Modification to the renneting functionality of casein micelles caused by nonionic surfactants

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Materials
  • Size Exclusion Chromatography
  • Gel Electrophoresis
  • Light Scattering
    • Dynamic Light Scattering
    • Diffusing Wave Spectroscopy
  • Rheology
  • Rennet Activity
  • Statistical Analysis
• Results and Discussion
  • Samples Characterization
  • Rennet Coagulation
• Conclusions
• References
• Copyright

Abstract 

Nonionic emulsifiers of small molecular weight such as polysorbates are widely used in dairy products. Nevertheless, the mechanism of interaction between these surfactants and milk proteins is not yet fully understood. This work investigated the effect of Tween 20 on casein micelles by studying the renneting behavior of skim milk in the presence of different amounts of surfactant. The presence of Tween accelerated both the first and second phase of renneting in skim milk. The gel obtained showed a higher elastic modulus than that of a skim milk gel, but also showed similar brittleness. By varying the size of the surfactant (Tween 20 or Tween 80) as well as the colloidal state of the proteins in solution, it was possible to demonstrate that the surfactant did not have a direct effect on the activity of the enzyme, but rather had a direct effect on the casein micelles. The effect of surfactant on the gelation point was reduced by increasing surfactant size. The presence of Tween caused an increase in the size of the micelles without affecting their stability. In addition, Tween did not alter the amount of caseins free in the serum phase. These findings can contribute to improving our ability to custom design final structures in rennet-induced gels, though further studies are needed to fully understand the mechanism at play when casein micelles are enzymatically cleaved in the presence of nonionic surfactants of small molecular weight.

Key words: skim milk, nonionic surfactant, rennet coagulation

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Introduction 

Dairy-based products and products containing dairy ingredients are complex mixtures in which milk proteins coexist in the same environment as other food components. In particular, nonionic surfactants of small molecular weight are widely used in dairy products such as ice cream and whipped cream to improve their physical and sensory characteristics. In colloidal systems, the presence of polysorbates can lead to changes in interfacial composition and structure and to an increase in interparticle interactions between components, which in turn may lead to desired (de)stabilization and improved mouth-feel.

Surfactants are amphiphilic molecules composed of a hydrophilic head and a lipophilic tail (McClements, 1999). This structure gives them the ability to reduce the interfacial tension between 2 immiscible liquids, facilitating droplet disruption and modifying structures at interfaces. Among the variety of surfactants available, polysorbates are widely used in dairy formulations, primarily because they are water-soluble and nonionic; the head group is a sorbitan etherified with polyoxyethylene chains and the tail is a fatty acid. They are classified on the basis of the tail group; among them, Tween 20 is polyoxyethylene sorbitan monolaurate (molecular weight=1,227 g/mol), and Tween 80 is polyoxyethylene sorbitan monooleate (molecular weight=1,310 g/mol; Boyd et al., 1971; Ayorinde et al., 2000).

Although much is known about the mechanism of displacement occurring at oil–water interfaces between surfactants of small molecular weight and milk proteins (Morris and Gunning, 2008), much less is known about the mechanism of interaction between polysorbates and milk proteins despite the frequent use of these emulsifiers in dairy products. It has been previously shown that Tween 20 is able to interact with β-LG (Wilde and Clarke, 1993) and that ionic surfactants can bind to CN micelles through electrostatic interactions (Fox and Hearn, 1978; Shalabi and Fox, 1982); however, to date, no evidence has been reported on interactions between nonionic surfactants of small molecular weight and CN micelles. It is generally assumed that the small molecules adsorb only to the milk fat surface and do not affect the structure and function of the CN micelles in the milk. The work reported in this paper provides evidence of changes induced by emulsifiers of small molecular weight to the functionality of CN micelles. In particular, we discuss the effects of the emulsifier on the kinetics of CN enzymatic aggregation.

Enzymatic coagulation by rennet is the basis of the cheesemaking process and is generally considered to be composed of 2 stages (McMahon and Brown, 1984; Hyslop, 2003). During the first stage, the proteolytic enzyme chymosin cleaves κ-CN, which mostly resides on the surface of the CN micelles and imparts colloidal stability to these protein particles. This κ-CN breakdown affects micellar charge and steric stabilization (Horne, 1986; de Kruif, 1999), and, when a sufficient amount has been cleaved, unstable CN micelles begin to aggregate. Aggregation is the second stage of the rennet coagulation process and leads to curd formation.

