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Rennet-induced aggregation of milk containing homogenized fat globules. Effect of interacting and non-interacting fat globules observed using diffusing wave spectroscopy
International Dairy Journal 21 (2011) 679e684
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Rennet-induced aggregation of milk containing homogenized fat globules. Effect of interacting and non-interacting fat globules observed using diffusing wave spectroscopy M. Corredig*, G. Ion Titapiccolo, Z. Gaygadzhiev, M. Alexander Department of Food Science, University of Guelph, Guelph, Ontario, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2010 Received in revised form 10 January 2011 Accepted 23 January 2011
The dynamics of the interactions between casein micelles and oil droplets during rennet-induced gelation were described using rheology and diffusing wave spectroscopy. Two different colloidal states were obtained by preparing WPI-stabilized oil droplets in either milk serum or low ionic strength buffer: flocculated and non-flocculated. These two colloidal states strongly affected the diffusion of the noninteracting droplets in the gel and the elasticity of the protein network. In homogenized milk, the droplets interact with the protein network and play a structuring role, causing an increased elastic modulus. However, after displacement of the protein from the interface using Tween 20, the interpretation of the rheological data would lead to incorrect conclusions without light scattering measurements. This work demonstrated that an in-depth understanding of the colloidal properties of the oil droplets is necessary to fine-tune the interactions between these particles and the casein matrix in dairy gels. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction During renneting of milk, k-casein is cleaved specifically by chymosin and, as k-casein plays a major role in imparting colloidal stability to casein micelles, the micelles progressively lose steric repulsion and start interacting with one another (Tuinier & De Kruif, 2002; Zoon, van Vliet, & Walstra, 1988). A three dimensional network is formed, with partly fused casein micelles (Lucey, Johnson, & Horne, 2003). In a rennet-induced gel, the Characteristics of the colloidal particles involved in the pre-gelation, gelation, and rearrangements processes will determine the final properties of the dairy matrix. In particular, fat globules play an important role in structuring of dairy products. It is well documented that the rheological behaviour of filled milk gels depends on the structural properties of the filler particles, the viscoelasticities of the matrix and the filler, the volume fraction of the components, the shape and size of the filler droplets and their interactions with the building blocks of the network (Sala, van Aken, Cohen Stuart, & van de Velde, 2007; van Vliet, 1988). Dairy matrices containing fat globules have been previously described in literature, mostly using model systems consisting of reconstituted milk containing specifically designed and well characterized oil droplets (Cho, Lucey, & Singh, 1999; Michalski, Cariou, Michel, & Garnier, * Corresponding author. Tel.: þ1 519 824 4120; fax: þ1 519 824 6631. E-mail address: [email protected] (M. Corredig). 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.01.010
2002). In general, when the oil droplets “actively” interact with the casein network, there is an increase in the gel modulus, while when oil droplets do not have affinity with the building blocks, there is very little or no contribution to the gel stiffness. However, if the fillers are sufficiently large, they will indirectly affect the formation of the network by limiting the rearrangements of the caseins during gelation, and this will create thicker gel strands with filler particles immobilized within the pores. This has been recently illustrated using microscopy in a dairy matrix containing starch granules (Azim, Alexander, Koxholt, & Corredig, 2010). Most of the reports so far have investigated the rheological and microstructural properties of gelling model systems containing milk proteins and fat globules. Fat globules covered with small molecular weight surfactants (Tween 20 or Tween 60) or whey proteins led to the formation of a weak acid gel with low elastic modulus (G0 ), compared with the same systems gelled with oil droplets which would interact with the network (Cho et al., 1999; van Vliet, 1988; van Vliet & Dentener-Kikkert, 1982). Most of the research in this field has been carried out on acid-induced gels and in reconstituted model systems. In whole non-homogenized milk, the milk fat globules do not participate in the formation of the casein network (Michalski et al., 2002) and behave as “inert” fillers. Homogenization reduces substantially the fat globules’ size, and caseins are the main proteins adsorbed onto the newly covered interface (Mulder & Walstra, 1974; Sharma & Dalgleish, 1993). Renneting of homogenized milk shows a faster release of the
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caseinomacropeptide, a shorter gelation time and high retention of moisture in the curd (Jana & Upadhyay, 1993; Peters, 1956; Robson & Dalgleish, 1984). It has been recently shown that using diffusing wave spectroscopy it is possible to follow the dynamics of oil droplets in mixed systems with casein micelles, as when present in milk, the oil droplets refractive index contrast is much higher than that of the micelles. The oil droplets therefore contribute most of the scattering signal (Gaygadzhiev, Corredig, & Alexander, 2008). The noninvasive nature of this light scattering technique therefore allows using oil droplets as tracer particles during the initial stages of the formation of the protein network. In combination with rheological measurements it is then possible to clearly describe the details of the interactions occurring in rennet-induced gels. The objective of this work was to identify the dynamics of the interactions between casein micelles and fat globules during the formation of a rennet-induced gel. A model system consisting of non-interacting droplets was first investigated to determine the effect of the colloidal state (flocculated and non-flocculated droplets) on the rheological properties and the aggregation behaviour of the casein network. In the second part of this manuscript, the behaviour of fat globules in homogenized milk was studied during renneting, and homogenized milk was compared with skim milk as well as homogenized milk with Tween 20.
by adding appropriate ratios of WPI-stabilized emulsions, concentrated milk (2) and permeate, to a final volume fraction of 5% and 10% for oil droplets and casein micelles, respectively. For control skim milk, 1 vol of 2 concentrated skim milk was diluted with milk permeate. For emulsions prepared in permeate, 1 vol of 2 concentrated milk was added to 1 vol of emulsion in permeate previously diluted in permeate. For the emulsions prepared with imidazole buffer, a 2 permeate was used for the dilution, as this ensured to obtain a similar ionic concentration of the serum phase, as this is a critical factor in rennet-induced milk gels. For the experiments with homogenized milk, fresh whole milk was homogenized at 350 bar for two passes as described above. Aliquots of homogenized milk were also treated with polyoxyethylene sorbitan monolaurate (2%, w/v) (Tween 20, 1227 g mol1 Sigma, St Louis, MI USA), and stirred for 6 h, to ensure protein displacement at the interface. The addition of this small molecular weight surfactant causes the displacement of most of the protein at the interface (Mackie, Gunning, Wilde, & Morris, 1999) resulting in a system containing the same volume fraction of fat globules, with comparable colloidal characteristics and ionic composition, but non-interacting properties of the droplets (Ion-Titapiccolo, Alexander, Corredig, 2010a).
