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HYDROCOLLOIDS
Food Hydrocolloids 21 (2007) 765775 www.elsevier.com/locate/foodhyd

FOOD

Acid-induced gelation of milk protein concentrates with added pectin: Effect of casein micelle dissociation
Lara Matia-Merino1, Harjinder Singh
Riddet Centre, Massey University, Private Bag 11 222, Palmerston North, New Zealand Received 16 May 2006; accepted 4 December 2006

Abstract The dynamics of the formation of the acid gel network for mixtures of milk protein concentrate (MPC) and low methoxyl amidated (LMA) pectin were studied using rheological measurements. The results as a function of pectin content and casein micelle integrity, from neutral pH to approximately pH 4.2, together with the microstructural changes observed in some of these systems, are presented. The gelation proles of a mixture of 4% w/v MPC and LMA pectin (00.075% w/v) after the addition of 1.2% w/v glucono-d-lactone showed a gradual decrease in the shear modulus with the incorporation of pectin. The effects of casein micelle integrity on caseinpectin interactions were studied, by preparing MPC dispersions containing various levels of micellar casein. A gradual change in the shear modulus, from a disrupting effect of pectin added to MPC, in which the casein micelles are intact, to a clear synergistic effect of pectin added to dissociated casein systems, was found in the acid-induced milk gels. r 2007 Elsevier Ltd. All rights reserved.
Keywords: Acid milk gels; Pectin; Milk protein concentrates; Rheology; Calcium; CSLM

1. Introduction The rheology and the structure of milk protein gels have been the subject of many studies during the last decade, as recently reviewed by a number of authors (Lucey, 2002; van Vliet, Lakemond, & Visschers, 2004). Whereas the aggregation and gelation of casein micelles as a result of acidication have been studied frequently (Lucey & Singh, 2003), the presence of hydrocolloids during the acidinduced gelation presents another degree of complexity of milk protein systems that needs to be further explored. The kinetics and the dynamics of the formation of casein gels can be controlled by varying the amount of glucono-dlactone (GDL) to produce different rates of acidication (Horne, 2001). The rst stages of acidication are critical for the development of the network and the aggregation process appears to be more complicated than simply
Corresponding author. Tel.: +64 6 350 4401; fax: +64 6 350 5655.

E-mail address: H.Singh@massey.ac.nz (H. Singh). Current address: Institute of Food, Nutrition and Human Health, Palmerston North, Massey University, New Zealand.
1

reaching a critical pH for the collapse of the hairy k-casein layer on the micelles, as believed until recently (De Kruif, 1997). Pectin is an anionic carboxylated polysaccharide that is frequently used in the food industry. The proportion of carboxyl groups esteried with methanol and with acid amide units and the ester distribution along the polymer determine the functionality of pectin (May, 2000; Morris, 1998; Voragen, Pilnik, Thibault, Axelos, & Renard, 1995). Pectin is widely used in many dairy products as a gelling/ thickening agent (acid and non-acid milk desserts) and as a stabilizer ingredient (acid milk drinks, milk/juice blends). In particular, the gelation of low methoxyl pectin, over a wide range of pH and solids content, is mainly the result of strong interactions between calcium ions and blocks of galacturonic acid (Braccini & Perez, 2001). Previous rheological and microstructural studies have shown that low methoxyl amidated (LMA) pectin is a challenging polysaccharide to investigate when it is present during the acidication of casein-based systems (MatiaMerino, Lau, & Dickinson, 2004). The fact that (1) ionic calcium is released into the serum phase during the