Polysorbates are very small molecules compared with CN micelles, and interactions between them could affect the production and final characteristics of dairy gels. The aim of this study was to explore the effect of these surfactants of small molecular weight on CN micelles by investigating their renneting behavior. A more detailed understanding of the microstructural development of this system could improve our ability to fine-tune structure formation in rennet gels.

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Materials and Methods 

Materials 

Fresh whole milk was obtained from the Elora Dairy Research Centre (Elora, Ontario, Canada), and sodium azide was immediately added at a concentration of 0.02% (wt/vol) to act as a bacteriostatic agent. Milk was skimmed by centrifugation at 4,000 × g for 20min at 4°C using a Beckman J2-21 centrifuge with JA-10 rotor (Beckman Coulter, Mississauga, Ontario, Canada) and subsequent filtration through Whatman glass fiber filters (Fisher Scientific, Whitby, Ontario, Canada); the filtration was repeated 4 times. Skim milk samples were stored at 4°C until analysis. Tween 20 or Tween 80 (Sigma Chemical Co., St Louis, MO) was added to skim milk in liquid form at 0.5% and 2% (wt/vol), respectively, at room temperature and stirred for 6h at the same temperate to ensure proper dilution. It should be noted here that these concentrations are above the critical micellar concentration of Tween.

For the sodium caseinate (NaCas) experiments, 2 solutions of different NaCas concentrations (Alace, Fonterra Inc., Camp Hill, PA) of 4% (wt/vol) were prepared by dissolving the powder in buffer (20mM imidazole, 5mM CaCl₂, pH 6.8) at room temperature and stirring for 4h. The sample was stored overnight at 4°C and then filtered at room temperature with a 0.45-μm membrane filter (Millipore Corporation, Bedford, MA). The sodium–calcium caseinate sample containing surfactant was prepared adding 2% Tween 20 to this solution and stirring for 6h at room temperature. Renneting of NaCas solutions were then carried out with or without Tween added (see below).

For the renneting experiments, Chymostar Single Strength rennet (Rhodia, Cranbury, NJ) was used at a concentration of 0.007% (0.018 international milk clotting units/mL) at a temperature of 30°C. The samples were stirred for 30s after rennet addition and immediately placed in the experimental equipment.

Size Exclusion Chromatography 

To investigate the differences in soluble proteins among the sera with and without surfactant, skim milk samples were centrifuged at 25,000 × g for 1h at 20°C in a Beckman Coulter Optima LE-80k ultracentrifuge with rotor type 70.1 Ti (Beckman Coulter Canada Inc., Mississauga, Ontario, Canada). The supernatants were removed from each centrifuge tube with a syringe and filtered using a 0.45-μm filter (Millipore Corporation, Bedford, MA). Supernatants were stored at 4°C and analyzed within 2 to 3 d.

Each supernatant was analyzed in size exclusion chromatography (SEC) using an AKTA purifier 10 (General Electric Company, Uppsala, Sweden) equipped with a 1-mL sample loop. An XK 16 empty column (GE Healthcare Bio Sciences Inc., Baie d’Urfe, Quebec, Canada) with a packed bed height of 67cm was used; the packing material was Sephacryl S-500 High Resolution gel (Amersham Biosciences Inc., Baie d’Urfé, Québec, Canada). The mobile phase was 20mM bis-tris-propane at pH 7.0 containing 0.02% of sodium azide. Samples were loaded into the column and eluted at a flow-rate of 1 mL/min. Eluted peaks were detected at 280nm for a total time of 180min. Fractions corresponding to each peak were collected and concentrated to 100 μL using a Centrivap cold trap (Labconco, Kansas City, MO) for 3h at 60°C to be analyzed in SDS-PAGE.

Gel Electrophoresis 

Sodium dodecyl sulfate-PAGE was performed using a Bio-Rad electrophoresis unit (Bio-Rad Power Pac HC, Hercules, CA). All the sera and the concentrated fractions obtained with the SEC were diluted 1:2 in sample buffer (containing 1 M Tris HCl, pH 6.8; 10% (wt/vol) SDS; 75% (vol/vol) glycerol; β-mercaptoethanol; and 1% (vol/vol) bromophenol blue) and heated for 5min at 95°C. The same sample buffer without β-mercaptoethanol was used to study the samples in nonreducing conditions. The sera of the samples prepared for SEC were diluted 1:2 with Milli-Q water (Millipore, Billerica, MA) prior the dilution with sample buffer.