2. Materials and methods
In all cases, gelation experiments were conducted at 30 C soon after homogenization (in the case of homogenized milk with Tween 20, after 6 h), by addition of rennet (Chymostar, single strength, Rhodia, Cranbury, NJ, USA) added at a concentration of 0.018 IMCU mL1. Immediately after the addition of rennet, the samples were subjected to simultaneous measurements using rheology and diffusing wave spectroscopy. Rheological measurements were conducted using a stresscontrolled rheometer (AR 1000, TA Instruments Ltd., New Castle, MA, USA) with a conical concentric cylinder geometry (5920 mm fixed gap, 15 mm radius). A time sweep test was performed for 180 min, with the following parameters: 0.01 strain, 0.1 Hz and an initial oscillation stress of 0.0018 Pa. The gelation time was determined as the time needed to reach tan d ¼ 1. In situ light scattering measurements were performed using diffusing wave spectroscopy (DWS). This technique is based on the measurement of multiple scattered light (Maret & Wolf, 1987). Rennet-induced coagulation was followed at 30 C. The sample was placed in a 5 mm optical glass cuvette (Hellma Canada Limited, Concord, Ontario, Canada). The details of the instrumentation can be found elsewhere (Gaygadzhiev, Corredig, & Alexander, 2009). The intensity of scattered light and autocorrelation functions were collected every 4 min, for a total experimental time of 180 min for the reconstituted milk, and 80 min in the case of homogenized milk. A standard latex suspension (260 nm diameter, Portland Duke Scientific, Palo Alto, CA, USA) was employed to calibrate the laser intensity. The mobility of the particles was obtained from the measurement of the particle diffusion coefficient, directly derived from the characteristic decay time of the correlation function. The mean square displacement (MSD) was also calculated to probe the changes occurring to the particle mobility (Weitz, Zhu, Durian, Gang, & Pine, 1993). In a free diffusing regime, the MSD increases linearly over time. Under these conditions the apparent radius can be calculated using the StokeseEinstein relation. When the relation between the MSD and time deviates from linearity, there is an arrest in particle mobility (Romer, Sheffold, & Shurtenberger, 2000). Experiments were analyzed using software developed by Mediavention Inc. (Guelph, Ontario, Canada). In addition to using diffusing wave spectroscopy, the apparent average diameter of the oil droplets was also measured using
2.1. Sample preparation Whole milk was collected at the research farm of the University of Guelph (Elora, Ontario, Canada) and sodium azide (0.02%, w/v) was immediately added to inhibit microbial growth. The skimmed milk was prepared by centrifuging at 4000 g for 20 min at 4 C (Beckman J2-21 centrifuge, Beckman Coulter, Mississauga, Ontario, Canada) and filtering four times through fibreglass filter (Whatman, Fisher Sci., Whitby, Ontario, Canada). This ensured near complete removal of fat. In the reconstituted milk experiments, a 2 concentrated milk was prepared by reducing its volume using ultrafiltration (PLGC, 10 kDa cut-off regenerated cellulose cartridge, Millipore, Bedford, MA, USA). In addition to the retentate (2 milk concentrate), the permeate from milk (this corresponds to the composition of the original milk serum) was collected. For the experiments with WPIemulsions prepared in buffer (see below), to ensure a similar content of soluble calcium in the final reconstituted milk, it was necessary to prepare a 2 permeate starting from skim milk reconstituted from skim milk powder (Parmalat Canada, London, Ontario, Canada) at 20% (w/v) solids. The whey protein-stabilized emulsions were prepared using whey protein isolate (WPI) (Land O’Lakes, St Paul, MN, USA). WPI was either dissolved in 5 mM imidazole buffer at pH 6.7, or in milk permeate. In both cases, the solutions were stirred for 2 h at room temperature and then stored at 4 C. WPI contained 92% protein, 2.7% ash, 1.6% lactose and <0.5% fat (as specified by the manufacturer). Anhydrous milk fat (Parmalat Canada) was pre-emulsified with a high speed blender (PowerGen 125, Fisher Sci.) with the protein solutions to a final 2% (w/w) and 25% (w/w) for protein and milk fat, respectively. The premix was then passed 4 times at 350 bar through a high pressure homogenizer (Emulsiflex C5, Avestin, Ottawa, Ontario, CA). The homogenizer was immersed in a temperature-controlled water bath (Versa-Bath, Fisher Sci.) at 45 C. When a WPI solution in buffer was used, less protein was necessary to stabilize the oil droplets (as shown by preliminary experiments which tested different concentrations of protein and particle size distributions), and 1% (w/w) protein and 20% (w/w) milk fat were used. Recombined milk samples were obtained
2.2. Gelation experiments
M. Corredig et al. / International Dairy Journal 21 (2011) 679e684
2.3. Statistical analysis
30
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dynamic light scattering (Zetasizer Nano, Malvern Instruments, Worcestershire, MA, UK). For these measurements, the samples were extensively diluted (approximately 2000 times) in permeate (milk serum), pre-filtered through a 0.22 mm filter (Millipore Canada, Missisagua, Ontario, Canada), to preserve as much as possible the ionic conditions. All samples were measured immediately after dilution.
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All experiments were conducted in triplicate (three separate batches of milk and emulsion). Analysis of variance and least significant difference were calculated to determine significant differences between treatments (at 95% confidence level) using SPlus 8.0 (Tibco, Somerville, MA, USA).
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3. Results and discussion Table 1 summarizes the average apparent radius measured for the WPI-stabilized droplets. When the radius was measured using DLS, under extensively diluted conditions, the emulsions prepared in buffer showed an apparent radius of 120 nm once diluted in milk serum (permeate). The apparent radius measured by DWS, under concentrated conditions, also indicated the presence of small droplets, with a value of about 160 nm. It was concluded that these emulsion droplets were stable to aggregation once reconstituted in an ionic environment similar to that of skim milk. On the other hand, when the WPI-emulsions were prepared with a solution prepared in permeate and not buffer, the apparent radius was larger than for the emulsions in buffer. This is clearly shown by the discrepancies in the radius values measured both by DWS and DLS. The results indicated that when the oil droplets were homogenized under relatively high ionic conditions, the droplets were aggregated in flocs. This effect of ionic strength on the stability of emulsion droplets has been previously discussed (Agboola & Dalgleish, 1995; Srinivasan, Singh, & Munro, 2000). Skim milk, concentrated 2 by ultrafiltration, was recombined with permeate and WPI-stabilized emulsions prepared in either imidazole buffer or permeate and diluted to a similar ionic composition using either 2 permeate or permeate, respectively (see Methods for details). Fig. 1 shows the changes in the rheological and light scattering parameters during gelation of skim milk (circles) and skim milk reconstituted with WPI-emulsion droplets, either stable or flocculated (squares or triangles, respectively). Fig. 1A shows the development of G0 as a function of time after the addition of rennet. In skim milk, the value of G0 remained very low until 95.2 (0.2) min, when it started to increase, indicating the formation of a gel. Comparing the rheological behaviour of the control skim milk and the recombined milk with WPI-stabilized oil droplets prepared in buffer, the progression of the G0 value over time showed an earlier gelation point (80.4 4.2 min) but the value of G0 at 180 min was not statistically different. In contrast, the system containing flocculated oil droplets showed a faster gelation and a dramatic increase of the elastic modulus. The recombined Table 1 Particle size of whey protein-stabilized emulsions prepared in either permeate or 5 mM Imidazole buffer, pH 6.7, and dispersed in permeatea. Emulsion (prepared in/dispersed in)
Apparent radius (DLS, nm)
Apparent radius (DWS, nm)
Buffer/permeate Permeate/permeate
120 13 582 18
163 3 217 11
a Radius obtained either using dynamic light scattering (DLS, diluted conditions) or diffusing wave spectroscopy (DWS, undiluted conditions). Values are reported as averages and standard deviations of three experiments.