0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.12.007

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acidication of casein micelles (Law & Leaver, 1998; Singh, Roberts, Munro, & Teo, 1996 ), creating favourable gelling conditions for the pectin, and (2) adsorption of pectin around the casein particles through electrostatic interaction seems to occur at or below pH 5.0 (Tuinier, Rolin, & de Kruif, 2002) makes casein micelles and pectin an interesting biopolymer mixture that could result in different gel networks upon acidication. Through its role in the ionization of the protein and pectin molecules, pH is the most signicant factor affecting the electrostatic proteinpectin interactions. At neutral pH, where both casein micelles and pectin carry negative charges, the interaction is minimal (Oakenfull & Scott, 1998) and the non-adsorbing pectin induces depletion occulation above a certain critical concentration. At a lower pH, when the pectin is adsorbed onto the casein micelles, the stability of the system may vary. Depending upon the amount of pectin, bridging occulation and depletion occulation can also induce phase separation of a casein and pectin mixture (Maroziene & de Kruif, 2000; Syrbe, Bauer, & Klostermeyer, 1998). In general, the relative concentration of a biopolymer mixture is crucial for a subsequent gelation process because the relative kinetics of phase separation and gelation will determine the morphology of a mixed gel (Morris, 1990; Tolstoguzov, 1990). This can be seen specically for LMA pectin added to caseinate-stabilized emulsion systems prior to acidication (Matia-Merino & Dickinson, 2004). It has been established that casein micelles may dissociate to various degrees when the pH of milk is decreased. The combined effect of low temperature and low pH is more than additive in causing dissociation of casein and solubilization of the colloidal calcium phosphate (CCP) from the casein micelles (Anema & Klostermeyer, 1997; Dalgleish & Law, 1988; Law & Leaver, 1998; Le Graet & Gaucheron, 1999; Singh et al., 1996; Udabage, McKinnon, & Augustin, 2000). CCP can be removed from milk in a controlled manner by using an acidication dialysis technique (Pyne & McGann, 1960) that involves the acidication of cold milk (5 1C) to pH values in the range 4.86.7, followed by equilibrium dialysis against excess skim milk. This results in milks that are reduced in CCP content (and consequently that have different levels of micellar casein), but otherwise are identical in composition to skim milk. The impact of these changes in casein micelle structure and CCP on the interactions of casein micelles with LMA pectin is unknown. In this paper, we report on the effect of casein micelle integrity, prior to acidication, on caseinpectin interactions. The dynamics of the formation of the acid gel network, along with the microstructural changes, were studied through rheological measurements of mixtures of milk protein concentrate (MPC) and LMA pectin as a function of pectin content and casein micelle integrity in MPC dispersions, from neutral pH to approximately pH 4.2.

2. Materials and methods 2.1. Materials A freshly made batch of MPC powder was obtained from Fonterra Co-operative Group Ltd, New Zealand. The typical composition of the MPC (% w/w) was: 82% total protein (71.3% casein, 10.9% whey protein), 1.5% fat, 2.3% lactose, 6.6% moisture and 7.0% ash (21.7 g calcium/kg, 13.4 g phosphorus/kg, 0.57 g sodium/kg and 0.95 g magnesium/kg). The pectin, GRINDSTEDs Pectin LA 410, was provided by Danisco; it is a low ester amidated pectin (approximately 31% degree of esterication and approximately 19% degree of amidation) with high calcium reactivity and is standardized with sugars. GDL and Rhodamine B were obtained from Sigma Chemical Co. Ltd (St. Louis, MO, USA). 2.2. Preparation of reconstituted MPC and compositional analysis Reconstituted MPC samples were prepared by dissolving MPC powder in Milli-Q water (Millipore Corp., Bedford, MA, USA) with continuous stirring at 6062 1C for 2 h. Sodium azide (0.02%) was added to the milk dispersion as a preservative. The samples were stirred overnight before use. Under these conditions, MPC was completely soluble in water and no denaturation of whey proteins was observed. The calcium levels in the milk powder samples, milk dispersions and serum phases were determined by inductively coupled plasma emission spectroscopy (Alkanani, Friel, Jackson, & Longerich, 1994). The total protein, casein and whey protein contents were calculated from the total nitrogen, non-casein nitrogen and non-protein nitrogen contents using methods described previously (Hill, 1993). 2.3. Adjustment of pH, dialysis and ultracentrifugation MPC samples (10% w/v) were cooled to approximately 8 1C and adjusted to pH 5.670.005 or pH 5.170.005 by the slow addition of 1 M/0.1 M HCl under vigorous stirring. A stable pH reading was achieved after approximately 2 h. Sub-samples (100 ml) were then dialysed against reconstituted MPC (20 times volume, three changes, 3648 h) to restore the soluble mineral components and the pH to levels similar to those in the original MPC dispersion (Pyne & McGann, 1960). After dialysis, all samples were allowed to equilibrate at room temperature and the pH was adjusted to 7.0 by the slow addition of 2 M NaOH. Particle size, protein and mineral content and rheological measurements were carried out on these samples. Dialysed MPC samples (or the original MPC dispersions) were also subjected to ultracentrifugation (100,000 g, for 1 h at 20 1C) in a Beckman L8-80 M ultracentrifuge,