For the electrophoresis analysis, the resolving gel contained 15% acrylamide in 1.5 M Tris HCl at pH 8.9 whereas the stacking gel contained 4% acrylamide in 0.1 M Tris PO₄ buffer at pH 6.7. The electrophoresis buffer was 3% (wt/vol) Tris HCl, 14.4% (wt/vol) glycine, and 1% (wt/vol) SDS at pH 8.3. Aliquots of 7 μL of the prepared samples were loaded into the gels, and the electrophoresis separation was performed at 200V for 40min. Gels were then stained with coomassie blue in 50% (vol/vol) methanol and 10% (vol/vol) acetic acid for 30min while shaking and destained in 2 steps, first with a solution of 45% (vol/vol) methanol and 10% (vol/vol) acetic acid for 1h, then overnight with the same solution diluted 1:1 in Milli-Q water. The gels were scanned the next day with a Sharp JX-330 scanner (Amersham Biosciences).

Light Scattering 

Dynamic Light Scattering 

Particle size distributions of the different milk samples were measured using dynamic light scattering (DLS; Zetasizer Nano, Malvern Instruments, Worcestershire, UK). The hydrodynamic size of the CN micelles was obtained from the average of 3 separate readings. Samples were diluted approximately 2,000 times in permeate (milk serum filtered with a 0.22-μm filter) to avoid multiple scattering while preserving the environmental conditions of the CN micelles, and were then placed in the spectrometer right after dilution.

Diffusing Wave Spectroscopy 

A volume of approximately 1.5mL of undiluted milk sample was placed into a flat-faced, optical glass cuvette with a 5-mm path length (Hellma Canada Ltd., Concord, Ontario, Canada) and the temperature of the equipment was equilibrated and maintained at 30°C with a water bath. The light source was a solid-state diode-pumped Nd:YAG laser (Coherent, Santa Clara, CA) with a wavelength of 532nm and power of 100 mW. The transmitted scattered light was collected by a single fiber optic that was then bifurcated and fed to 2 matched photomultipliers (HC120–03, Hamamatsu, Loveland, OH) and a correlator (FLEX2K-12 × 2, Correlator.com, Bridgewater, NJ). Standard latex spheres with a diameter of 260nm (Portland Duke Scientific, Palo Alto, CA) were used to calibrate the laser intensity daily. Correlation functions and intensity of the transmitted scattered light were measured at intervals of 2min for 80min (for samples with rennet). Data were analyzed using specialized software (DWS-Fit, Mediavention Inc., Guelph, Ontario, Canada). Each experiment was replicated at least 3 times, starting from different batches of fresh skim milk.

Diffusing wave spectroscopy (DWS) is based on the measurement of temporal fluctuations of light that has been multiple-scattered by particles in a sample (ten Grotenhuis et al., 2000). In recent years, it has been employed in the investigation of destabilization mechanisms in food systems (ten Grotenhuis et al., 2000; Dalgleish et al., 2004). An exhaustive description of the equipment and theory can be found elsewhere (Alexander and Dalgleish, 2004). Briefly, DWS relies on many scattering events happening as a photon of light traverses a colloidal dispersion and the light propagation is approximated by a random path distribution. Diffusing wave spectroscopy can yield information on the static properties (positional correlations) of a system via the photon transport mean free path (l*), as well as dynamic properties via the decay time (τ) and the mean square displacement (MSD). Similarly to DLS, DWS measures the intensity fluctuations of the scattered light caused by motion of the colloidal particles. In general, the intensity autocorrelation function of multiple scattered light, g₂(t), can be described as a function of time by

where β is determined by the optics setup; P(s) is the probability of a given path followed by the photon; s is the path length; k₀ = 2πn/λ_o (where n is the refractive index of the medium and λ_o is the wavelength of laser light in vacuum); and 〈Δr²(t)〉 is the MSD, which, for free diffusing particles, is related to the diffusion coefficient, D = 6t/〈Δr²(t)〉. The value of l* can be determined experimentally by dividing the average intensity of the transmitted light of the sample, T, by that of a calibrating sample with well-determined l* (Weitz et al., 1993). At very short correlation times, 〈Δr²(t)〉 has been proven to be generally described by Krall and Weitz (1998):