Radius (μm)
5 4 3 2 1 0
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20
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60
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Time (min) Fig. 1. Development of the storage moduli, G0 , measured by rheology (A), and the radius, measured by diffusing wave spectroscopy (B), during the rennet coagulation process of skim milk (C) and recombined milk containing whey protein isolatestabilized oil droplets prepared in permeate (:) and in low ionic strength buffer (-).
milk systems contained non-interacting emulsion droplets, with identical volume fractions of caseins and fat. DWS measurements confirmed the rheological observations. As shown in Fig. 1B, there were clear differences between the treatments in the development of the apparent radius versus renneting time. These results demonstrated in more details the differences in the aggregation behaviour between the two reconstituted milk systems. It should be pointed out that the scattering signal for recombined milk is heavily dominated by the oil droplets, and the results shown here depict the aggregation of the oil droplets as influenced by the presence of a protein gel developing around them. In skim milk, the aggregation point (the point of onset of the radius increase) was in agreement with the gelation point measured with rheology. This was expected from what was reported in previous literature (Sandra, Alexander, & Dalgleish, 2007). A pronounced difference in the particle size change over time was shown between the two reconstituted milk systems. In the milk containing stable oil droplets (those prepared with buffer, square symbols) the dynamic properties of the colloidal particles (the oil droplets) present in the system remained very similar to the initial properties, in spite of the aggregation of the casein micelles around them (a similar G0 value was shown for this sample compared with the skim milk control). In contrast, the recombined milk containing flocculated droplets (those homogenized in milk serum) showed extensive aggregation, with an aggregation time significantly shorter than that of the control sample, indicating a shorter onset of aggregation for these samples. It should be
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pointed out however, that the apparent size obtained by DWS after colloidal mobility is restricted does not represent a true size, but it can still be used as an indication of the decreased mobility. The start of the increase in size happened earlier in the reconstituted milk than for skim milk control, suggesting that the aggregation of the colloidal particles occurred earlier. This effect can not be fully attributed to an increase in volume fraction, as a recent study has demonstrated that gelation time is not affected by concentration (Sandra, Cooper, Alexander, & Corredig, in press). To better determine the changes in mobility over time between the samples, the changes in MSD versus time are shown in Fig. 2. While at the initial time (Fig. 2A) both recombined milk systems and skim milk control showed a free diffusing behaviour, after 180 min of the addition of rennet (Fig. 2B), there were significant differences between the samples. The skim milk control (circles) showed clear signs of arrested motion, as the casein micelles become increasingly entrapped in their own network strands. The recombined milk with aggregated oil droplets (triangles) also showed arrest in motion of the particles, and to a larger extent than for milk control. In this case, the fat globules were hindered in their motion, and were not able to probe all their available space. On the other hand, the WPI-covered droplets prepared in buffer (squares) still showed free diffusing behaviour after 180 min of renneting, even though the rheological parameters indicated that gelation had occurred (Fig. 1A). It was concluded that although the viscoelastic parameters could suggest the presence of “active” interacting filler particles in the case of WPI-stabilized emulsions prepared in permeate, these oil droplets did not participate in the formation of the network, but showed a dramatic decrease in mobility. This was caused by the
6e-5
progressive constraint experienced by the fat globules (present in aggregated form) within the pores of the casein network. The increase in the storage modulus was due to a continuous thickening of the strands around the pores, as influenced by the presence of the oil droplets. In reconstituted milk containing stable WPIemulsion droplets, on the other hand, although there was an increase in storage modulus, the droplets still did not appear to interact with the casein network but were free and small enough to continue to probe the entire space within the pores and not affecting the value of G0 . The increase in apparent particle size shown in Fig. 1B could be attributed to the slowing down of the mobility just due to hydrodynamic effects (crowding). The formation of structure in milk containing homogenized oil droplets is also a clear example of how the rennet gel is affected by the colloidal properties of the fat globules as well as by the nature of the interface. For this reason, the dynamics of the changes occurring during renneting of homogenized milk were studied by rheology and diffusing wave spectroscopy. It is established that homogenized fat globules actively participate in the formation of the rennet-induced gel network, as the caseins adsorbed on the oil droplets interact with those forming the network in the continuous phase (Lucey et al., 2003; Michalski et al., 2002). As a consequence, the elastic modulus of the gel increased dramatically compared with skim milk, as clearly shown in Fig. 3. In this figure, the storage modulus (A) and apparent hydrodynamic radius (Fig. 3B) for skim milk control (circles), homogenized milk (filled squares), as well as homogenized milk with Tween 20 added (empty squares) are shown as a function of renneting time. Tween 20 was added to homogenized milk after homogenization, to displace the proteins 140
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<Δr2(t)> (μm2)
100 4e-5 3e-5 2e-5
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Radius (μm)
<Δ r2(t)> (μm2)
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Time (s) Fig. 2. Mean square displacement (MSD) as a function of correlation time for the control skim milk (C) and recombined milks containing whey protein isolate-stabilized oil droplets prepared in permeate (:) and in low ionic strength buffer (-). Panel A, immediately after addition of rennet; panel B, 180 min after addition of rennet.
0
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Time (min) Fig. 3. Development of the storage moduli, G0 , measured by rheology (A), and the radius, measured by diffusing wave spectroscopy (B), during the rennet coagulation process of skim milk (C), homogenized milk (-) and homogenized milk with Tween 20 (,).
M. Corredig et al. / International Dairy Journal 21 (2011) 679e684
from the fat interface, leading to a system containing non-interacting fat globules, with comparable colloidal properties to the original homogenized milk. The gelation point of milk, as measured by rheology (Fig. 3A) was clearly faster in the presence of fat globules. While skim milk gelled at 37 4 min, homogenized milk showed a gelation point at 25.6 3 min. Surprisingly, although the presence of Tween 20 should create non-interacting oil droplets, a faster gelation point was measured (14.7 2.5 min) compared with that of homogenized milk without Tween 20. The elastic modulus after 45 min from the gelation point was also significantly higher for the homogenized milk with Tween 20, compared with homogenized milk or skim milk control. These differences were explained by a direct effect of Tween 20 molecules on the rennet reactivity of the casein micelles. Indeed, it has been previously demonstrated that when Tween 20 is added to skim milk, both the first stage and the second stage of rennet-induced gelation are modified with an increase in the storage modulus (Ion Titapiccolo, Corredig, & Alexander, 2010b). The aggregation behaviour of the three samples is also clearly shown in Fig. 3B, where the results of apparent particle size measured by DWS supported the rheological data. The sudden change in apparent radius occurred earlier for the homogenized milk containing Tween 20, and for the homogenized milk, compared with that of skim milk. However, the samples showed very different rates of increase in size (or, rather, the rate of decrease in the mobility of the particles), because of differences in the interactions with the casein matrix and in their mobility (see below). While homogenized milk and skim milk clearly showed a fast onset of aggregation, in the case of homogenized milk containing Tween 20, after a small change in size (most likely due to crowding of the droplets into the casein pockets) at about 10 min, the apparent radius did not further increase, reaching a plateau at about 1 mm, suggesting a lack of aggregation for the oil droplets. To better clarify the differences in particle mobility between homogenized milk with or without Tween 20, the changes occurring in the MSD versus time curves were recorded, as shown in Fig. 4. The plots for homogenized oil droplets (filled symbols) and homogenized oil droplets containing Tween 20 (empty symbols) are drawn for different times during the gelation process (reaction time increases with arrow). As previously shown for recombined milk (Fig. 2), the initial stages immediately after renneting showed a linear dependence of the MSD with correlation time, indicating free diffusive motion (Romer et al., 2000; Weitz et al., 1993). As the casein
2
<Δr (t)> (μm2)
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micelles become more unstable, because of the cleavage of the kcasein, the fat globules in homogenized milk are no longer able to probe the accessible space range, resulting in an asymptotic behaviour of the MSD curve versus correlation time (clearly seen for the bottom two curves). On the other hand, the homogenized milk droplets treated with Tween 20 still showed free diffusive motion, as the samples kept their linear behaviour for the whole renneting experimental time (w80 min). The changes in slope for the initial stages of the renneting reaction are due to the initial aggregation of the fat globules before casein coagulation and arrested motion occurred. It was therefore concluded that, as expected, homogenized fat globules are an active part of the rennet gel network, and gelation occurs faster. On the other hand, when the protein is displaced from the interface (with the addition of Tween 20) the oil droplets do not participate in the network, and they are trapped in the network cages, unaggregated and free to move around within the pores of the protein network. 4. Conclusions This work clearly demonstrated that with careful design of the colloidal state of the fat globule in combined gels, the interactions with the gel network can be controlled and ultimately affect the gel structure, mechanical and sensory properties of composite gels. In addition to using rheological measurements, we can get further insight into these gelling systems by observing the dynamic behaviour of their components using light scattering. In skim milk containing WPI-covered oil droplets, it was clearly demonstrated that although the oil droplets do not participate in the formation of the gel network, dramatic differences in microstructural characteristics can be obtained by modifying the colloidal state of the fat globules. Small, non-interacting droplets will reside within the pockets formed by the casein network, and will not modify the viscoelasticity of the system. However, when the same droplets are flocculated, their actual size will approximate that of the floc size, and the rate of gel formation and the stiffness of the gel will increase. As the casein gel develops, and the network grows, the flocculated droplets become trapped inside the formed cages. Due to their state of flocculation, the droplets have much less available space that in the non-flocculated case and their mobility is progressively squeezed as the casein gel pores continue to stiffen. This research also confirmed the numerous reports on homogenized oil droplets as fillers in rennet gels. As hypothesized, Tween 20-coated globules behave as “inactive” fillers, while the homogenized globules, coated with casein micelles, interact with the like protein network. This work, however, demonstrated that by better understanding the beginning stages of aggregation we may be able to apply approaches such as controlled (modifying composition of the cream phases, for example) homogenization processes tailored to increase curd yields, improve body and texture and fat release, and design novel dairy gels.