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Ti-80 rotor (Beckman Instruments Inc., Palo Alto, CA, USA). The clear supernatants were carefully removed from the centrifuge tubes with a syringe and the protein and mineral contents were determined as described above. To use concise and clear terms in this paper, the MPCs subjected to acidication to pH 5.6 or pH 5.1+dialysis+ nal pH adjustment are referred to as MPC-treated-pH 5.6 and MPC-treated-pH 5.1. 2.4. Calcium ion activity The ionic calcium levels in the reconstituted MPC samples were measured using a Radiometer (Copenhagen, Denmark) F2112Ca calcium-specic electrode coupled with an Orion (Beverly, Massachusetts, USA) 90-02 double junction reference electrode with 0.01 M KCl in the outer chamber. Electrode potentials were measured by recording the mV response after dipping the electrodes in the samples. A stable mV response was obtained after 1 min. The ionic calcium levels were calculated from the recorded potentials by using a calibration curve obtained from standard (0.253.0 mM) CaCl2 2H2O in 0.01 M KCl. 2.5. Particle size analysis Casein micelle size was measured by photon correlation spectroscopy using a Malvern Zetasizer 4 instrument and the associated ZET5110 particle sizing cell (Malvern Instruments Ltd, Malvern, Worcs., UK) at 2070.5 1C. MPC samples were dispersed in Ca-imidazole buffer (20 mM imidazole, 5 mM CaCl2, 30 mM NaCl, pH 7.0) as described previously (Anema, 1997). 2.6. Preparation of MPC and pectin mixtures A 2% w/v LMA pectin stock solution was prepared at 65 1C for 2 h. Different volumes of pectin were diluted into Milli-Q water and enough volume of MPC solution was then added under continuous stirring to achieve nal pectin concentrations between 0.01 and 0.18% w/v (or up to 0.3% w/v for visual observations) and a constant concentration of 4% w/v MPC. 2.7. Preparation of acid milk gels and rheological measurements Appropriate amounts of GDL (1.20.855% w/v) were added to the protein or protein+pectin mixtures (equilibrated at 25 1C) to reach approximately pH 4.2 after 8 h. After mixing thoroughly for 2 min, 17 ml of sample was introduced into a CC Z3 DIN measurement cell (concentric cylinder geometry) to study the development of viscoelasticity in situ by dynamic oscillatory rheometry using a Paar Physica rheometer UDS 200 (Ostldern, Germany). A thin layer of low viscosity silicone oil was layered on the surface of the sample to prevent evaporation. The development of complex modulus (G*), storage modulus (G0 ),

loss modulus (G00 ) and tan d was monitored in the linear region (0.5% strain) at a constant frequency of 1 Hz at 25 1C for a period of 7 h. The decrease in pH was monitored in parallel samples using a pH meter (Jenway 3310, Essex, UK). All gels were characterized by a storage modulus that was greater than the loss modulus and close in value to the complex modulus, which was the rheological parameter used throughout this study. The gelation point was dened as the time of sharp increase in the shear modulus G* from the baseline, this being signalled by the detection of a modulus above the instrument noise level (ca. 1 Pa). 2.8. Confocal laser scanning microscopy A Leica DM RBE confocal laser scanning microscope LS 510 (Leica Lasertechnik GmbH, Heidelberg, Germany) was used to examine the gel microstructures, with 100x and 40x oil immersion objectives (numerical apertures 1.4 and 1.3, respectively). MPC solution was stained with Rhodamine B (0.05% w/v) prior to acidication. After GDL addition to the MPC (with or without the added pectin), approximately 80 ml of the dyed protein solution was placed into a laboratory-made welled slide. A cover slide was then placed over the sample, which was observed under the microscope at room temperature (similar to the temperature used during the rheological measurements, 25 1C). An Ar/Kr mixed gas laser was used at an excitation wavelength of 568 nm. Images were scanned at 1015 mm below the coverslip. The uorescence collected from the sample was an average of eight scans that were used to create each image (512 pixels 512 pixels). Experiments were done in duplicate. The gelation process of some samples was observed in situ. In all gels the nal structure was observed after 15 h of gelation, in order to maximize the differences between the nal networks. From previous experience, we chose 15 h of gelation as a convenient representative period for comparison since the differences in the microstructure of gels occurring during the rst hours of gelation always resulted in large differences within a 15 h period. 3. Results and discussion 3.1. MPC and pectin mixtures Dispersions containing 4% w/v MPC and a range of pectin concentrations (00.3% w/v) were examined visually prior to acidication, with the aim of identifying any phase separation or pre-gelation phenomenon. Mixtures containing MPC were homogeneous without any signs of gelation at up to 0.09% w/v added pectin over the observation period of 5 days. At between 0.1% and 0.2% w/v pectin, the mixtures were uniform and liquid during the rst day, but non-homogeneous mixtures (gelled material suspended in the solution) were formed after 2 days in cold storage. At X0.3% w/v pectin, the mixtures gradually gelled after

