Interactions between Denatured Milk Serum Proteins and Casein

The acid-induced aggregation of casein micelles from milk, in the presence of different whey protein preparations from heated and unheated milk, has b...
1 downloads 0 Views
11380

Langmuir 2005, 21, 11380-11386

Interactions between Denatured Milk Serum Proteins and Casein Micelles Studied by Diffusing Wave Spectroscopy Marcela Alexander and Douglas G. Dalgleish* Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received July 22, 2005. In Final Form: September 13, 2005 The acid-induced aggregation of casein micelles from milk, in the presence of different whey protein preparations from heated and unheated milk, has been studied using diffusing wave spectroscopy (DWS). In particular, the study focused on the turbidity (or l*) parameter obtainable from DWS, which can give information on the interactions between particles in aggregating systems. The experiments provided evidence that the presence of small, soluble, whey protein/κ-casein aggregates derived from heated milk gave rise to interactions with both heated and unheated casein micelles over a pH range of 5.6 down to 5.2. Comparison of heated and unheated milks, together with milks whose sera had been exchanged, showed that direct interactions were indeed occurring, even between untreated casein micelles and soluble whey protein complexes. Comparison of the behavior of the whey protein aggregates in emulsion preparations where they could not interact with the large particles confirmed that the effect was specific to the presence of casein micelles and could not arise simply from the aggregation of the whey proteins themselves.

1. Introduction Acidified milk and milk drinks have been known for thousands of years. Acidification of milk by chemical acidulants or by lactic acid bacteria induces destabilization of the colloidal suspension of casein micelles, which eventually aggregate irreversibly to form a particulate gel. There are several hypotheses regarding the mechanisms by which aggregation occurs.1,2 The most straightforward theory is to treat the casein micelles of the milk as sterically stabilized colloids, so that, on acidification of unheated milk, the slightly negatively charged surface layer of κ-casein on the micelle surface is neutralized, resulting in a partial collapse of its extended conformation.3 This results in a simultaneous loss of both electrostatic and steric stabilization, allowing the micelles to approach closely and eventually to aggregate and gel.4 It has also long been established that the strength (as measured for example, by the elastic modulus, G′), of the acid gel is greatly enhanced by a prior heat treatment of the milk at temperatures above 80 °C.5-7 In heat-treated milk, the pH where gelation occurs is increased, the coagulation time is reduced, and the gel is strengthened.8 The heating process denatures the whey proteins in the milk serum,8,9 which then interact among themselves and with the micellar κ-casein to create soluble whey protein/ κ-casein aggregates or to bind to the micellar surface.10-12 * To whom correspondence should be addressed. E-mail: [email protected]. (1) Heertje, I.; Visser, J.; Smits, P. Food Microstruct. 1985, 4, 267277. (2) Horne, D. S.; Davidson, C. M. International Dairy Federation, Special Issue 1993, 9303, 267-276. (3) De Kruif, C. G.; Zhulina, E. B. Colloids Surf. A 1996, 117, 151159. (4) Lucey, J. A.; Singh, H. Food Res. Int. 1998, 30, 529-542. (5) Mulvihill, D. M.; Grufferty, M. B. In Heat-Induced Changes in Milk; Fox, P. F., Ed.; IDF Special Issue No. 9501; IDF: Brussels, 1995; pp 188-205. (6) Van Vliet, T.; Keetels, C. J. A. M. Neth. Milk Dairy J. 1995, 49, 27-35. (7) Lucey, J. A.; Munro, P. A.; Singh, H. Int. Dairy J. 1999, 9, 275279. (8) Vasbinder, A. J.; van Mil, P. J. J. M.; Bot, A.; de Kruif, C. G. Colloids Surf. B 2001, 21, 245-250. (9) Corredig, M.; Dalgleish, D. G. Food Res. Int. 1996, 29, 49-55. (10) Anema, S. G.; Li, Y. J. Agric. Food Chem. 2003, 51, 1640-1646.