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Acknowledgements
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This work was funded by the Ontario Dairy Council and Natural Sciences and Engineering Council of Canada through the Industrial Research Chair program. References
0 0
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Time (s) Fig. 4. Mean square displacement (MSD) as a function of correlation time for homogenized milk (C) and the same milk with Tween 20 added (B). The renneting reaction progresses in the direction of the arrow (experimental time 80 min).
Agboola, S., & Dalgleish, D. G. (1995). Calcium-induced destabilization of oil-inwater emulsions stabilized by caseinate of by b-lactoglobulin. Journal of Food Science, 60, 399e404. Azim, Z., Alexander, M., Koxholt, M., & Corredig, M. (2010). Influence of cross-linked waxy maize starch on the aggregation behaviour of casein micelles during fermentation. Food Biophysics, 5, 227e237.
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Cho, Y. H., Lucey, J. A., & Singh, H. (1999). Rheological properties of acid milk gels as affected by the nature of the fat globule surface material and heat treatment of milk. International Dairy Journal, 9, 537e545. Gaygadzhiev, Z., Corredig, M., & Alexander, M. (2008). Diffusing wave spectroscopy study of the colloidal interactions occurring between casein micelles, emulsion droplets and their mixtures: a comparison to hard sphere behaviour. Langmuir, 24, 3794e3800. Gaygadzhiev, Z., Corredig, M., & Alexander, M. (2009). The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennetinduced gelation of milk. Colloids and Surfaces B: Biointerfaces, 68, 154e162. Ion Titapiccolo, G., Corredig, M., & Alexander, M. (2010a). Modification to casein micelles renneting functionality caused by non-ionic surfactants. Journal of Dairy Science, 93, 506e514. Ion Titapiccolo, G., Alexander, M., & Corredig, M. (2010b). Rennet-induced aggregation of homogenized milk, effect of the interacting oil droplets. Dairy Science and Technology, 90, 623e639. Jana, A. H., & Upadhyay, K. G. (1993). A comparative study of the quality of mozzarella cheese obtained from unhomogenized and homogenized buffalo milks. Cultured Dairy Products Journal, 28, 16e22. Lucey, J. A., Johnson, M. E., & Horne, D. S. (2003). Invited review: perspectives on the basis of the rheology and texture properties of cheese. Journal of Dairy Science, 86, 2725e2743. Mackie, A. R., Gunning, A. P., Wilde, P. J., & Morris, V. J. (1999). Orogenic displacement of proteins from the air/water interface by competitive adsorption. Journal of Colloid Interface Science, 210, 157e166. Maret, G., & Wolf, P. E. (1987). Multiple light scattering from disordered media. The effect of Brownian motion of scatterers. Zeitschrift für Physik B - Condensed Matter, 65, 409e413. Michalski, M. C., Cariou, R., Michel, F., & Garnier, C. (2002). Native vs. damaged milk fat globules: membrane properties affect the viscoelasticity of milk gels. Journal of Dairy Science, 85, 2451e2461. Mulder, H., & Walstra, P. (1974). The milk fat globule-emulsion science as applied to milk products and comparable foods. Farnham Royal, Buckinghamshire, UK: Commonwealth Agricultural Bureaux.
Peters, I. I. (1956). Cheddar cheese made from pasteurized milk homogenized at various pressure. Journal of Dairy Science, 39, 1083e1088. Robson, E. W., & Dalgleish, D. G. (1984). Coagulation of homogenized milk particles by rennet. Journal of Dairy Science, 80, 1901e1907. Romer, S., Sheffold, S., & Shurtenberger, P. (2000). Sol-gel transition of concentrated colloidal suspensions. Physical Review Letters, 85, 4980e4983. Sala, G., van Aken, G. A., Cohen Stuart, M. A., & van de Velde, F. (2007). Effect of droplet-matrix interactions on large deformation properties of emulsion-filled gels. Journal of Texture Studies, 38, 511e535. Sandra, S., Alexander, M., & Dalgleish, D. G. (2007). The rennet coagulation mechanism of skim milk observed by diffusing wave spectroscopy. Journal of Colloid Interface Science, 308, 364e373. Sandra, S., Cooper, C., Alexander, M., & Corredig, M. Coagulation properties of ultrafiltered milk retentates measured using rheology and diffusing wave spectroscopy. Food Research International, in press, doi:10.1016/j.foodres.2011.02.018. Sharma, R., & Dalgleish, D. G. (1993). Interactions between milk serum proteins and synthetic fat globule membrane during heating of homogenized whole milk. Journal of Agricultural and Food Chemistry, 41, 1407e1412. Srinivasan, M., Singh, H., & Munro, P. A. (2000). The effect of sodium chloride on the formation and stability of sodium caseinate emulsions. Food Hydrocolloids, 14, 497e507. Tuinier, R., & De Kruif, C. G. (2002). Stability of casein micelles in milk. Journal of Chemical Physics, 117, 1290e1295. van Vliet, T. (1988). Rheological properties of filled gels. Influence of filler matrix interaction. Colloid and Polymer Science, 266, 518e524. van Vliet, T., & Dentener-Kikkert, A. (1982). Influence of the composition of the milk fat globule membrane on the rheological properties of acid milk gels. Netherlands Milk and Dairy Journal, 36, 261e265. Weitz, D. A., Zhu, J. X., Durian, D. J., Gang, H., & Pine, D. J. (1993). Diffusing-wave spectroscopy: the technique and some applications. Physica Scripta, T49B, 610e621. Zoon, P., van Vliet, T., & Walstra, P. (1988). Rheological properties of rennet-induced skim milk gels. 3. The effect of calcium and phosphate. Netherlands Milk and Dairy Journal, 42, 295e312.