The MSD is the average of the square of the distance traveled by the particles at a given time. Therefore, the rate of growth of the mean square displacement depends on how often the particles encounter other particles in their paths. In a free-diffusing regimen, the motion is described as a random walk for which the MSD increases linearly with time (the exponent p in equation [2] is equal to 1). In a gel, for example, the particles will be able to move only a certain distance from their average position before being restricted in motion by their interactions with other particles or by an existing network around them. Therefore, their displacement with time will be shorter and the exponent p in equation [2] will be less than 1 (Krall and Weitz, 1998).

Rheology 

Rheological experiments were performed with a stress-controlled rheometer (AR 1000, TA Instrument Ltd., New Castle, DE) using a conical concentric cylinder geometry (5,920-μm fixed gap; 15-mm radius; 14-mm motor outlet radius; 42-mm cylinder immersed height). Rennet coagulation was monitored at 30°C using an external water bath (Isotemp 3016, Fisher Scientific, Whitby, Ontario, Canada) connected to the rheometer to ensure constant temperature during the experiments. Each sample underwent a series of successive tests. After the addition of rennet, the sample was immediately placed in the rheometer and a time sweep was run at 0.01 controlled strain, 1.0Hz frequency, and 0.1mN initial torque. The first time sweep was ended at the time of the crossover between storage modulus (G′) and loss modulus (G″); this time was considered as the gelation point of the sample. A second time sweep was performed with the same parameters immediately after the first to monitor gel development. After 45min, a frequency sweep was run from 10 to 0.01Hz at a controlled strain of 0.01. Last, a strain sweep was performed at 1Hz to ensure that the parameters applied during the previous tests were within the linear viscoelastic region of the sample.

Rennet Activity 

The release of the CN macro peptide (CMP) from κ-CN in milk and sodium–calcium caseinate samples during gelation was quantified by reverse-phase HPLC. After addition of rennet, each sample was divided in aliquots in different test tubes. Every 5min, 4% trichloroacetic acid (TCA) was added to the tubes to stop the enzymatic reaction, resulting in a final concentration of 2% TCA in the sample. The experiment was performed at a constant temperature of 30°C for 45min. After TCA addition, each sample was mixed using a vortex mixer and stored overnight at 4°C. The samples were then equilibrated at room temperature and centrifuged at 4,461 × g for 15min using an Eppendorf 5415D centrifuge (Brinkmann Instruments Ltd., Mississauga, Ontario, Canada). The supernatants were collected, filtered through 0.45-μm Millex-GV filter units (Millipore Corporation), and analyzed by reverse phase HPLC.

Reverse-phase HPLC was carried out with a Finnigan SpectraSystem LC unit (ThermoFinnigan, Burlington, Ontario, Canada) equipped with degasser, pump, autosampler with a 100-μL loop, and UV detector (set at 210nm). The column was a Pharmacia Biotech μRPC C2/C18 ST 4.6/100 (Piscataway, NJ) with a C18 guard column (Phenomenex, Torrance, CA). A nonlinear gradient was run between solvent A (0.1% trifluoroacetic acid in water) and solvent B (0.1% trifluoroacetic acid in 90% acetonitrile). Total peak areas were integrated using ChromQuest software (v. 4.1, ThermoFinnigan) from approximately 10 to 38min on the chromatograms. The graphs obtained plotting the area of each peak (% CMP release) versus time after addition of rennet were fitted with a first-order equation:

Statistical Analysis 

All experiments were carried out in triplicate. To determine significant differences between treatments, ANOVA was performed. Least significant difference computations were carried out to determine significant differences between samples. Statistical analyses were conducted using SPlus 8.0 (Tibco, Palo Alto, CA) at 95% confidence level.

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Results and Discussion 

Samples Characterization 

The presence of Tween 20 [0.5 and 2% (wt/vol)] caused a significant increase in the size of the CN micelles, as measured by DLS and DWS. When 2% Tween 20 was added to milk, the CN micelles showed a diameter increase of 5±3.1nm with DLS and a diameter increase of approximately 20±4.4nm with DWS. The difference observed between the 2 instruments can arise from the use of a different concentration of the samples, which leads to different diffusion properties of the particles in the system. On the other hand, the presence of Tween did not have a marked effect on the pH of the milk, which decreased from 6.7 to 6.4.