The acid-induced destabilization of this heated milk can no longer be described in the simple colloid-chemical terms used for unheated milk,13 because not only are the casein micelles themselves altered, but there are potentially reactive particles dispersed in the serum, whose concentration significantly affects the gelation of the heated milk.10,14 These soluble complexes of serum proteins appear to act as bridges between the casein micelles and to facilitate aggregation.15,16 However, it is not wellestablished whether this involves a two-step reaction involving first the coagulation of the soluble whey protein complexes themselves, followed by interactions with the casein micelles, or whether both interactions (whey complex-whey complex and whey complex-casein micelle) occur simultaneously. It has been reported that, in rheological measurements of milk during acidification, there is a two-stage development of the elastic modulus around the coagulation point.4 The first stage of this process has been attributed to the gelation of the whey protein aggregates,12 but there is yet no direct evidence of this “whey-gel”. Nor, indeed, is there any direct evidence for the interactions between the soluble whey proteins and the casein micelles, whether the latter came from heated or unheated milk, although the results from rheology may suggest that these interactions do indeed occur.16,17 To try to gain further insight into the respective functions of soluble whey protein/κ-casein complexes and native or heat-treated casein micelles, we have studied the acid coagulation of milk preparations using diffusing wave spectroscopy (DWS). This technique allows the investigation of aggregation phenomena in a noninvasive way, and most importantly, without the need to dilute the (11) Vasbinder, A. J.; de Kruif, C. G. Int. Dairy J. 2003, 13, 669-677. (12) Guyomarc’h, F.; Law, A. J. R.; Dalgleish, D. G. J. Agric. Food Chem. 2003, 51, 4652-4660. (13) Horne, D. S. Colloids Surf., A 2003, 213, 255-263. (14) Guyomarc’h, F.; Queguiner, C.; Law, A. J. R.; Horne, D. S.; Dalgleish, D. G. J. Agric. Food Chem. 2003, 51, 7743-7750. (15) Lucey, J. A.; Teo, C. T.; Munro, P. A.; Singh, H. J. Dairy Res. 1997, 64, 591-600. (16) Schorsch, C.; Wilkins, D. K.; Jones, M. G.; Norton, I. T. J. Dairy Res. 2001, 68, 471-481. (17) Lucey, J. A.; Teo, C. T.; Munro, P. A.; Singh, H. Food Hydrocolloids 1998, 12, 159-165.

10.1021/la0519958 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/13/2005

Interactions between Denatured Proteins and Micelles

Langmuir, Vol. 21, No. 24, 2005 11381

samples. Especially, we have used the photon transport mean free path (l*) parameter to follow interparticle interactions as the acidification progressed. These measurements were made in parallel with measurements of apparent particle radii, up to the point where the milks coagulated. In addition to studying milks, we have used different combinations of casein micelles and sera to define the behavior of all of the constituents of heated and unheated milks. 2. Diffusing Wave Spectroscopy Light scattering has been used for many years in the study of colloidal systems. It is relatively inexpensive, easy to use, and the theory is well understood. However, the need to dilute the sample to reach the single scattering regime has hampered its utilization in more industrially realistic (for example, turbid and gelling) samples. In recent years, DWS has been gaining acceptance as a method for studying highly turbid media such as milk.8,18,19 The basic principle of DWS is that of dynamic light scattering; however, the path of the photon of light in DWS is treated as a random walk through the sample. This requires the turbidity of the sample to be such that there is very little transmitted light and, therefore, no nonscattered or singly scattered photons on the detection side. In such a case, the field autocorrelation function of light passing through a turbid sample can be written as20

g(1)(t) ≈

(

(l*L + 34) x6tτ

) [x] x

L 8t 1+ sinh 3τ l*

4 6t + τ 3

[x]

6t L cosh τ l*

6t τ

(1)

where τ ) (Dko2)-1, where D is the particle diffusion coefficient, ko ) 2πn/λ is the wave vector of the light, and n is the refractive index of the milk serum. L is the thickness of the sample being measured. The factor l* is the photon transport mean free path, related to the mean free path of photons between scattering events. This correlation function ( eq 1) is only valid when the thickness of the sample L . l* (i.e., L/l* > 10) and t , τ. For this case, it is very nearly exponential in time and has a characteristic decay time of τ(l*/L)2. The quantity l* can be defined as the length scale over which the direction of the scattered light has been completely randomized, and it is directly related to the total scattered intensity of the system. It can be calculated from the equation:

Ti )

I 5l*/3L ) I0 1 + 4l*/3L

(2)

For completely noninteracting scatterers that are completely uncorrelated spatially, the value of l* depends on particle size, particle concentration, and index of refraction of the scatterers and the dispersion medium and the wavelength of the laser light. In concentrated and optically dense suspensions, however, the spatial positions of the particles and their correlation may become significant. These correlations in position affect the angular distribution of the scattered light and hence the (18) Vasbinder, A. J.; van de Velde, F.; de Kruif, K. G. J. Dairy Sci. 2004, 87, 1167-1176. (19) Hemar, Y.; Singh, H.; Horne, D. S. Curr. Appl. Phys. 2004, 4, 362-365. (20) Weitz, D. A.; Pine, D. J. In Dynamic Light Scattering, The Methods and Some Applications; Brown, W., Ed.; Oxford University Press: Oxford, 1993; p 652.