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International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Rennet-induced aggregation of milk containing homogenized fat globules. Effect of interacting and non-interacting fat globules observed using diffusing wave spectroscopy M. Corredig*, G. Ion Titapiccolo, Z. Gaygadzhiev, M. Alexander Department of Food Science, University of Guelph, Guelph, Ontario, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 June 2010 Received in revised form 10 January 2011 Accepted 23 January 2011
The dynamics of the interactions between casein micelles and oil droplets during rennet-induced gelation were described using rheology and diffusing wave spectroscopy. Two different colloidal states were obtained by preparing WPI-stabilized oil droplets in either milk serum or low ionic strength buffer: flocculated and non-flocculated. These two colloidal states strongly affected the diffusion of the noninteracting droplets in the gel and the elasticity of the protein network. In homogenized milk, the droplets interact with the protein network and play a structuring role, causing an increased elastic modulus. However, after displacement of the protein from the interface using Tween 20, the interpretation of the rheological data would lead to incorrect conclusions without light scattering measurements. This work demonstrated that an in-depth understanding of the colloidal properties of the oil droplets is necessary to fine-tune the interactions between these particles and the casein matrix in dairy gels. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction During renneting of milk, k-casein is cleaved specifically by chymosin and, as k-casein plays a major role in imparting colloidal stability to casein micelles, the micelles progressively lose steric repulsion and start interacting with one another (Tuinier & De Kruif, 2002; Zoon, van Vliet, & Walstra, 1988). A three dimensional network is formed, with partly fused casein micelles (Lucey, Johnson, & Horne, 2003). In a rennet-induced gel, the Characteristics of the colloidal particles involved in the pre-gelation, gelation, and rearrangements processes will determine the final properties of the dairy matrix. In particular, fat globules play an important role in structuring of dairy products. It is well documented that the rheological behaviour of filled milk gels depends on the structural properties of the filler particles, the viscoelasticities of the matrix and the filler, the volume fraction of the components, the shape and size of the filler droplets and their interactions with the building blocks of the network (Sala, van Aken, Cohen Stuart, & van de Velde, 2007; van Vliet, 1988). Dairy matrices containing fat globules have been previously described in literature, mostly using model systems consisting of reconstituted milk containing specifically designed and well characterized oil droplets (Cho, Lucey, & Singh, 1999; Michalski, Cariou, Michel, & Garnier, * Corresponding author. Tel.: þ1 519 824 4120; fax: þ1 519 824 6631. E-mail address: [email protected] (M. Corredig). 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.01.010
2002). In general, when the oil droplets “actively” interact with the casein network, there is an increase in the gel modulus, while when oil droplets do not have affinity with the building blocks, there is very little or no contribution to the gel stiffness. However, if the fillers are sufficiently large, they will indirectly affect the formation of the network by limiting the rearrangements of the caseins during gelation, and this will create thicker gel strands with filler particles immobilized within the pores. This has been recently illustrated using microscopy in a dairy matrix containing starch granules (Azim, Alexander, Koxholt, & Corredig, 2010). Most of the reports so far have investigated the rheological and microstructural properties of gelling model systems containing milk proteins and fat globules. Fat globules covered with small molecular weight surfactants (Tween 20 or Tween 60) or whey proteins led to the formation of a weak acid gel with low elastic modulus (G0 ), compared with the same systems gelled with oil droplets which would interact with the network (Cho et al., 1999; van Vliet, 1988; van Vliet & Dentener-Kikkert, 1982). Most of the research in this field has been carried out on acid-induced gels and in reconstituted model systems. In whole non-homogenized milk, the milk fat globules do not participate in the formation of the casein network (Michalski et al., 2002) and behave as “inert” fillers. Homogenization reduces substantially the fat globules’ size, and caseins are the main proteins adsorbed onto the newly covered interface (Mulder & Walstra, 1974; Sharma & Dalgleish, 1993). Renneting of homogenized milk shows a faster release of the
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caseinomacropeptide, a shorter gelation time and high retention of moisture in the curd (Jana & Upadhyay, 1993; Peters, 1956; Robson & Dalgleish, 1984). It has been recently shown that using diffusing wave spectroscopy it is possible to follow the dynamics of oil droplets in mixed systems with casein micelles, as when present in milk, the oil droplets refractive index contrast is much higher than that of the micelles. The oil droplets therefore contribute most of the scattering signal (Gaygadzhiev, Corredig, & Alexander, 2008). The noninvasive nature of this light scattering technique therefore allows using oil droplets as tracer particles during the initial stages of the formation of the protein network. In combination with rheological measurements it is then possible to clearly describe the details of the interactions occurring in rennet-induced gels. The objective of this work was to identify the dynamics of the interactions between casein micelles and fat globules during the formation of a rennet-induced gel. A model system consisting of non-interacting droplets was first investigated to determine the effect of the colloidal state (flocculated and non-flocculated droplets) on the rheological properties and the aggregation behaviour of the casein network. In the second part of this manuscript, the behaviour of fat globules in homogenized milk was studied during renneting, and homogenized milk was compared with skim milk as well as homogenized milk with Tween 20.
by adding appropriate ratios of WPI-stabilized emulsions, concentrated milk (2) and permeate, to a final volume fraction of 5% and 10% for oil droplets and casein micelles, respectively. For control skim milk, 1 vol of 2 concentrated skim milk was diluted with milk permeate. For emulsions prepared in permeate, 1 vol of 2 concentrated milk was added to 1 vol of emulsion in permeate previously diluted in permeate. For the emulsions prepared with imidazole buffer, a 2 permeate was used for the dilution, as this ensured to obtain a similar ionic concentration of the serum phase, as this is a critical factor in rennet-induced milk gels. For the experiments with homogenized milk, fresh whole milk was homogenized at 350 bar for two passes as described above. Aliquots of homogenized milk were also treated with polyoxyethylene sorbitan monolaurate (2%, w/v) (Tween 20, 1227 g mol1 Sigma, St Louis, MI USA), and stirred for 6 h, to ensure protein displacement at the interface. The addition of this small molecular weight surfactant causes the displacement of most of the protein at the interface (Mackie, Gunning, Wilde, & Morris, 1999) resulting in a system containing the same volume fraction of fat globules, with comparable colloidal characteristics and ionic composition, but non-interacting properties of the droplets (Ion-Titapiccolo, Alexander, Corredig, 2010a).