To ensure that the presence of Tween did not affect the stability of the system with time, samples of skim milk containing 0.5, 1, and 2% (wt/vol) of Tween 20 were monitored with DWS over a 5-h period and compared with the CN size at time=0s of skim milk. Figure 1 shows A) the development of the turbidity parameter, 1/l*, and B) the radii of the samples. No significant changes in either parameter were observed over time; moreover, the presence of surfactant, even at high concentrations, did not lead to any significant increase in 1/l* value compared with the control. To determine whether the presence of surfactant affected the release of soluble CN from the micelles, the serum phase of skim milk containing 0.5% and 2% (wt/vol) of Tween 20 was analyzed using SEC and SDS-PAGE. Figure 2 shows the SEC chromatograms of the sera obtained by centrifugation of the 3 samples; they all show the characteristic 5-peak pattern of milk serum (Guyomarc’h et al., 2003). Only the first 3 peaks were of interest in this work because peaks 4 and 5 corresponded to orotic acid and hydrolyzed fragments of DNA, respectively. Peak 1 corresponded to residual fat in solution, and it decreased slightly with increasing concentration of Tween. It can be speculated that the presence of surfactant displaced CN proteins adsorbed at the interface of the fat globules, allowing these to aggregate and be washed off with the pellet, thereby decreasing their solubility with increasing Tween content. Peak 2 corresponded to whey protein–κ-CN complexes as well as soluble CN. Because this work dealt with unheated milk, the number of complexes was expected to be negligible, as is shown in Figure 2. The height and width of this peak remained unchanged in the presence of surfactant, indicating that there was no change in the amount of soluble proteins present in the serum. Peak 3 corresponded to soluble whey proteins, and this peak also seemed unaffected by the presence of Tween. It can be concluded, then, that the presence of surfactant did not affect the quantity or composition of soluble proteins in skim milk serum. To analyze the differences in protein concentration between the sera more precisely, the sera and fractions obtained from SEC for the first 3 peaks were also analyzed for protein composition using SDS-PAGE (data not shown). Both reducing and nonreducing conditions were used to better identify eventual complexes and proteins involved. The fractions isolated showed similar composition, confirming that the presence of surfactant did not have any effect on the solubility of CN micelles.

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• Figure 1. 

  Stability of skim milk with 0.5% Tween 20 (●; Sigma Chemical Co., St. Louis, MO), 1% Tween 20 (■), and 2% Tween 20 (▴) monitored with diffusing wave spectroscopy: A) turbidity parameter (1/l*) and (B) radius over time. Also shown are the initial values for skim milk (♦).

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• Figure 2. 

  Size exclusion chromatograms of the sera of skim milk (solid line), skim milk with 0.5% Tween 20 (dashed line; Sigma Chemical Co., St. Louis, MO), and skim milk with 2% Tween 20 (dotted line).

Rennet Coagulation 

The effect of Tween 20 (0.5 and 2%) in the first phase of the rennet coagulation was observed by measuring the release of CMP from the surface of the CN micelles (Figure 3). The presence of surfactant led to a faster removal of CMP as measured by the time taken to reach 95% release (the maximum CMP released from the control skim milk was taken as 100%). The control sample reached 95% of CMP released in 33min, whereas skim milk with Tween reached the same point in 25 and 20min for 0.5% and 2% Tween, respectively. The 3 samples were statistically different (Table 1), and all samples were able to reach 100% CMP released. This suggests that all κ-CN was still accessible to the cleaving action of the enzyme even in the presence of Tween.

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• Figure 3. 

  Percentage casein macro peptide (CMP) release versus time after addition of rennet in skim milk (●), skim milk with 0.5% Tween 20 (■; Sigma Chemical Co., St. Louis, MO), and skim milk with 2% Tween 20 (▴). See Table 1 for statistical significance.

Table 1. Rennet coagulation parameters obtained with diffusing wave spectroscopy, casein macro peptide (CMP) release, and rheometer for skim milk (SM) and SM with different amounts of Tween 20 (Tw20)¹

a–cMeans within a column with different superscripts are significantly different (P < 0.05).

1Values are the means of 3 replicate experiments. Tween 20 is from Sigma Chemical Co., St. Louis, MO. 1/l* = the transport mean free path; G′ = storage modulus.

