% WPIþ 0.25% kC (b); 3.9% WPIþ 0.50% kC (c); 3.9% WPIþ 0.75% kC(d); 3.9% WPIþ 1.0% kC (e).

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of the mixtures was governed by kC, because of the low degree ofinteraction with whey proteins due to phase separation underneutral pH conditions. This leads to enhanced protein aggregationresulting in stiffer interconnected networks with each macromol-ecule in its own domain. Fig. 4 shows the variation of G’ withtemperature for 3.9% WPI gels and their mixtures with differentconcentrations of kC. The gelation temperature, Tgel, taken as thatfor which tan d¼ 1 was about 76 �C for individual WPI, but the

Fig. 5. CLSM images of WPI and kC and colocaliz

presence of kC accelerates aggregation of WPI decreasing Tgel up toabout 70 �C for mixtures with 1.0% kC. Below this temperature,a large increase in G0 (and G00, not shown) with a maximum at about40 �C occurs for mixtures with 0.75 and 1.0% kC. This can beattributed to kC dissolution followed by a decrease in viscosity onheating (Tecante & Doublier, 1999). This maximum is not observedfor low kC concentrations, i.e. 0.25 and 0.5%. According to Table 2,phase separation occurs for all mixtures with 3.9%WPI irrespectiveof kC concentration. Between 40 �C and Tgel, protein gelation occurswith the effect of increasing G0. During the isothermal period at90 �C, G0 tend to reach a constant value. When mixtures are cooledto 30e40 �C, G0 (and G00) increase further due to kC gelation.Therefore, carrageenan gels separate of WPI in its own domain.Fig. 5 shows the colocalized image of a 2.0% WPIþ 0.75% kCmixture, which according to Table 2 forms a two-phase system.Protein aggregates and kC can be observed. Gaaloul, Corredig, et al.(2009), Gaaloul, Turgeon, et al. (2009) obtained the phase diagramfor WPIekC mixtures. However, the thermal treatment conditionsand concentrations of components are very different from ours,which yields significantly different results.

Fig. 6 shows the variation of G0 at 6.28 rad/s with CH10concentration for different kC concentrations. Two-phase systemsare formed in which swollen granules are surrounded by kC andreinforce the kC network. The viscoelastic properties of suchcomposite gels depend essentially on 4 of the swollen granules(Tecante & Doublier, 1999). Fig. 7 shows the CLSM images of CH10,kC and a 1.0% CH10 and 0.75% kC mixture. The starch granules lookgreen as expected, but they also look red when the fluorochromefor kC was excited. The colocalized image looks yellowish, indi-cating that tagged kCmight be adsorbed onto the surface of swollenstarch granules or in the vicinity around them. However, this isconsistent with the rheological behavior of this mixture.

ed image of a 2.0% WPIþ 0.75% kC mixture.

Fig. 6. Variation of G0 at 6.28 rad/s with CH10 concentration for kC concentrations of:0 (circles), 0.25 (squares), 0.50 (triangles), 0.75 (inverted triangles) and 1.0% (dia-monds). Lines are included as a guide.

Fig. 7. CLSM images of CH10, kC and colocalized image of a 1% CH10þ 0.75% kC mixture. SwCH10þ 0.75% kC mixture. (For interpretation of the references to color in this figure legend

A. Espinosa-Dzib et al. / Food Hydrocolloids 28 (2012) 248e257 253

3.3. Viscoelastic behavior and microstructure of three-componentmixtures

Fig. 8(a) and (b) shows the viscoelastic behavior of three-component mixtures containing 1.0% CH10, 0.75% kC and 2.0 and3.5% WPI, respectively. Increasing WPI concentration from 2.0 to3.5% does not change noticeably the viscoelastic behavior of thegels. The dynamic moduli increases about 10% and gels with 3.5%WPI are slightly more elastic (lower tan d) over a large interval ofangular frequency. According to Table 2, phase separation occursfor these kC and WPI concentrations. The mixtures are three-phase systems in which gelatinized starch granules are sur-rounded by a two-phase medium of kC and WPI. The viscoelasticbehavior of each one of the mixtures shown in Fig. 8a andb corresponds well to a strong gel with G0 >G00 over the entirefrequency range and tan d< 0.1. Some of the two- and all of thethree-component mixtures display such behavior. In three-component mixtures, kC forms gels regardless of its concentra-tion, while WPI forms either aggregates or gels, i.e., networks ofaggregates, depending on its concentration. CH10 does not formgels, but swollen granules which have the effect discussed inprevious paragraphs.