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mixing, going from a thick viscous uid to a rm gel that supported its own weight after 2 days. The interaction between casein micelles and pectin is expected to be minimal at neutral pH, when both protein and polysaccharide carry a negative charge (Oakenfull & Scott, 1998). The phase diagram established by Maroziene and de Kruif (2000) showed that about 0.2% w/v LMA pectin is needed to induce phase separation by depletion occulation effects in skim milk. Our observations indicated that, at this concentration of LMA pectin, signs of gelation occurred before any sign of macrophase separation was detected at the xed protein level used in this study (approximately 3.2% total protein). With respect to the observed gelation of the mixtures over time at X0.1% w/v pectin, previous studies on mixtures of casein micelles and LMA pectin have attributed an increase in the shear modulus under quiescent aging to an increased availability of diffusible calcium ions from within the casein micelles (Abbasi & Dickinson, 2002). Calcium ion activity was measured after adding pectin to the MPC dispersions. A gradual slight reduction in ionic calcium with increasing pectin was detected for the mixtures containing MPC. In general, no dramatic changes in ionic calcium with increasing amounts of pectin were

observed, even at concentrations above 0.1% w/v pectin. Let us now turn to the acidication of these systems. 3.2. Gelation of MPC and LMA pectin The gelation proles of a mixture of 4% MPC and LMA pectin (00.075% w/v) after acidication with 1.2% w/v GDL are shown as a function of time in Fig. 1a. On acidication, there was a decrease in the complex shear modulus with the incorporation of pectin (from ca. 400 Pa in the absence of pectin to ca. 280 Pa at 0.05% w/v pectin). At X0.06% w/v pectin, the spontaneous gel shrinkage within the measurement cell during the rheological test led to the loss of the stress signal. This indicates the threshold amount of pectin at which a spontaneous syneresis occurs. The reduction in the shear modulus of the gels containing pectin is in agreement with previous results on the acidication of mixtures of sodium caseinate containing calcium+low LMA pectin concentrations (Matia-Merino et al., 2004) and is discussed later. The development of tan d in the same system is shown in Fig. 1b. In all cases, there was a maximum in tan d(t) after the gelation point, indicating a new liquid-like character

450 400 350 300

7.5 7 6.5 6 5.5 5 4.5 4 0 1 2 3 4 t (h) 5 6 7 8 pH 0 1 2 3 t (h) 4 5 6 7

G* (Pa)

250 200 150 100 50 0

0.6

0.5

tan

0.4

0.3

0.2

Fig. 1. Development of complex shear modulus (a) and tan d (b) at 25 1C in a mixture of 4% MPC and LMA pectin (w/v) (0% (K); 0.025% (); 0.035% (m); 0.045% (x); 0.05% (E); 0.06% (+); 0.075 (&)) after acidication with 1.2% w/v GDL. The pH is plotted (a) for mixtures with no added pectin ( ) and for mixtures containing 0.05% w/v pectin ().