turbidity and l*. In general, l* is a function of the scattering form factor, F(q), and the structure factor, S(q):20



l* ∝ ( F(q)‚S(q)‚q3 dq)-1

(3)

The form function, F(q), is related to the size, shape, and contrast of the scatterers, as is used in the Mie21 and Rayleigh-Gans-Debye22 descriptions of dilute suspensions of scattering particles. The structure factor, S(q), describes the positional correlations between the scatterers. In highly diluted systems, such as those used in traditional static or dynamic light scattering, S(q) is generally assumed to be equal to 1 since the particles are far apart and completely uncorrelated in their positions. However, for concentrated suspensions such as milk or food emulsions, where there is a substantial volume fraction (φ g 0.1) of dispersed phase, then the dispersed particles are relatively close to one another (for a suspension of φ ) 0.1 of monodisperse particles, the distances between adjacent particles are approximately 0.75 of the particle diameter). In this case, spatial correlations may become important in the light scattering, especially if the particles are charged or can otherwise affect one another. In such a case, assuming that the volume fraction and refractive index contrast between the particles and the continuous phase are constant throughout a process, a change in the value of l* will indicate some degree of increase in the interactions between particles because interparticle interactions will define S(q). This will occur when the system tends to aggregate, gel, or crystallize. We have shown previously that, for the milk system as it is acidified, some changes in l* can be explained by correlations between particles.23 In other words, although it is not possible at this stage to calculate the magnitudes of interparticle interactions directly from the measured l*, nevertheless changes in this parameter can be taken as indications of changing organization within the suspension. 3. Materials and Methods 3.1. Milks, Milk/Serum Mixtures, and Emulsions. Fresh milk was collected from the Ponsonby Research Station of the University of Guelph (Guelph, Ontario), and sodium azide (0.02% w/v) was immediately added to prevent bacterial growth. The raw milk was skimmed at 6000g for 20 min at 5 °C in a BeckmanCoulter centrifuge (model J2-21, Beckman Coulter, Mississauga, Ontario, Canada) and filtered three times through Whatman glass fiber filters (Fisher Scientific, Mississauga, Canada). Heat treatment, when necessary, was performed on samples of milk in glass vials in a water bath at 85 °C for 20 min (time to attain temperature was 3 min), followed by immediate cooling in an ice bath. These milks were each separated into two batches. One batch of each (heated and unheated milk) was stored in the refrigerator until needed, while 20 g of the other batches (also heated and unheated milk) were centrifuged at 22 592g at 20 °C for 60 min in a rotor type 45Ti (Beckman Coulter, Mississauga, Ontario, Canada). This treatment is known to be sufficient to sediment the casein micellar fraction in the milks but to leave the native serum proteins and the serum protein/κ-casein complexes in the serum.12 The micellar pellets were removed from the sera and labeled UP and HP for unheated and heated pellet, respectively. The sera (labeled US and HS) were similarly collected. Different mixtures of the casein micelles and sera were then made by adding all the US to the HP and all of the HS to the HP, to give HP/US and UP/HS mixtures, respectively. The entire serum and micelle fractions were used to ensure as much (21) Mie, G. Ann. Phys. 1908, 25, 377-445. (22) Van de Hulst, H. C. Light Scattering by Small Particles; John Wiley: New York, 1957. (23) Alexander, M.; Corredig, M.; Dalgleish, D. G. Food Hydrocolloids 2006, 20, 325-331.