ollen starch granules appear green and kC appears red. Colocalized CLSM image of a 1%, the reader is referred to the web version of this article.)

Fig. 8. Variation of dynamic moduli with angular frequency for 1.0% CH10þ 2.0%WPIþ 0.75% kC (a); 1.0% CH10þ 3.5% WPIþ 0.75% kC (b). G0 (black symbols); G00 (whitesymbols).

Fig. 9. Variation of G0 at 6.28 rad/s with kC concentrations for different WPI concen-trations; 0.5% (circles), 2.0% (triangles), 3.5% (inverted triangles) and 3.9% (stars), and1.0% CH10 (a); 2.0% CH10 (b); 4.0% CH10 (c).

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In three-component systems, it is possible to represent thecontribution of each component to the overall viscoelasticresponse of the mixture in several ways. In this paper, the analysisis performed considering the effect of kC because this componentexerts the most significant influence; i.e., the overall response ofthe mixtures is governed by kC, as its effect is stronger than thoseof CH10 and WPI. Fig. 9 shows the variation of G0 at 6.28 rad/s forWPIekC mixtures with different CH10 concentrations. Thisfrequency was selected because it is a good compromise betweenlow and high values and because it is representative of the overalltrend of the viscoelastic behavior of the mixtures. The overalltrends for the loss moduli of the mixtures are essentially thesame; therefore, the analysis is performed considering only thevariation in G0.

The G0 values of WPIekC mixtures with 1.0% CH10 are shown inFig. 9(a). In the absence of kC, the G0 values correspond to the two-component mixtures containing 1.0% CH10 and different concen-trations of WPI. These values were lower than those correspondingto mixtures containing kC. For example, for the 1.0% CH10þ 0.5%WPImixture,G0 was about 0.50 Pa. Addition of 0.25% kC increasedG0

to about 20 Pa, and the modulus gradually increased with theincrease in kC concentration such that for 1.0% kC, G0 was about4000 Pa. Addition of WPI resulted in a significant increase in thesemoduli. For example, by reference again toG0 ¼ 20 Pa for themixture1.0% CH10þ 0.5% WPIþ 0.25% kC, the mixture with 1.0%

CH10þ 3.9%WPIþ 0.25% kC had aG0 of about 500 Pa, because at thisWPI concentrationwheyproteins formagel. In theabsenceof kC, thevalues of G0 corresponds to those for the two-component mixtureswith 1.0, 2.0 and 4.0% CH10 and different WPI concentrations. The

Fig. 10. Individual and colocalized CLSM images for 1.0% CH10þ 2.0% WPIþ 0.75% kC (a); 1.0% CH10þ 3.5% WPIþ 0.75% kC (b). S indicates untagged swollen starch granules.

Fig. 11. Variation of G’(Pa) during heating (continuous line, temperature profile) for1.0% WPIþ 0.25% kC (a); 4.0 CH10%þ 1.0% WPIþ 0.25% kC (b); 4.0% CH10þ 2.0%WPIþ 0.75% kC (c); 4.0% CH10þ 3.0% WPIþ 0.75% kC (d); 4.0% CH10þ 3.9% WPIþ 1.0%kC (e).

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convergence of G0 for the different WPI concentrations for 1.0% kCconcentrations can be explained by the gelation of kC. In this region,kC governs the rigidity of the gels over WPI.