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emerging as a result of further acidication. The more pectin added, the lower were the values of tan d at the gelation point and at the subsequent maximum. A maximum in tan d has previously been observed during the formation of acid-set gels produced from heated skim milk (Lucey, Tamehana, Singh, & Munro, 1998; WalshOGrady, OKennedy, Fitzgerald, & Lane, 2001). This maximum has been related to a partial loosening of the weak initial gel network due to solubilization of CCP from the casein micelle in the pH range 6.05.3. A minimum followed by a maximum in tan d has also been observed during the acidication of micellar casein systems within a range of milk salt concentrations (Auty, OKennedy, Allan-Wojtas, & Mulvihill, 2005), which has been attributed to rearrangements of the casein protein. Consistent with the above study, it appears that MPC acid-induced gels undergo extensive rearrangements during the early stages of gelation. In order to discuss the observed effect of pectin on the gelation of MPCs, it is important to state the changes that are likely to occur during the acidication of the mixtures used in this study. (1) There will be solubilization of the CCP plus dissociation of the casein from the casein micelles along with a simultaneous decrease in the net negative charge on the caseins. Consequently, casein particles begin to aggregate to form a gel around pH 5.1 where the solubility of casein diminishes as previously observed during the acidication of milk (Horne, 2001). (2) As the pH is reduced, an increased amount of the calcium ions will be available for binding to the pectin, hence promoting its aggregation/gelation. (3) The carboxylic groups of LMA pectin become less ionized under acidication, promoting conformational ordering and intermolecular association (Gilsenan, Richardson, & Morris, 2000). It is also well established that pectin will adsorb onto the casein micelles as a result of an electrostatic interaction at around and below pH 5.0 between the net positively charged casein and the negatively charged pectin (Maroziene & de Kruif, 2000; Tuinier et al., 2002). Similarly, whey proteinpectin electrostatic interactions will also take place under acidication as shown for pure protein systems (Girard, Turgeon, & Gauthier, 2002). Therefore, the nal acid-

induced network will be the result of all these processes taking place at the same time. Measurements of calcium ion activity during the acidication process showed that the ionic calcium content gradually increased in the serum phase from 1.0 mM to approximately 7.7 mM, with the values becoming relatively stable at around pH 4.5. However, the variations in the release of calcium ions within the systems containing pectin were not signicantly different for the range used in this study, making it difcult to draw conclusions on the contribution of ionic calcium to the weakening/strengthening effect on the network while binding to pectin and/or casein. Confocal micrographs of acid-induced MPC gels at three different LMA pectin levels are shown in Fig. 2. There was a tendency towards a more open microstructure in the acid gels with increasing pectin concentration. That is, in the absence of pectin, the network of protein strands appeared to be denser, more interconnected and had relatively small pore size. On the contrary, the microstructures detected at higher pectin concentrations (0.050.1%) showed larger average pore size and the protein clusters seemed to have a lower degree of interconnectivity. This further supports the rheological data, which showed the weakening of the acid gel network in the presence of increasing pectin concentrations. This is consistent with the literature since a relationship between the average pore size and gel strength has been previously demonstrated (Pereira, Matia-Merino, Jones, & Singh, 2006; Pugnaloni, Matia-Merino, & Dickinson, 2005). Evidence of extensive syneresis due to gel shrinkage above a critical amount of added pectin in the acid-induced MPC gels is shown in Fig. 3. The photographs were taken 24 h after the addition of GDL. At high pectin concentrations, the curd tended to oat in the aqueous phase, indicating its lower density, which was probably due to the increasing amount of pectin in the serum phase which increased the viscosity and density of the serum phase. In agreement with the rheological measurements, the critical amount of pectin to cause the retraction of the curd corresponded to values 40.05% w/v pectin for the MPC gels. Spontaneous shrinkage has previously been observed

Fig. 2. Confocal micrographs of acid-induced MPC gels (4% w/v) with different concentrations of LMA pectin taken 15 h after acidication (pH approximately 4.24.1). Scale bar 10 mm.

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during the acidication of mixtures of caseinate containing added calcium and LMA pectin over a certain range of concentrations (Matia-Merino et al., 2004). However, in this study, in contrast to caseinate gels, the MPC gels could not be reverted to one single gel phase by increasing the amount of polysaccharide and forming a dominating pectin gel. For casein micelles, probably stronger interactions due to higher calcium levels released during acidication (up to 7 mM Ca2+) make the curd shrink easily when reaching certain pH/calcium levels, above a critical pectin concentration.