2. Materials and methods
In all cases, gelation experiments were conducted at 30 C soon after homogenization (in the case of homogenized milk with Tween 20, after 6 h), by addition of rennet (Chymostar, single strength, Rhodia, Cranbury, NJ, USA) added at a concentration of 0.018 IMCU mL1. Immediately after the addition of rennet, the samples were subjected to simultaneous measurements using rheology and diffusing wave spectroscopy. Rheological measurements were conducted using a stresscontrolled rheometer (AR 1000, TA Instruments Ltd., New Castle, MA, USA) with a conical concentric cylinder geometry (5920 mm fixed gap, 15 mm radius). A time sweep test was performed for 180 min, with the following parameters: 0.01 strain, 0.1 Hz and an initial oscillation stress of 0.0018 Pa. The gelation time was determined as the time needed to reach tan d ¼ 1. In situ light scattering measurements were performed using diffusing wave spectroscopy (DWS). This technique is based on the measurement of multiple scattered light (Maret & Wolf, 1987). Rennet-induced coagulation was followed at 30 C. The sample was placed in a 5 mm optical glass cuvette (Hellma Canada Limited, Concord, Ontario, Canada). The details of the instrumentation can be found elsewhere (Gaygadzhiev, Corredig, & Alexander, 2009). The intensity of scattered light and autocorrelation functions were collected every 4 min, for a total experimental time of 180 min for the reconstituted milk, and 80 min in the case of homogenized milk. A standard latex suspension (260 nm diameter, Portland Duke Scientific, Palo Alto, CA, USA) was employed to calibrate the laser intensity. The mobility of the particles was obtained from the measurement of the particle diffusion coefficient, directly derived from the characteristic decay time of the correlation function. The mean square displacement (MSD)
2.1. Sample preparation Whole milk was collected at the research farm of the University of Guelph (Elora, Ontario, Canada) and sodium azide (0.02%, w/v) was immediately added to inhibit microbial growth. The skimmed milk was prepared by centrifuging at 4000 g for 20 min at 4 C (Beckman J2-21 centrifuge, Beckman Coulter, Mississauga, Ontario, Canada) and filtering four times through fibreglass filter (Whatman, Fisher Sci., Whitby, Ontario, Canada). This ensured near complete removal of fat. In the reconstituted milk experiments, a 2 concentrated milk was prepared by reducing its volume using ultrafiltration (PLGC, 10 kDa cut-off regenerated cellulose cartridge, Millipore, Bedford, MA, USA). In addition to the retentate (2 milk concentrate), the permeate from milk (this corresponds to the composition of the original milk serum) was collected. For the experiments with WPIemulsions prepared in buffer (see below), to ensure a similar content of soluble calcium in the final reconstituted milk, it was necessary to prepare a 2 permeate starting from skim milk reconstituted from skim milk powder (Parmalat Canada, London, Ontario, Canada) at 20% (w/v) solids. The whey protein-stabilized emulsions were prepared using whey protein isolate (WPI) (Land O’Lakes, St Paul, MN, USA). WPI was either dissolved in 5 mM imidazole buffer at pH 6.7, or in milk permeate. In both cases, the solutions were stirred for 2 h at room temperature and then stored at 4 C. WPI contained 92% protein, 2.7% ash, 1.6% lactose and <0.5% fat (as specified by the manufacturer). Anhydrous milk fat (Parmalat Canada) was pre-emulsified with a high speed blender (PowerGen 125, Fisher Sci.) with the protein solutions to a final 2% (w/w) and 25% (w/w) for protein and milk fat, respectively. The premix was then passed 4 times at 350 bar through a high pressure homogenizer (Emulsiflex C5, Avestin, Ottawa, Ontario, CA). The homogenizer was immersed in a temperature-controlled water bath (Versa-Bath, Fisher Sci.) at 45 C. When a WPI solution in buffer was used, less protein was necessary to stabilize the oil droplets (as shown by preliminary experiments which tested different concentrations of protein and particle size distributions), and 1% (w/w) protein and 20% (w/w) milk fat were used. Recombined milk samples were obtained
2.2. Gelation experiments
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2.3. Statistical analysis
30
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dynamic light scattering (Zetasizer Nano, Malvern Instruments, Worcestershire, MA, UK). For these measurements, the samples were extensively diluted (approximately 2000 times) in permeate (milk serum), pre-filtered through a 0.22 mm filter (Millipore Canada, Missisagua, Ontario, Canada), to preserve as much as possible the ionic conditions. All samples were measured immediately after dilution.
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All experiments were conducted in triplicate (three separate batches of milk and emulsion). Analysis of variance and least significant difference were calculated to determine significant differences between treatments (at 95% confidence level) using SPlus 8.0 (Tibco, Somerville, MA, USA).
15 10 5 0 -5
B
3. Results and discussion Table 1 summarizes the average apparent radius measured for the WPI-stabilized droplets. When the radius was measured using DLS, under extensively diluted conditions, the emulsions prepared in buffer showed an apparent radius of 120 nm once diluted in milk serum (permeate). The apparent radius measured by DWS, under concentrated conditions, also indicated the presence of small droplets, with a value of about 160 nm. It was concluded that these emulsion droplets were stable to aggregation once reconstituted in an ionic environment similar to that of skim milk. On the other hand, when the WPI-emulsions were prepared with a solution prepared in permeate and not buffer, the apparent radius was larger than for the emulsions in buffer. This is clearly shown by the discrepancies in the radius values measured both by DWS and DLS. The results indicated that when the oil droplets were homogenized under relatively high ionic conditions, the droplets were aggregated in flocs. This effect of ionic strength on the stability of emulsion droplets has been previously discussed (Agboola & Dalgleish, 1995; Srinivasan, Singh, & Munro, 2000). Skim milk, concentrated 2 by ultrafiltration, was recombined with permeate and WPI-stabilized emulsions prepared in either imidazole buffer or permeate and diluted to a similar ionic composition using either 2 permeate or permeate, respectively (see Methods for details). Fig. 1 shows the changes in the rheological and light scattering parameters during gelation of skim milk (circles) and skim milk reconstituted with WPI-emulsion droplets, either stable or flocculated (squares or triangles, respectively). Fig. 1A shows the development of G0 as a function of time after the addition of rennet. In skim milk, the value of G0 remained very low until 95.2 (0.2) min, when it started to increase, indicating the formation of a gel. Comparing the rheological behaviour of the control skim milk and the recombined milk with WPI-stabilized oil droplets prepared in buffer, the progression of the G0 value over time showed an earlier gelation point (80.4 4.2 min) but the value of G0 at 180 min was not statistically different. In contrast, the system containing flocculated oil droplets showed a faster gelation and a dramatic increase of the elastic modulus. The recombined Table 1 Particle size of whey protein-stabilized emulsions prepared in either permeate or 5 mM Imidazole buffer, pH 6.7, and dispersed in permeatea. Emulsion (prepared in/dispersed in)
Apparent radius (DLS, nm)
Apparent radius (DWS, nm)
Buffer/permeate Permeate/permeate
120 13 582 18
163 3 217 11
a Radius obtained either using dynamic light scattering (DLS, diluted conditions) or diffusing wave spectroscopy (DWS, undiluted conditions). Values are reported as averages and standard deviations of three experiments.