To try to further elucidate the origin of the variations in CMP release and to determine whether the effect of Tween is related to an increased activity of the enzyme per se, renneting experiments were performed using sodium–calcium caseinate solutions. Two solution with 4% protein with and without Tween were prepared and the amount of CMP released was determined as described above. As shown in Figure 4, the presence of Tween did not cause any significant changes in the rate of release of CMP in the NaCas solution. Unlike the case of skim milk, Tween did not have any effect on CMP removal by the enzyme. It can then be concluded that the presence of Tween did not accelerate the enzyme activity, thus supporting the hypothesis that the presence of Tween affected the structure of the CN micelle, hence its rennetability.

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• Figure 4. 

  Casein macro peptide (CMP) release of a 4% caseinate solution versus time after addition of rennet for sodium–calcium caseinate (●) and sodium–calcium caseinate with 2% Tween 20 (○; Sigma Chemical Co., St. Louis, MO).

To follow the kinetics of gelation of all samples, DWS and rheology experiments were also performed (Figure 5). Figure 5A shows the development of 1/l* as a function of time after the addition of rennet. All the samples showed a delay phase before a sudden increase in 1/l*. This behavior has been reported before in milk renneting systems (Sandra et al., 2007) and is related to the development and later formation of a gel. After this delay, 1/l* started to increase, reflecting the beginning of interparticle interactions; the time of increase appeared to be inversely proportional to the amount of Tween. After the initial 1/l* increase, there was a second change in slope, which corresponded directly to the beginning of the aggregation of the micelles. The coagulation time manifested itself as a sudden change in apparent radius, as clearly shown in Figure 5B. In agreement with the 1/l* data, the coagulation time was inversely proportional to the amount of Tween present in milk. The results of the slope of a log-log graph of the mean square displacement curves (exponent p of equation 2) are shown in Figure 4C. These results confirmed the distinct arrest of motion of the CN micelles at the time of coagulation, as shown by a decrease in the value of p from its initial, free diffusing value of 1. The time at which this slope decreased from 1 was consistent with the time of the aggregation of the CN micelles shown in Figure 5B and consistently decreased with increasing amount of Tween 20. Last, Figure 5D summarizes the changes of G′ as a function of renneting time for all 3 samples. The coagulation times shown in these rheology experiments were longer than those obtained by DWS for each respective amount of Tween 20; this effect has been observed before (Sandra et al., 2007) and it is related to the different length scale probed by the 2 instruments. Nevertheless, the rheology data also confirmed that the gelation point of skim milk was accelerated by the presence of an increasing amount of Tween 20.

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• Figure 5. 

  Rennet coagulation process monitored by diffusing wave spectroscopy and rheometer of skim milk (●), skim milk with 0.5% Tween 20 (■; Sigma Chemical Co., St. Louis, MO), and skim milk with 2% Tween 20 (▴): A) the transport mean free path (1/l*), B) radius, C) mean square displacement (MSD) slope, and D) storage modulus versus time after addition of rennet. See Table 1 for statistical significance.

The results shown above can be explained by the changes caused by Tween 20 during the primary stages of rennet aggregation (i.e., the CMP release kinetics shown in Figure 3). Increasing the amount of Tween 20 in skim milk had the effect of accelerating the release of the CN macropeptide into solution during rennet action. This, in turn, meant that the stabilizing ability of the κ-CN hairs was reduced rapidly in the presence of Tween. As the destabilization process accelerated, aggregation and flocculation happened sooner in time after rennet addition.

Because the kinetics of CMP release were affected by the presence of Tween in milk, it is important to compare the behavior of 1/l* and G′ parameters as a function of CMP release (Figure 6). The turbidity parameter 1/l* increased at around 80% of CMP release for the control sample and 0.5% Tween. These results indicate that at least (approximately) 80% of κ-CN needed to be removed to induce a noticeable increase in intermicellar interactions. The increase in G′ happened later, at about 95% of CMP released, as determined by their viscoelastic properties. This implies that in skim milk, κ-CN removal of approximately 80% was sufficient to modify the long-range interparticle interactions and affect the positional distribution of the CN micelles. However, a full 95% needed to be removed from the micelle surface before the stability energy barrier was removed and aggregation could take place. These results are in accordance with previous DWS studies on rennet aggregation of skim milk (Alexander and Dalgleish, 2004; Sandra et al., 2007). For the sample with 2% Tween, 1/l* and G′ increased earlier than the control, at about 70% and 90% CMP released, respectively. This indicates that a lower level of proteolysis was required in the 2% Tween sample to initiate the interparticle interactions and coagulation of CN micelles. The presence of 2% Tween 20 not only accelerated the first phase of renneting but also the second, possibly increasing surface hydrophobicity of the micelles. It is not clear at this point what was the exact mechanism taking place in this process. It could be speculated that the interactions between Tween and the CN micelles were disrupting the micellar formation of Tween (which was present above the critical micellar concentration point); otherwise, Tween would not affect the secondary stage of the aggregation, although it could still affect the renneting activity.