Overall, the addition of LMA pectin to acid-induced MPC gels did not contribute to an increase in the strength of the acid network but decreased the viscoelastic parameters during the rheological measurements of the gelation process. At X0.06% pectin, shrinkage of the network occurs resulting in extensive syneresis. 3.3. Effect of dissociation of casein micelles on gelation of MPCs The method of Pyne and McGann (1960) was used to remove different proportions of CCP from the MPC dispersions. Evidence for the change in the physicochemical properties of the casein micelles in the MPC samples is given in Fig. 4. Clearly, the colloidal calcium was depleted from the casein micelles with the acidicationdialysis treatment; the colloidal calcium in the MPCtreated-pH 5.1 sample was reduced to approximately 100 mg/kg from almost 2000 mg/kg in the control MPCpH 7.0 sample. The obvious dissociation of the casein micelles was reected in the decrease in casein micelle size (from 214 nm to approximately 148 nm) and in the progressive increase in casein in the serum phase. The extent of micellar casein dissociated varied from 47% in the MPC-treated-pH 5.6 sample to 94% in the MPC-treatedpH 5.1 sample. The average casein micelle size for the control MPC was similar to values detected previously in unheated milk (215 nm) measured at 20 1C (Anema, Lowe, & Lee, 2004). The dissociation of casein from the micelles also caused a marked decrease in the turbidity of the MPC samples because of the smaller casein micelles. The calcium ion activity was also measured in nontreated MPC at pH 7.0, with values ranging between approximately 0.6 mM Ca2+ and approximately 1.0 mM Ca2+ (after 2 days of storage). Similar values of around 0.7 mM ionic calcium were found for both treated samples (measured at the end of the dialysis period).
220 100

Fig. 3. Visual appearance of gels formed at 25 1C at 24 h after the addition of GDL (1.2% w/v): (a) 4% w/v MPC solutions+LMA pectin (00.3% w/ v); (b) detail of the extensive syneresis. The arrow indicates the starting point of gel shrinkage and the release of serum.

2000 200 Colloidal calcium (mg/Kg) Casein micelle size (nm) 1500 180 1000 160 80 % Soluble casein (wt/wt)

60

40

500

140

20

0 Control-pH 7.0 Treated-pH 5.6 Type of MPC Treated-pH 5.1

120

Fig. 4. Level of colloidal calcium (mg/kg) ( ), casein micelle size (nm) () and percentage of soluble casein (% w/w) (&) measured in MPC at neutral pH (control), in MPC-treated-pH 5.6 and in MPC-treated-pH 5.1.

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When these MPC dispersions were acidied by GDL, the major problem encountered was the change in their buffering capacity. It has been shown previously (Lucey, Hauth, Gorry, & Fox, 1993) that normal milk has a buffering peak at approximately pH 5.1 that is attributed to solubilization of CCP, which results in the formation of phosphate ions that can combine with H+, resulting in buffering. The buffering capacity is reduced in re-formed milks (i.e. milk acidied at low temperature to pH 5.0 or 4.6 followed by neutralization) (Lucey et al., 1996). It was suggested that little re-formation of CCP occurs with neutralization, and that other types of calcium phosphates that have different acidbase buffering properties are formed. In our study, dialysis, which removes a considerable proportion of the minerals, including phosphates, was used after acidication. The CCP-altered MPC samples showed a reduction in the buffering properties, the pH being lowered to a greater extent (down to pH 3.73.8 after 8 h) upon the addition of the same amount of GDL (1.2% w/v) as that used for the control MPC dispersion. After adjustment of the acidulant concentration, reasonably similar curves were produced for all systems (Fig. 5a).

The gelation proles, following the development of the shear modulus G*, for the systems studied without any added pectin were plotted as a function of time (Fig. 5a) and as a function of pH (Fig. 5b). The treated MPC gels had a lower shear modulus G* than the control MPC gels. The modulus after 7 h of acidication decreased with an increase in the level of dissociation of micellar casein, from ca. 400 Pa for the control MPC (non-dissociated sample) down to ca. 250 and ca. 100 Pa, for the MPC sample with 47% and 94% dissociated micellar casein, respectively. The gelation pH of all samples fell between pH 5.1 and pH 5.2, although the kinetics of the process were signicantly different. Between pH 5.1 and 4.6, the rate of formation of network connections appeared to be faster for the MPC sample with 47% dissociated micellar casein, even above the control MPC sample (Fig. 5b). In order to explore possible reasons for the loss of strength of the acid gels developed from the treated MPC samples, the gelation process was observed under the confocal microscope. Sequential images of the gelation process of the control MPC and the MPC-treated-pH 5.1 were taken. The control MPC sample followed the typical