Radius (μm)
5 4 3 2 1 0
0
20
40
60
80
100
120
140
160
180
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Time (min) Fig. 1. Development of the storage moduli, G0 , measured by rheology (A), and the radius, measured by diffusing wave spectroscopy (B), during the rennet coagulation process of skim milk (C) and recombined milk containing whey protein isolatestabilized oil droplets prepared in permeate (:) and in low ionic strength buffer (-).
milk systems contained non-interacting emulsion droplets, with identical volume fractions of caseins and fat. DWS measurements confirmed the rheological observations. As shown in Fig. 1B, there were clear differences between the treatments in the development of the apparent radius versus renneting time. These results demonstrated in more details the differences in the aggregation behaviour between the two reconstituted milk systems. It should be pointed out that the scattering signal for recombined milk is heavily dominated by the oil droplets, and the results shown here depict the aggregation of the oil droplets as influenced by the presence of a protein gel developing around them. In skim milk, the aggregation point (the point of onset of the radius increase) was in agreement with the gelation point measured with rheology. This was expected from what was reported in previous literature (Sandra, Alexander, & Dalgleish, 2007). A pronounced difference in the particle size change over time was shown between the two reconstituted milk systems. In the milk containing stable oil droplets (those prepared with buffer, square symbols) the dynamic properties of the colloidal particles (the oil droplets) present in the system remained very similar to the initial properties, in spite of the aggregation of the casein micelles around them (a similar G0 value was shown for this sample compared with the skim milk control). In contrast, the recombined milk containing flocculated droplets (those homogenized in milk serum) showed extensive aggregation, with an aggregation time significantly shorter than that of the control sample, indicating a shorter onset of aggregation for these samples. It should be
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pointed out however, that the apparent size obtained by DWS after colloidal mobility is restricted does not represent a true size, but it can still be used as an indication of the decreased mobility. The start of the increase in size happened earlier in the reconstituted milk than for skim milk control, suggesting that the aggregation of the colloidal particles occurred earlier. This effect can not be fully attributed to an increase in volume fraction, as a recent study has demonstrated that gelation time is not affected by concentration (Sandra, Cooper, Alexander, & Corredig, in press). To better determine the changes in mobility over time between the samples, the changes in MSD versus time are shown in Fig. 2. While at the initial time (Fig. 2A) both recombined milk systems and skim milk control showed a free diffusing behaviour, after 180 min of the addition of rennet (Fig. 2B), there were significant differences between the samples. The skim milk control (circles) showed clear signs of arrested motion, as the casein micelles become increasingly entrapped in their own network strands. The recombined milk with aggregated oil droplets (triangles) also showed arrest in motion of the particles, and to a larger extent than for milk control. In this case, the fat globules were hindered in their motion, and were not able to probe all their available space. On the other hand, the WPI-covered droplets prepared in buffer (squares) still showed free diffusing behaviour after 180 min of renneting, even though the rheological parameters indicated that gelation had occurred (Fig. 1A). It was concluded that although the viscoelastic parameters could suggest the presence of “active” interacting filler particles in the case of WPI-stabilized emulsions prepared in permeate, these oil droplets did not participate in the formation of the network, but showed a dramatic decrease in mobility. This was caused by the
6e-5
progressive constraint experienced by the fat globules (present in aggregated form) within the pores of the casein network. The increase in the storage modulus was due to a continuous thickening of the strands around the pores, as influenced by the presence of the oil droplets. In reconstituted milk containing stable WPIemulsion droplets, on the other hand, although there was an increase in storage modulus, the droplets still did not appear to interact with the casein network but were free and small enough to continue to probe the entire space within the pores and not affecting the value of G0 . The increase in apparent particle size shown in Fig. 1B could be attributed to the slowing down of the mobility just due to hydrodynamic effects (crowding). The formation of structure in milk containing homogenized oil droplets is also a clear example of how the rennet gel is affected by the colloidal properties of the fat globules as well as by the nature of the interface. For this reason, the dynamics of the changes occurring during renneting of homogenized milk were studied by rheology and diffusing wave spectroscopy. It is established that homogenized fat globules actively participate in the formation of the rennet-induced gel network, as the caseins adsorbed on the oil droplets interact with those forming the network in the continuous phase (Lucey et al., 2003; Michalski et al., 2002). As a consequence, the elastic modulus of the gel increased dramatically compared with skim milk, as clearly shown in Fig. 3. In this figure, the storage modulus (A) and apparent hydrodynamic radius (Fig. 3B) for skim milk control (circles), homogenized milk (filled squares), as well as homogenized milk with Tween 20 added (empty squares) are shown as a function of renneting time. Tween 20 was added to homogenized milk after homogenization, to displace the proteins 140
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<Δr2(t)> (μm2)
100 4e-5 3e-5 2e-5
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Time (s) Fig. 2. Mean square displacement (MSD) as a function of correlation time for the control skim milk (C) and recombined milks containing whey protein isolate-stabilized oil droplets prepared in permeate (:) and in low ionic strength buffer (-). Panel A, immediately after addition of rennet; panel B, 180 min after addition of rennet.
0
0
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40
60
80
Time (min) Fig. 3. Development of the storage moduli, G0 , measured by rheology (A), and the radius, measured by diffusing wave spectroscopy (B), during the rennet coagulation process of skim milk (C), homogenized milk (-) and homogenized milk with Tween 20 (,).
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from the fat interface, leading to a system containing non-interacting fat globules, with comparable colloidal properties to the original homogenized milk. The gelation point of milk, as measured by rheology (Fig. 3A) was clearly faster in the presence of fat globules. While skim milk gelled at 37 4 min, homogenized milk showed a gelation point at 25.6 3 min. Surprisingly, although the presence of Tween 20 should create non-interacting oil droplets, a faster gelation point was measured (14.7 2.5 min) compared with that of homogenized milk without Tween 20. The elastic modulus after 45 min from the gelation point was also significantly higher for the homogenized milk with Tween 20, compared with homogenized milk or skim milk control. These differences were explained by a direct effect of Tween 20 molecules on the rennet reactivity of the casein micelles. Indeed, it has been previously demonstrated that when Tween 20 is added to skim milk, both the first stage and the second stage of rennet-induced gelation are modified with an increase in the storage modulus (Ion Titapiccolo, Corredig, & Alexander, 2010b). The aggregation behaviour of the three samples is also clearly shown in Fig. 3B, where the results of apparent particle size measured by DWS supported the rheological data. The sudden change in apparent radius occurred earlier for the homogenized milk containing Tween 20, and for the homogenized milk, compared with that of skim milk. However, the samples showed very different rates of increase in size (or, rather, the rate of decrease in the mobility of the particles), because of differences in the interactions with the casein matrix and in their mobility (see below). While homogenized milk and skim milk clearly showed a fast onset of aggregation, in the case of homogenized milk containing Tween 20, after a small change in size (most likely due to crowding of the droplets into the casein pockets) at about 10 min, the apparent radius did not further increase, reaching a plateau at about 1 mm, suggesting a lack of aggregation for the oil droplets. To better clarify the differences in particle mobility between homogenized milk with or without Tween 20, the changes occurring in the MSD versus time curves were recorded, as shown in Fig. 4. The plots for homogenized oil droplets (filled symbols) and homogenized oil droplets containing Tween 20 (empty symbols) are drawn for different times during the gelation process (reaction time increases with arrow). As previously shown for recombined milk (Fig. 2), the initial stages immediately after renneting showed a linear dependence of the MSD with correlation time, indicating free diffusive motion (Romer et al., 2000; Weitz et al., 1993). As the casein
2
<Δr (t)> (μm2)
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micelles become more unstable, because of the cleavage of the kcasein, the fat globules in homogenized milk are no longer able to probe the accessible space range, resulting in an asymptotic behaviour of the MSD curve versus correlation time (clearly seen for the bottom two curves). On the other hand, the homogenized milk droplets treated with Tween 20 still showed free diffusive motion, as the samples kept their linear behaviour for the whole renneting experimental time (w80 min). The changes in slope for the initial stages of the renneting reaction are due to the initial aggregation of the fat globules before casein coagulation and arrested motion occurred. It was therefore concluded that, as expected, homogenized fat globules are an active part of the rennet gel network, and gelation occurs faster. On the other hand, when the protein is displaced from the interface (with the addition of Tween 20) the oil droplets do not participate in the network, and they are trapped in the network cages, unaggregated and free to move around within the pores of the protein network. 4. Conclusions This work clearly demonstrated that with careful design of the colloidal state of the fat globule in combined gels, the interactions with the gel network can be controlled and ultimately affect the gel structure, mechanical and sensory properties of composite gels. In addition to using rheological measurements, we can get further insight into these gelling systems by observing the dynamic behaviour of their components using light scattering. In skim milk containing WPI-covered oil droplets, it was clearly demonstrated that although the oil droplets do not participate in the formation of the gel network, dramatic differences in microstructural characteristics can be obtained by modifying the colloidal state of the fat globules. Small, non-interacting droplets will reside within the pockets formed by the casein network, and will not modify the viscoelasticity of the system. However, when the same droplets are flocculated, their actual size will approximate that of the floc size, and the rate of gel formation and the stiffness of the gel will increase. As the casein gel develops, and the network grows, the flocculated droplets become trapped inside the formed cages. Due to their state of flocculation, the droplets have much less available space that in the non-flocculated case and their mobility is progressively squeezed as the casein gel pores continue to stiffen. This research also confirmed the numerous reports on homogenized oil droplets as fillers in rennet gels. As hypothesized, Tween 20-coated globules behave as “inactive” fillers, while the homogenized globules, coated with casein micelles, interact with the like protein network. This work, however, demonstrated that by better understanding the beginning stages of aggregation we may be able to apply approaches such as controlled (modifying composition of the cream phases, for example) homogenization processes tailored to increase curd yields, improve body and texture and fat release, and design novel dairy gels.