11382

Langmuir, Vol. 21, No. 24, 2005

as possible that a constant volume fraction of the casein micelles in solution was maintained. These mixtures were premixed for 1 min using a high-speed blender (PowerGen 125, Fisher Scientific, Co., Nepean, Ontario, Canada) and then gently stirred for 30 min to ensure dispersion of the casein micelles and were stored at 4 °C until required. This mixing allowed us to have four different samples, containing different protein materials: (i) untreated milk (UP/ US) containing native casein micelles suspended in a serum containing native whey proteins, (ii) heated milk (HP/HS), containing casein micelle/whey protein complexes suspended in a serum containing soluble whey protein/κ-casein complexes; in this case no undenatured serum protein was present, (iii) the HP/US mixture, containing casein micelle/whey protein complexes suspended in a serum containing only native whey proteins, and (iv) the UP/HS mixture, where native casein micelles were suspended in a serum containing soluble whey protein/κ-casein complexes. The different complexes present in the sera of the milks in the absence of casein micelles cannot be studied simply by removing the casein micelles because the light scattering of sera from either unheated or heated milks is much too low to allow their study by DWS. Therefore, to study their behavior in the absence of potentially interacting micellar material, they were mixed with droplets of an emulsion with which we believed they did not interact. This created suspensions of the necessary turbidity but that contained no casein micelles. The emulsion was prepared by homogenizing 20% (w/w) soybean oil with an aqueous solution of 1% Tween-20 (Sigma-Aldrich Canada Ltd, Oakville, Ontario, Canada) in a high-pressure homogenizer (Emulsiflex C5, Avestin, Ottawa, Ontario, Canada) at a pressure of 15 000 psi (100 MPa). The emulsion was prepared by passing a pre-emulsion through the homogenizer two times. The emulsion was used after storage at 4 °C for 24 h. The average particle size (d32) of the emulsion droplets was 430 nm as measured by a Mastersizer X (Malvern Instruments, Southboro, MA). To make the mixtures with the serum proteins, the emulsion was centrifuged at 20 992g at 20 °C for 30 min. The upper layer of emulsion droplets was collected, and 1.78 g was added to 18.02 g of serum prepared from either heated or unheated milks by centrifugation to give a volume fraction of emulsion droplets of 0.1. These mixtures were gently stirred for 30 min and used immediately. 3.2. Acidification. Acidification of the milk samples was achieved by the addition of 1.5% solid glucono-δ-lactone (GDL), at a temperature of 30 °C. Sufficient sample quantities were made to fill the sample cell of the DWS spectrometer and to allow a separate batch to be used for determining the pH. The pH of both milk and emulsion samples was measured and related to time as described previously.24 For the acidification of the emulsion-based samples, which have a lower buffer capacity than the milks and milk mixtures, a concentration of GDL of 1.0% was used. 3.3. Diffusing Wave Spectroscopy. Measurements were made using an apparatus constructed in the laboratory, as described previously.25 Light of wavelength 488 nm from a 30 mW solid-state laser (Model 532-100MBS, Omnichrome, Chino, CA) was passed through the sample that was contained in a rectangular flat-faced glass cuvette, with a path length L ) 4 mm, immersed in a tank of water (1 L) maintained at 30 °C. The scattered light was collected and fed via photomultipliers to a correlator, which generated the l* values and the correlation functions from which the values of τ were calculated. The theoretical analysis and calculation of the 1/l* parameter and subsequent radius computation have been explained in detail elsewhere.25 It should be noted that the radii that we quote are calculated from the measured diffusion coefficients by means of the Stokes-Einstein equation. This implies that any changes in diffusion coefficient are caused by changes in size of the particles. To make the measurements, the milk and emulsion samples were pre-equilibrated for 10 min in a water bath at 30 °C. The (24) Dalgleish, D. G.; Alexander, M.; Corredig, M. Food Hydrocolloids 2004, 18, 747-755. (25) Alexander, M.; Dalgleish, D. G. Colloids Surf. B 2004, 38, 8390.

Alexander and Dalgleish

Figure 1. Apparent radii of particles in the milks and mixtures of serum and casein micelles during acidification by 1.5% GDL. The different curves show the behavior of UP/US (filled circles), HP/HS (filled squares), UP/HS (open circles), and HP/US (open squares). defined amount of solid GDL was added to a stock of milk or emulsion that was gently shaken to disperse and dissolve the acidulant. The sample cell was filled and immediately placed in the DWS spectrometer. The time that elapsed between the addition of GDL to the start of the first run (always less than 2 min) was noted. Collection of the correlation functions was then started immediately. Each measurement took 3 min, and measurements were continued every 2 min up to a total collection time of between 2 and 4 h. All of the results shown are averages of two independent experiments.