450 400

7 6.5

350 300 G* (Pa) 250 200 150 100 50 0 0 1 2 3 4 t (h) 5 6 7 8 4.5 4 5.5 5 6 pH

350 300 250

G* (Pa)

200 150 100 50 0 5.5 5.4 5.3 5.2 5.1 5 pH 4.9 4.8 4.7 4.6 4.5 4.4

Fig. 5. Development of shear modulus G* with time (a) and with pH (b) at 25 1C for: MPC at pH 7.0 after the addition of 1.2% w/v GDL (K); MPCtreated-pH 5.6 after the addition of 1.05% w/v GDL (m); MPC-treated-pH 5.1 after the addition of 0.855% w/v GDL (). The open symbols in (a) correspond to pH values for the three systems.

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Fig. 6. Confocal micrographs taken during the gelation process at 25 1C after addition of: (a) 1.2% w/v GDL to MPC (control) and (b) 0.855% w/v GDL to MPC-treated-pH 5.1. Scale bar 10 mm. White arrow indicates the point at which it was necessary to refocus.

aggregation process of GDL-induced gelation of skim milk (Auty, Fenelon, Guinee, Mullins, & Mulvihill, 1999). At the onset of gelation, a visible protein network was formed, at which point much of the stained protein became immobilized in the focus plane under study (Fig. 6a). In contrast, after the onset of gelation in the MPC-treated-pH 5.1 (with 94% dissociated micellar casein), the aggregation process was more difcult to follow because of the continuous loss of the focal plane. The connections of the aggregating milk protein exhibited a tendency for strand breakage and rearrangements during the acidication process until the protein network was xed (Fig. 6b). This could account for the weaker network formed from the treated MPC samples. It seems reasonable to suggest that the difference in the dissociated casein micelle systems during the aggregation process can also be attributed to the different building blocks forming the aggregating networksmaller remaining casein micelles and greater amounts of casein in the serum phasewhich do not aggregate in the same way to form a strong network as the original micelles. Finally, let us turn to the acidication of these treated MPC systems in the presence of LMA pectin. As previously demonstrated, the rate of acidication (or the amount of GDL) is one of the factors that inuences the gel properties (Horne, 2001). In our study, acidication was carried out after the addition of (1) 1.2% w/v GDL and (2) a lower amount of GDL calculated to give a similar rate of acidication to that described above. The gelation curves obtained are shown in Fig. 7 for the MPC with 47% dissociated micellar casein (or MPC-treated-pH 5.6) and in Fig. 8 for the MPC with 94% dissociated micellar casein (or MPC-treated-pH 5.1).

The contribution of pectin to the development of the network was clear in both treated MPC samples and at two rates of acidication. In Figs. 7a and 8a, the gelation of MPC without added pectin showed the typical overacidication effectthe pH being lowered to a value beyond the isoelectric pointwhich is detected rheologically as a maximum in the storage modulus beyond the critical gelation time (Horne, 2001; Koh, Matia-Merino, & Dickinson, 2002; Pugnaloni et al., 2005). The incorporation of pectin changed the kinetics of gelation. In the control MPC systems, addition of pectin caused a considerable decrease in G* (Fig. 1a), whereas the G* values increased markedly when the same amount of pectin was incorporated into MPC-treated-pH 5.1 (Fig. 8b). This trend was observed until shrinkage of the network took place (i.e. up to 0.05% w/v pectin for the control MPC and up to 0.1% or 0.18% w/v pectin for the MPC-treated-pH 5.1 depending on the rate of acidication, fast or slow respectively). When the casein micelles were disaggregated to intermediate values (MPC with 47% dissociated micellar casein and 197 nm casein micelle size), the effect of pectin started to reverse, showing the beginning of the synergistic effect. That is, during the experimental time scale the values of G* with added pectin were still lower than the G* values without added pectin. However, the proles indicated that the systems with added pectin may reach greater G* values beyond the 7 h of the experiment, especially at a slower rate of acidication (Fig. 7b). Additionally, there were no substantial changes in gelation times with the incorporation of pectin (up to 0.06% w/v). Independently of the rate of acidication, the synergistic effect of pectin became more obvious when the casein

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350 300 250

G* (Pa)

200 150 100 50 0 0 1 2 3 4 t (h) 5 6 7 8 7 6.5 6 5.5 150 100 50 0 0 1 2 3 4 t (h) 5 6 7 8 5 4.5 4 pH

b
G* (Pa)