4e-5
Acknowledgements
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This work was funded by the Ontario Dairy Council and Natural Sciences and Engineering Council of Canada through the Industrial Research Chair program. References
0 0
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4e-5
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Time (s) Fig. 4. Mean square displacement (MSD) as a function of correlation time for homogenized milk (C) and the same milk with Tween 20 added (B). The renneting reaction progresses in the direction of the arrow (experimental time 80 min).
Agboola, S., & Dalgleish, D. G. (1995). Calcium-induced destabilization of oil-inwater emulsions stabilized by caseinate of by b-lactoglobulin. Journal of Food Science, 60, 399e404. Azim, Z., Alexander, M., Koxholt, M., & Corredig, M. (2010). Influence of cross-linked waxy maize starch on the aggregation behaviour of casein micelles during fermentation. Food Biophysics, 5, 227e237.
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Cho, Y. H., Lucey, J. A., & Singh, H. (1999). Rheological properties of acid milk gels as affected by the nature of the fat globule surface material and heat treatment of milk. International Dairy Journal, 9, 537e545. Gaygadzhiev, Z., Corredig, M., & Alexander, M. (2008). Diffusing wave spectroscopy study of the colloidal interactions occurring between casein micelles, emulsion droplets and their mixtures: a comparison to hard sphere behaviour. Langmuir, 24, 3794e3800. Gaygadzhiev, Z., Corredig, M., & Alexander, M. (2009). The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennetinduced gelation of milk. Colloids and Surfaces B: Biointerfaces, 68, 154e162. Ion Titapiccolo, G., Corredig, M., & Alexander, M. (2010a). Modification to casein micelles renneting functionality caused by non-ionic surfactants. Journal of Dairy Science, 93, 506e514. Ion Titapiccolo, G., Alexander, M., & Corredig, M. (2010b). Rennet-induced aggregation of homogenized milk, effect of the interacting oil droplets. Dairy Science and Technology, 90, 623e639. Jana, A. H., & Upadhyay, K. G. (1993). A comparative study of the quality of mozzarella cheese obtained from unhomogenized and homogenized buffalo milks. Cultured Dairy Products Journal, 28, 16e22. Lucey, J. A., Johnson, M. E., & Horne, D. S. (2003). Invited review: perspectives on the basis of the rheology and texture properties of cheese. Journal of Dairy Science, 86, 2725e2743. Mackie, A. R., Gunning, A. P., Wilde, P. J., & Morris, V. J. (1999). Orogenic displacement of proteins from the air/water interface by competitive adsorption. Journal of Colloid Interface Science, 210, 157e166. Maret, G., & Wolf, P. E. (1987). Multiple light scattering from disordered media. The effect of Brownian motion of scatterers. Zeitschrift für Physik B - Condensed Matter, 65, 409e413. Michalski, M. C., Cariou, R., Michel, F., & Garnier, C. (2002). Native vs. damaged milk fat globules: membrane properties affect the viscoelasticity of milk gels. Journal of Dairy Science, 85, 2451e2461. Mulder, H., & Walstra, P. (1974). The milk fat globule-emulsion science as applied to milk products and comparable foods. Farnham Royal, Buckinghamshire, UK: Commonwealth Agricultural Bureaux.
Peters, I. I. (1956). Cheddar cheese made from pasteurized milk homogenized at various pressure. Journal of Dairy Science, 39, 1083e1088. Robson, E. W., & Dalgleish, D. G. (1984). Coagulation of homogenized milk particles by rennet. Journal of Dairy Science, 80, 1901e1907. Romer, S., Sheffold, S., & Shurtenberger, P. (2000). Sol-gel transition of concentrated colloidal suspensions. Physical Review Letters, 85, 4980e4983. Sala, G., van Aken, G. A., Cohen Stuart, M. A., & van de Velde, F. (2007). Effect of droplet-matrix interactions on large deformation properties of emulsion-filled gels. Journal of Texture Studies, 38, 511e535. Sandra, S., Alexander, M., & Dalgleish, D. G. (2007). The rennet coagulation mechanism of skim milk observed by diffusing wave spectroscopy. Journal of Colloid Interface Science, 308, 364e373. Sandra, S., Cooper, C., Alexander, M., & Corredig, M. Coagulation properties of ultrafiltered milk retentates measured using rheology and diffusing wave spectroscopy. Food Research International, in press, doi:10.1016/j.foodres.2011.02.018. Sharma, R., & Dalgleish, D. G. (1993). Interactions between milk serum proteins and synthetic fat globule membrane during heating of homogenized whole milk. Journal of Agricultural and Food Chemistry, 41, 1407e1412. Srinivasan, M., Singh, H., & Munro, P. A. (2000). The effect of sodium chloride on the formation and stability of sodium caseinate emulsions. Food Hydrocolloids, 14, 497e507. Tuinier, R., & De Kruif, C. G. (2002). Stability of casein micelles in milk. Journal of Chemical Physics, 117, 1290e1295. van Vliet, T. (1988). Rheological properties of filled gels. Influence of filler matrix interaction. Colloid and Polymer Science, 266, 518e524. van Vliet, T., & Dentener-Kikkert, A. (1982). Influence of the composition of the milk fat globule membrane on the rheological properties of acid milk gels. Netherlands Milk and Dairy Journal, 36, 261e265. Weitz, D. A., Zhu, J. X., Durian, D. J., Gang, H., & Pine, D. J. (1993). Diffusing-wave spectroscopy: the technique and some applications. Physica Scripta, T49B, 610e621. Zoon, P., van Vliet, T., & Walstra, P. (1988). Rheological properties of rennet-induced skim milk gels. 3. The effect of calcium and phosphate. Netherlands Milk and Dairy Journal, 42, 295e312.