4. Results and Discussion 4.1. Overall Features of the Reaction. Figure 1 shows the apparent radii of the different micelle/serum mixtures as functions of pH during the acidification reactions. These show the well-known gelation properties of the casein micelles as a result of acidification. On the large scale, in unheated milk, the radius of the casein micelles remained approximately constant (but see below) until a pH of about 5, below which there was a rapid increase, associated with the extensive aggregation of the particles and the gelation of the milk. The heated milk, by contrast, showed the dramatic increase in apparent particle size at a considerably higher pH (5.4). These observations are similar to those of Vasbinder et al.8 and agree with what is known of the rheology of the gelation of heated milk.7,14,26 Interestingly, the two mixed systems, HP/US and UP/HS, behaved very similarly in respect of their radii, showing the large increase in size starting at pH 5.3. Vasbinder et al. 27 have shown that the extent of denaturation of the serum proteins shifts the pH of coagulation, but our results show in addition that either component of heated milk (the micellar or the serum complex fraction) can enhance the onset of gelation equally. Heated casein micelles are known to have some denatured whey proteins attached to their surfaces.8-10 These proteins will tend to interact with those present on neighboring micelles to cause aggregation (i.e., they effectively change the isoelectric point of the casein micelles).16 However, the situation with unheated casein micelles is different. Generally, these are stable to aggregation down to a pH of around 5.0. It has generally (26) Horne, D. S. Int. Dairy J. 1999, 9, 261-268. (27) Vasbinder, A. J.; Alting, A. C.; de Kruif, K. G. Colloids Surf. B 2003, 31, 115-123.

Interactions between Denatured Proteins and Micelles

Figure 2. Development of 1/l* as a function of system pH during acidification by 1.5% GDL. Symbols are the same as in Figure 1. Results are the averages of two independent runs for each sample. For clarity, error bars are not shown, but maximum errors are (0.07 mm-1 until the systems become non-ergodic at pH values below pH 5.2 for the systems containing heated components and below pH 4.9 for the UP/US milk.

been assumed that they did not interact with whey proteins in solution, although it is known that the presence of denatured whey proteins in the serum does increase the strength of acid gels.7 There seemed in these aggregation results to be no difference between the two different mixtures, so that as long as either the micelles or the serum came from heated milk (at least if the heating had been done at the natural pH of the milk), the aggregation potential seemed to be the same. However, it will be seen later that the two mixtures behaved very differently in other respects. Figure 2 shows the behavior of the l* parameter (expressed as 1/l*) as the pH decreased in the four samples. From this, it can be seen that there is a marked difference between the samples containing heated micelles and those containing unheated micelles. First, the samples containing heated casein micelles had a larger starting value of 1/l* than those containing unheated micelles. Second, the former show a more steady increase in 1/l* as the acidification proceeds, whereas the latter are more flat to start with and then show a rapid increase in 1/l*. However, for all of the mixtures, the values of 1/l* increased from their initial values in the course of the acidification reaction until a pH of about 5.8 was reached, when three out of the four formed a plateau, or tended to do so. Below pH 5.8, differences in behavior between the different samples became apparent. In both of the mixtures that contain heated serum (UP/HS and HP/HS), there was a small but reproducible discontinuity of the curve that was not present in the samples containing unheated serum. The region of these effects is marked by arrows in the figure. This is most evident when the results for unheated milk and UP/HS are compared. The measurements in UP/HS show that there seems to be an increase in 1/l*, starting at about pH 5.5, superimposed upon the trend shown by the unheated milk. A similar, but less evident, difference appears when the HP/HS and HP/US mixtures are compared. Evidently, heated and unheated micelles show very different patterns of behavior when they are acidified, and effects of the serum on 1/l* seem to be smaller, although by no means negligible. It is of course expected that the scattering behavior of the milks and mixtures will be dominated by the casein micelles because they constitute 80% or more of the particulate material and