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• Figure 6. 

  Development of storage modulus (G′; open symbols) and the transport mean free path (1/l*; filled symbols) versus casein macro peptide (CMP) release of skim milk (●,○), skim milk with 0.5% Tween 20 (■,□; Sigma Chemical Co., St. Louis, MO), and skim milk with 2% Tween 20 (▴,▵).

Table 1 summarizes the key features shown in Figure 5 and their statistical significance. The values of this table were obtained by determining the intersection point of 2 best-fit lines on the points before and after a noticeable data change. This table clearly shows that the presence of Tween significantly affected the coagulation point as well as the time of initial interparticle interactions (onset of 1/l* change), and that the extent of this effect was proportional to the concentration of Tween in solution. Table 1 also shows the G′ values of the milk gels 45min after the gelation point and the strain at break; the latter was calculated as a change of 5% in the G′ measured during the strain sweep. The presence of surfactant leads to the formation of stiffer gels. The stiffness increased from 27.0Pa of skim milk to 43.7Pa of skim milk with 0.5% of Tween, and almost tripled in the presence of 2% surfactant. This effect was not related to the coagulation time of the samples because the stiffness was measured for all samples 45min after the gelation point. Strain sweep experiments also indicated that the presence of Tween 20 did not alter the brittleness of milk gels. In all cases, the gels showed strain at break values similar to those of skim milk without Tween. However, the presence of 2% Tween 20 induced a biphasic behavior of the strain sweep, with a first change in G′ (around 2% change) at approximately 3.5% strain (data not shown), whereas the strain at break was approximately 24%, same as for skim milk.

To determine whether the size of the surfactant had any bearing on the extent of these changes, gelation experiments were also carried out using Tween 80. Tween 80 has a molecular weight of 1,310 g/mol compared with 1,227 g/mol for Tween 20. Tween 20 is composed of 50% lauric acid and the other 50% contains a mixture of myristic, palmitic, and stearic acids. All the fatty acids in Tween 20 are saturated so they have a linear structure (albeit of different lengths), whereas in Tween 80 the oleic acid has a double bond that creates an angle (kink) in the structure. Figure 7 shows the development of G′ as a function of time after the addition of rennet for the control and for skim milk with 2% (vol/wt) Tween 20 and Tween 80. The addition of Tween 80 caused a statistically significant reduction in the effect on the gelation point of the CN micelles compared with Tween 20. In fact, both the decrease in gelation point and the increase in the absolute value of G′ were less pronounced when using Tween 80 instead of Tween 20, although statistically significantly different than the values of the control. Whereas milk containing Tween 20 showed a gelation point at 18.0±0.99min, skim milk containing Tween 80 showed a gelation point of 24.3±0.23 and skim milk containing no Tween showed a gelation point of 39.3±2.52min. When 2% of Tween 80 was added to milk, the G′ at 45min reached 55.8±0.25Pa, a value different from that of control milk and 2% Tween 20 but closer to that of milk containing 0.5% Tween 20, even if statistically different (Table 1).

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• Figure 7. 

  Increase in storage modulus (G′) values as rennet coagulation occurs in skim milk (●), skim milk with 2% Tween 20 (□; Sigma Chemical Co., St. Louis, MO), and skim milk with 2% Tween 80 (▵).