350 300 250 200

Fig. 7. Development of shear modulus G* with time at 25 1C, after the addition of (a) 1.2% w/v GDL and (b) 1.05% w/v GDL to MPC-treated-pH 5.6 plus different amounts of LMA pectin (% w/v): (K) 0%; (m) 0.03%; (B) 0.06%; (+) 0.08%.

micelles had been further dissociated (MPC with 94% dissociated micellar casein and 148 nm casein micelle size). In both cases of fast acidication (or over-acidied systems) (Fig. 8a) and slow acidication (or steady acid gels) (Fig. 8b), added pectin helped in the development of a rmer network structure up to the critical pectin concentration. It is interesting to observe that the effect of pectin inclusion on gel strength was found to depend on the acidication kinetics. For the faster acidication, the maximum in gel strength was found at 0.08% pectin (Fig. 8a) whereas the pectin level increased up to 0.13% to reach a maximum in gel strength at slower acidication (Fig. 8b). It seems reasonable to suggest that a slower rate of acidication promotes a more homogeneous distribution of the pectin within the matrix that allows pectin molecules to interact with casein particles in an organized manner. On the contrary, at a faster rate of acidication, the electrostatic complexation pectincasein (at around pH 5.0) may occur more randomly and consequently the shrinkage effect manifests itself at a lower amount of added pectin. The microstructures observed under the confocal microscope showed the change from a more open structure of the milk gels from the control MPC with 0.05% w/v added pectin (Fig. 9a), to a denser and more homogeneous gel,

with increased interconnectivity of the network, for the MPC-treated-pH 5.6 (Fig. 9b) and the MPC-treated-pH 5.1 (Fig. 9c). In general, the behaviour of these treated MPC systems is probably related to the different network connections created during acidication. Dissociation of the casein micelles (or an increase in the soluble casein), along with a reduction in the amount of colloidal calcium while re-equilibrating the mineral content in the serum phase prior to acidication, seems to lead to a synergistic effect between the casein protein and the pectin while forming an acid gel network, which is stronger and more connected. On lowering the pH, gelling pectin could cause interferences to the network connections in the control MPC which has higher levels of colloidal calcium. It seems fair to speculate that the rate of acidication during gelation and, the balance between (1) the release of calcium from the micelles, (2) the amount of pectin present and (3) the relative proportions of the caseins, in the micellar or soluble states, are critical in the pectincalciumcasein interactions and, therefore, in the development of the network. It seems that, if the micelles are more dissociated, the pectincalciumcasein interactions during acidication can improve the interconnectivity of the nal network.

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400 350 300

G* (Pa)

250 200 150 100 50 0 0 1 2 3 4 t (h) 5 6 7 7 6.5 6 5.5 5 4.5 4 0 1 2 3 4 t (h) 5 6 7 8 pH 8

400 350 300

G* (Pa)

250 200 150 100 50 0

Fig. 8. Development of shear modulus G* with time at 25 1C, after the addition of (a) 1.2% w/v GDL and (b) 0.855% w/v GDL to MPC-treated-pH 5.1 plus different amounts of LMA pectin (% w/v): (K) 0%; (J) 0.04%; (m) 0.06%; (B) 0.08%; (- -) 0.1%; (x) 0.13%; (+) 0.18%.

Fig. 9. Effect of 0.05% w/v LMA pectin on the microstructure of acid milk gels made from (a) control MPC at pH 7.0, (b) MPC-treated-pH 5.6 and (c) MPC-treated-pH 5.1, after the addition of 1.2, 1.05 and 0.855% w/v GDL, respectively. The confocal images were taken after 15 h of gelation. Scale bar 10 mm.

In summary, the rheological gelation prole and the developing microstructure after the acidication of mixtures of MPC and LMA pectin can be attributed to a combination of effects. The potential to generate different types of gel matrices, by controlling rate of acidication, casein micelle integrity, calcium levels and pectin concentrations to obtain different synergistic or antagonistic effects, gives a way to manipulate texture. The possible

formation of caseincalciumpectin complexes with the casein micelles or in the serum phase, along with caseincasein and pectinpectin interactions (through calcium) and caseinpectin complexes formed during acidication, are responsible for the development of the acid-induced three-dimensional network. Further work should be carried out to identify which of these interactions are dominant in controlling the dynamics of these mixed systems.

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