On the other hand, 2.0% CH10 shown in Fig. 9(b), representsa boundary value between diluted and semi-diluted CH10 disper-sions. As a consequence, 2.0% CH10 might represent a transitionvalue with respect to starch concentration, below which thevolume fraction is so low that addition of even small amounts of kCand WPI have a tremendous effect on the moduli for the three-component mixtures. This increase could be associated withphase separation that occurs between kC and WPI before gelationof WPI as discussed in reference to Fig. 4.

Fig. 9(c) shows the effect of a further increase to 4.0% CH10. Inthis case, in the absence of kC, G0 corresponds to the two-component mixtures with 4.0% CH10 and different WPI concen-trations. Taking into account that WPI does not form gels forconcentrations of 0.5 and 1.5%, the increase in G0 could be attrib-uted to the reinforcing effect of kC. However, addition of 0.25% kCto 3.0 and 3.9% WPI, results in a storage modulus very close to thatfor the two-component mixtures. The contribution of WPI to therigidity of the mixtures is somehow weakened by kC. Addition of0.25% kC to CH10eWPI mixtures with 4.0% starch concentrationsdoes not enhance the rigidity over that of the two-componentsystems. The same effect is gradual for 2.0 and 3.0% CH10 (datanot shown). For 0.5, 0.75 and 1.0% kC, increases in WPI concen-tration do not enhance the rigidity of the gel substantially. For 4%CH10, G0 values are almost independent of WPI concentration. It islikely that the high volume fractions of swollen granules hindercomplete formation of kC and WPI networks or interrupts theircontinuity.

Fig. 10(a) and (b) shows CLSM images for three-componentmixtures with 1.0% CH10, 0.75% kC, and 2.0% and 3.5% WPI,respectively. Swollen starch granules (S) were embedded in a two-phase medium formed by WPI and kC. In Fig. 10(a), aggregates of

WPI and kC are present. However, for 3.5%WPI, shown in Fig. 10(b),the phasewas predominantly formed by a protein network, with kClikely reinforcing it. Aggregates are appreciated in the colocalizedimages. Therefore, high concentrations of WPI produce mixtures inwhich the interaction between starch and WPI prevail over theinteraction between starch and kC. Visually, three-component gelswere whitish and consisted of well dispersed starch granules sur-rounded by the protein gel with visible kC gel “lumps” of differentsizes entrapped and dispersed all over the complete volume of the

Fig. 12. Mixtures after thermal treatment and centrifugation: 1.0% WPIþ 0.25% kC (a); 4.0% CH10þ 1.0% WPIþ 0.25% kC (b); 4.0% CH10þ 2.0% WPIþ 0.75% kC (c); 4.0% CH10þ 3.0%WPIþ 0.75% kC (d); 4.0% CH10þ 3.9% WPIþ 1.0% kC (e).

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sample. The size of theWPI aggregates is limited by the presence ofswollen starch granules (S) and the association between kC chains;this effect is promoted by the total content of sodium and potas-sium ions present in WPI and kC in addition to the ionic environ-ment of the solution (100 mM NaCl). Mangione et al. (2005) affirmthat if the concentrations of potassium and sodium are high enough(20 mM/100 mM), a synergistic behavior between the two ions willexist. For all of our three-component mixtures, the ratio of [Kþ]/[Naþ] was less than 0.33 which is the threshold suggested byMangione et al. (2005); therefore, the gelation mechanism is gov-erned by the sodium ion.

Swelling of starch granules can also be affected by the avail-ability of water. The studied three-component mixtures arecomplex because of the simultaneous presence of aggregatedparticles of protein, self-associated chains of kC and swollen starchgranules. Each component contributes to the macroscopic visco-elastic response of the mixtures, but their contribution depends ontheir relative concentration in the mix. Interactions between WPIaggregates and kC; WPI and swollen CH10 granules; and kC withswollen CH10 granules can exist. These can also contribute to theviscoelastic behavior. kC has a larger effect on the rigidity of the gelsthanWPI, evenwhen the concentration of the latter is high enoughfor proteins to form gels, and starch granules reinforce the gelmatrix either when WPI and kC form a single phase or when theyconstitute a two-phase medium. Sodium ions are required by wheyproteins to diminish electrostatic repulsions, but they are alsorequired to stabilize the structure of kC.