Langmuir, Vol. 21, No. 24, 2005 11383

because they are considerably larger than the complexes formed by the serum proteins, even in heated milk. It will be noted that not all of the graphs in Figure 2 extend to the lowest pH values. This is because, once the milks truly gel, the systems become non-ergodic and the measurements of l* tend to become very noisy. 4.2. Behavior of the Casein Micelles during the First Stage of Acidification. When they are studied in detail, the changes in the apparent radii for the different milk systems (Figure 3) all show a similar pattern, namely that the radii decrease with decreasing pH, level off, and then increase. In all cases, the increase begins at pH values about 0.4 larger than the pH where rapid increase in the particle size is observed, and that correspond to the onset of gelation as measured by rheology.14 The sizes of the decreases in radius before the onset of the increase of aggregation are approximately the same for all of the casein micelles, no matter whether they are heated or not. On average, the micellar radius decreases by 8.9 nm from its initial value of 96.1 nm. This is approximately consistent with the presence of a hairy layer of κ-casein macropeptide28 that is collapsed as a result of titration.1,29 However, rather than being quite sudden, as suggested by other results,8 our observations suggest that the decrease in radius seems to start early in the acidification process and to proceed gradually. Also, it is evident from the results that the binding of serum protein to the casein micelles during heating does not appear to interfere with the hairy layer or its properties since the change in radius during acidification is the same for heated and unheated casein micelles. These results shown in Figures 2 and 3 suggest that there may be different stages in the changing of the 1/l* parameter during the acidification. The first stage occurs between pH values of 6.7 and about 5.8. During this period, the value of 1/l* increases to a plateau, as is clearly seen in the behavior in the mixtures containing unheated casein micelles (US/UP and US/HP), and strongly suggested in the HP/HS sample. In experiments performed with less GDL, where the speed of the reaction was slower (results not shown here), this plateau was very visible, even for heated samples. Since the radius was seen to decrease during this stage of the reaction, there is no possibility that the increase in 1/l* is caused by aggregation of the particles; furthermore, there is no evidence from other techniques that we know of to suggest that casein micelles interact at these pH values. It therefore seems probable that there is a change in the optical properties of the casein micellar particles as a function of pH; that is, that the early changes in 1/l* are caused by a change in F(q) in eq 3. All of the casein micelles (heated and unheated) behave rather similarly over this range of pH, and this may be taken as an indication that the acidification is causing internal changes in the particles that result in the changes in the scattering parameters. It is difficult to model the light scattering of casein micelles because of their complex structures. The particles consist of a central core, composed of many calcium phosphate nanoclusters surrounded by casein proteins,30 the whole being surrounded by a diffuse layer of macropeptides from κ-casein.31 Neither the “core” nor the (28) Holt, C.; Dalgleish, D. G. J. Colloid Interface Sci. 1986, 114, 727-734. (29) Horne, D. S.; Davidson, C. M. Colloid Polym. Sci. 1986, 264, 727-734. (30) Holt, C.; Timmins, P. A.; Errington, N.; Leaver, J. Europ. J. Biochem. 1998, 252, 73-78. (31) De Kruif, C. G.; Holt, C. In Advanced Dairy Chemistry, Vol.1 Proteins; Fox, P. F., McSweeney, P. L. H., Eds.; Kluwer Academic/ Plenum Publishers: New York, 2003, pp 233-276.

11384

Langmuir, Vol. 21, No. 24, 2005

Alexander and Dalgleish

Figure 3. Plots showing the details of the early stages of the changes in radius, as well as the changes in 1/l*, of the particles in the different mixtures. All of the graphs are shown on the same scales. In all of the plots, the filled squares refer to the measurements of radius and the open squares refer to the measurements of 1/l*.

“hairy layer” are completely rigid, and both are relatively highly hydrated.31,32 During acidification, the surface layer is thought to be collapsed as it is neutralized,1,29 as shown by the decrease in the hydrodynamic radius, and at the same time, the value of 1/l* increases. However, simply removing the hairy layer by the use of rennet does not cause a significant change in 1/l*, although the hydrodynamic radius decreases by about 7 nm.24 The scattering of the casein micelles is therefore dominated by the core, and the early increase in 1/l* is probably the result of changes in the core of the casein micelles as the acidification proceeds. Calculation33 shows that a change in the refractive index of the core by only 0.002 is sufficient to produce the observed changes in 1/l*. This could be produced by collapsing the hairy layer on to the core (and therefore increasing its mass), or by slightly shrinking the core itself. That is, we believe that the early changes in the 1/l* parameter are a result of changes in F(q) in eq 3. A similar explanation may be used for the differences in 1/l* between the heated and unheated casein micelles before the acidification starts. The HP/HS and HP/US samples show similar measurements of 1/l* in this stage of the acidification, and so changes in 1/l* do not arise from the different sera in these samples but must be from the micellar particles. It is established that the composition and probably surface structure of the casein micelles is altered as a result of heating, and so it is perhaps not unexpected that their optical properties would be slightly altered. Again, the differences in the refractive index of (32) Tunier, R.; de Kruif, C. G. J. Chem. Phys. 2002, 117, 12901295. (33) Alexander, M.; Rojas-Ochoa, L. F.; Leser, M.; Schurtenberger, P. J. Colloid Interface Sci. 2002, 253, 35-46.