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Conclusions 

This research demonstrated, for the first time, that the presence of a small molecular weight emulsifier, Tween 20, affected the renneting behavior of CN micelles, making them more susceptible to rennet hydrolysis. The results of this study also demonstrated that the stability and solubility of CN micelles were not affected by the presence of Tween, though their sizes increased slightly. Tween 20 accelerated both the first and second phase of renneting. Moreover, the gel obtained in the presence of Tween was more rigid than a skim milk gel, though both had the same brittleness 45min after gelling. It is possible to hypothesize that Tween did not have a direct effect on the activity of the enzyme, but rather had a direct effect on the structure of the CN micelles. It is not clear whether these interactions happened on the surface or the interior of the micelles. The nature of the interactions could be of hydrophobic nature because of the nonionic nature of the surfactant molecules; however, the size (saturation level) of the surfactant's hydrophobic tail seems to be important.

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References 

1. Alexander M, Dalgleish DG. Application of diffusing wave spectroscopy to the study of gelation of milk by acidification and rennet. Colloids and Surfaces B. 2004;38:83–90
   • View In Article
2. Ayorinde FO, Gelain SV, Johnson JH, Wan LW. Analysis of some commercial polysorbate formulations using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2000;14:2116–2124
   • View In Article
   • MEDLINE
   • CrossRef
3. Boyd J, Parkinson C, Sherman P. Factors affecting emulsion stability, and the HLB concept. J. Colloid Interface Sci. 1971;41:359–370
   • View In Article
   • CrossRef
4. Dalgleish D, Alexander M, Corredig M. Studies of the acid gelation of milk using ultrasonic spectroscopy and diffusing wave spectroscopy. Food Hydrocoll. 2004;18:747–755
   • View In Article
5. de Kruif CG. Casein micelles interactions. Int. Dairy J. 1999;9:183–188
   • View In Article
6. Fox PF, Hearn CM. Heat-stability of milk—Influence of denaturable proteins and detergents on pH sensititvity. J. Dairy Res. 1978;45:159–172
   • View In Article
   • CrossRef
7. Guyomarc’h F, Law AJR, Dalgleish DG. Formation of soluble and micelle-bound protein aggregates in heated milk. J. Agric. Food Chem. 2003;51:4652–4660
   • View In Article
   • MEDLINE
   • CrossRef
8. Horne DS. Steric stabilization and casein micelle stability. J. Colloid Interface Sci. 1986;111:250–260
   • View In Article
   • CrossRef
9. Hyslop DB. Enzymatic coagulation of milk. In Advanced Dairy Chemistry. In: 3rd ed..  Fox PF,  McSweeney PLH editor. Proteins. Vol. 1:New York, NY: Kluwer Academic/Plenum Publishers; 2003;
   • View In Article
10. Krall AH, Weitz DA. Internal dynamics and elasticity of fractal colloidal gels. Phys. Rev. Lett. 1998;80:778–781
    • View In Article
    • CrossRef
11. McClements DJ. Food Emulsions. Boca Raton, FL: CRC Press LLC; 1999;
    • View In Article
12. McMahon DJ, Brown RJ. Enzymatic coagulation of casein micelles: A review. J. Dairy Sci. 1984;67:919–929
    • View In Article
    • Abstract
    • Full-Text PDF (992 KB)
    • CrossRef
13. Morris VJ, Gunning AP. Microscopy, microstructure and displacement of proteins from interfaces: Implications for food quality and digestion. Soft Matter. 2008;4:943–951
    • View In Article
14. Sandra S, Alexander M, Dalgleish DG. The rennet coagulation mechanism of skim milk as observed by transmission diffusing wave spectroscopy. J. Colloid Interface Sci. 2007;308:364–373
    • View In Article
    • MEDLINE
    • CrossRef
15. Shalabi SI, Fox PF. Heat stability of milk: Influence of cationic detergents on pH sensitivity. J. Dairy Res. 1982;49:597–605
    • View In Article
    • MEDLINE
    • CrossRef
16. ten Grotenhuis E, Paques M, van Aken GA. The application of diffusing wave spectroscopy to monitor the phase behavior of emulsion-polysaccharide systems. J. Colloid Interface Sci. 2000;227:495–504
    • View In Article
    • CrossRef
17. Weitz DA, Zhu JX, Durian DJ, Gang H, Pine DJ. Diffusing-wave spectroscopy: The technique and some applications. Phys. Scr. T. 1993;49B:610–621
    • View In Article
18. Wilde PJ, Clarke DC. The competitive displacement of β-lactoglobulin by Tween 20 at the oil-water interface. J. Colloid Interface Sci. 1993;155:48–54
    • View In Article
    • CrossRef