To understand the observed differences in viscoelastic behavior,CLSM observations were performed to characterize the phases of thecomposite gels by labeling the components. It was possible to visu-alize the kC-rich zones in the mixture. In some cases CH10 was nottagged, while covalent labeling of kC was always required. ANS isknown to become fluorescent in the UV light range when it isadsorbedontohydrophobic zones. This allowsproteins tobeobservedwithout covalent labeling. kCwas covalently labeledwith RhodamineB so that the three-componentmixtures could be observed. Fig. 10(a)and (b) shows that starch granules (S) conserved their integrity, anda two-phase medium is formed by WPI aggregates and kC.

On the other hand, the variation of G0 with temperature isshown in Fig.11 for differentWPI concentrations. A big difference instorage modulus exists between two-component mixtures of 1.0%WPIþ 0.25% kC (a) and three-component mixtures containing 4.0%CH10 plus 1.0% WPIþ 0.25% kC (b), 2.0% WPIþ 0,75% kC (c), 3.0%WPIþ 0.75% kC (d) and 3.9% WPIþ 1.0% kC (e). The maximum G0

due to kC occurs at around 38 �C, whereas Tgel for WPI, about 74 �C,is very near CH10 gelatinization. During cooling kC gelation takesplaces around 21 �C. The reinforcing effect of swollen starch gran-ules in the mixtures is noticeable. The storage modulus alsoincreases because of the WPI concentration. This increase can beattributed to a segregative phase separation as can be observed inthe images shown in Fig. 12. The mixture 1.0% WPIþ 0.25% kC doesnot exhibit phase separation. Addition of 4.0% CH10 to this mixtureproduced a two-phase system in which swollen granules wereembedded in a single continuous phase. All of the other three-component mixtures resulted in three-phase systems in whichCH10 was surrounded by two-phase media consisting of WPI andkC. In these three-phase mixtures, phase separation between WPIand kC probably occurs before starch gelatinization and WPI gela-tion with kC gelation taking place upon cooling from about 21 to35 �C, depending of its concentration in the mixtures.

4. Conclusions

Cross-linked waxy corn starch, whey proteins and k-carra-geenan form two- and three-phase mixtures with a wide range ofviscoelastic behaviors. Swollen starch granules are embedded ina single phase ofWPI and kCwhen the concentration of protein andcarrageenan is approximately below 2.0% and 0.50%, respectively.Above these concentrations, phase separation occurs between WPIand kC leaving starch granules immersed in a two-phase medium.The role of CH10 granules is to reinforce the rigidity of thesurrounding phase. In general, kC governs the rigidity of gelsalthough in some cases WPI exerts also a weaker influence. Theratio of [Kþ]/[Naþ] is under 0.33 and confirms that for the majorityof the mixtures containing WPI and kC, gelation of the latter isgoverned by sodium ions. The occurrence of phase separation

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between whey proteins and kC has a determinant influence on themicrostructures of the mixtures, and plays an important role intheir final mechanical behavior. This information could be usefulfor a better understanding of the role played by each of thesemacromolecular components in the actual application and devel-opment of novel foods, in which all of them are present as part ofthe main ingredients.

Acknowledgments

We appreciate financial support from DGAPA-UNAM (Grant IN208609). Alejandra Espinosa-Dzib wishes to acknowledge theConsejo Nacional de Ciencia y Tecnología (CONACYT, México) forthe scholarship (177364) granted and the Programa de Maestría yDoctorado en Ingeniería-UNAM for additional financial support.Thanks are also due to Karina Jiménez-Durán for her technicalsupport with CLSM, Professor E. Jaime Vernon-Carter for giving usaccess to the Malvern Master SizerS analyzer, Margarita Portillo-Bauza for the ion content analysis by atomic absorption andRodrigo Orozco-Alcántara for taking the photographs of themixtures.

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