the micellar cores required to give the observed changes in 1/l* are very small. The second stage of the changes in 1/l* arises from the interaction and aggregation of the particles at pH values below a pH of about 5.5. The results from the UP/US and UP/HS mixtures demonstrate that the first-stage changes in 1/l* are complete by a pH of 5.8 because the curves of 1/l* against pH level off at this point. We therefore believe that it is legitimate to subtract these from the total (Figure 4). The results on the samples containing heated casein micelles are less clear, but we have similarly subtracted the changes seen in the unheated micelles from those of the heated micelles. When this is done, a consistent picture emerges in all the milks and mixtures that, for any of the systems, 1/l* and the apparent radius begin to increase significantly at the same point, although the pH where this happens is of course dependent on the composition of the samples (pH 5.4, 5.6, 5.8, and 5.7 for UP/US, UP/ HS, HP/HS, and HP/US, respectively). This is consistent with the beginning of interactions between the particles, which alter their motion, diffuse more slowly, and at the same time alter the S(q) parameter in eq 3. As the pH drops, the repulsion potential between the casein micelles decreases, the tendency to aggregate (and to change spatial distribution) increases, and we see changes in both the apparent radius and the 1/l* parameters. 4.3. Aggregation Behavior of Milks and Mixtures. In unheated milk (UP/US) at a pH of approximately 5.3, the apparent radius begins to increase, although, as has been shown in Figure 1, the true gelation of the milk (as defined by the very rapid increase in the apparent radius) is not apparent until pH 5.0. It is evident, however, that the value of 1/l* also starts to increase at pH 5.3, in parallel with the change in apparent radius. Essentially the same

Interactions between Denatured Proteins and Micelles

Langmuir, Vol. 21, No. 24, 2005 11385

Figure 4. Plots showing the details of the early stages of the changes in apparent radius, as in Figure 3, and the changes in 1/l* with the first-stage contribution subtracted (it is assumed that this effect is constant at pH values below 5.6). The plot for HP/US, where the plateau in 1/l* in Figure 3 was not observed, was corrected by subtracting the results for HP/HS in the pH range 6.7-5.8. The arrows in the different plots indicate the pH values where 1/l* and particle radius begin to increase simultaneously.

pattern was seen with the UP/HS mixture, although for this, the point where radius and 1/l* started to increase was about pH 5.5. Since the casein micelles in both of these samples (UP/US and UP/HS) derive from unheated milk, the only cause of the early and parallel changes in 1/l* and radius in the UP/HS sample can be the behavior of the whey protein complexes present in the heated serum. Since we know that unheated casein micelles do not begin to aggregate and show an increase of 1/l* until a pH of 5.3 and since the serum protein complexes cannot be seen on their own (see below), we believe that the only way that the changes in radius in the UP/HS mixture can be explained is by direct interaction between the whey protein complexes and the casein micelles. While this has been inferred from the gelation behavior,16,34 our results constitute direct evidence for these interactions. The analysis leaves us with further questions. The change of 1/l* during aggregation of the milks and mixtures depends strongly on the presence of heated casein micelles, as shown in Figures 2 and 4. In the mixtures containing the heated micelles, the overall increase during acidificationis only about half of that seen in those containing unheated casein micelles. Part of this change comes from the change in F(q) (see above). However, the later changes in 1/l* must reflect the structural organization of the particles in solution as they associate to form gels, especially if F(q) does not change during the later acidification period. The “final” value of 1/l* (final in our (34) Lucey, J. A.; Tamehana, M.; Singh, H.; Munro, P. A. J. Dairy Res. 1998, 65, 555-567.

case refers to the point just prior to true gelation when the system stops being ergodic) corresponding to the heated pellets is significantly smaller than that for unheated pellets (Figure 2), and the rate of change of 1/l* in the later stages of acidification is much smaller. We believe that this implies that the spatial distributions of heated and unheated casein micelles are different as they gel and that the former is more similar to the distribution in the original milk. In the acid gels, the casein micelles are aggregating and “clumping” together, and a larger change in 1/l* would suggest a more compact distribution of the particles or a more compact gel. 4.4. Effects of the Heated Serum Protein Complexes. In both of the samples containing serum from heated milks, the plot of 1/l* against pH shows a contribution in the region of pH 5.5 that is absent from the plots of samples containing unheated serum (indicated by arrows in Figure 4). These additional contributions are obviously attributable to the presence of the protein complexes in the heated serum. The question to be established is whether they relate to the behavior of the serum complexes on their own or whether they can be taken to show that there are interactions between the complexes and the casein micellar particles. It can be shown that interactions are a likely explanation. If no cross-interaction occurs between the casein micelles and the whey protein complexes, so that micelles can interact only with micelles and soluble complexes only with soluble complexes, then the mixtures can be considered as containing two species of scattering, spatially uncorrelated

11386

Langmuir, Vol. 21, No. 24, 2005

Alexander and Dalgleish

Figure 5. Subtraction of the 1/l* values according to eqs 6a and 6b. The filled circles correspond to the subtraction of the values for UP/US from UP/HS, and the open circles the subtraction of HP/US from HP/HS. The open squares show the subtraction emulsion/US from emulsion/HS that does not show a similarity to either of the other subtractions but is expected to show the true difference between the sera.

particles, where the total transport mean free path, l*tot, is given by20

1 1 1 ) + l*tot l*1 l*2

(4)

where l*1 and l*2 are the transport mean free paths of the individual species. For our heated milk (HP/HS) sample, eq 4 will be

1 1 1 ) + l*HM l*HP l*HS

(5)

And analogous equations can be written for the UP/US, HP/US, and UP/HS mixtures. Therefore, it is in principle possible to study the separate effects of serum and micelles in l* by subtraction of the measured values in our four milk samples. That is, we can isolate effects arising from the serum by the subtraction:

(

) (

)

1 1 1 1 1 1 ) + + ) l*UP/HS l*UP/US l*UP l*HS l*UP l*US 1 1 (6a) l*HS l*US By the same reasoning, we should be able to measure the same difference by a different subtraction:

1 1 1 1 ) l*HP/HS l*HP/US l*HS l*US

(6b)

Figure 5 shows the results of these two subtraction processes. For noninteracting particles, as shown in eqs 6a and 6b, the two subtractions should give the same results, and so the two curves shown in Figure 5 should superimpose. It is clear that this is not the case; there is a divergence between pH 5.6 and 5.2, which is where the effect of heated serum is seen in the original data. Analogous results were obtained by calculating 1/l*HP - 1/l*UP by subtracting the 1/l* values of UP/US from those of HP/US and those from UP/HS subtracted from

HP/HS. Again, the curves did not superimpose (not shown). Since the curves in Figure 5 do not behave similarly, it appears that the assumption that the particles are not interacting is wrong and that there must be “cross-term” interactions between the proteins in the serum and the casein micelles. In an attempt to test whether the aggregation of the soluble complexes on their own could produce the effects in the value of 1/l*, experiments were made with a noninteracting emulsion suspended in the sera from unheated and heated milks and then acidified. The emulsion is believed to have minimum interactions with proteins because the surfactant Tween 20 is known to displace caseins from the emulsion surface.35 Although the values of 1/l* were higher for this preparation, because the particles were larger and had a larger refractive index contrast than the casein micelles, it was possible to show that, although there were some small changes in the value of

(

) (

)

1 1 1 1 1 1 ) + + l*HS l*US l*EM l*HS l*EM l*US

they did not resemble those seen at higher pH values in the milk systems (Figure 5). This may be taken as an additional proof that the interactions of the particles in the serum cannot be solely responsible for the observed discontinuities in the plots of 1/l* against pH. We should point out that the contribution to l* arising from the micelles is much larger than that arising from the soluble aggregates (as already pointed out). This means that, within the 1/l* subtraction, there are inherent errors associated with the different contributions. However, these errors are comparable both for the casein micelle and the emulsion case; therefore, we can conclude that the differences detected in both systems are indeed real. That is, these seem to arise from interactions between the soluble complexes and the casein micellar particles. While this is not unexpected for the behavior of the heated milk, it is less so for the unheated micelles. 5. Conclusion This work has established by direct measurements that for an acid milk gel to occur it requires the interactions between the soluble denatured whey proteins and casein micelles. It is therefore clear that the milk gels are a network comprising both whey proteins and casein micelles and not a system with two distinct and separate gelling proteins. We have also been able to show directly that denatured soluble whey protein/κ-casein complexes are able to interact with intact casein micelles and form a gel. Furthermore, the dynamics of gel formation for this gel seem to be the same as for that of heated casein micelles (albeit without soluble denatured whey protein). It is our belief that DWS shows promise as a technique to study details of particle interactions in milks and other complex suspensions at their normal concentrations. Acknowledgment. The authors thank Professor Ross Hallett, of University of Guelph, for the loan of the laser used in the DWS equipment. The research was funded by the Ontario Dairy Council and the Natural Sciences and Engineering Research Council of Canada. LA0519958 (35) Dalgleish, D. G.; Srinivasan, M.; Singh, H. J. Agric. Food Chem. 1995, 43, 2351-2355.