Colloids and Surfaces B: Biointerfaces 118 (2014) 49–56

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␣s -Casein—PE6400 mixtures: Surface properties, miscibility and self-assembly Anne Kessler a , Orquidéa Menéndez-Aguirre b , Jörg Hinrichs b , Cosima Stubenrauch c , Jochen Weiss a,∗ a Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 21/25, 70599 Stuttgart, Germany b Department of Dairy Science and Technology, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstraße 25, 70599 Stuttgart, Germany c Institut für Physikalische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany

a r t i c l e

i n f o

Article history: Received 2 December 2013 Received in revised form 8 March 2014 Accepted 8 March 2014 Available online 27 March 2014 Keywords: ␣s -Casein PEO13 –PPO30 –PEO13 Mixed micelle Surface properties Miscibility gap ␨-Potential

a b s t r a c t Surface properties, miscibility and self-assembly of mixtures of a food-grade ␣s -casein and the triblock copolymer PE6400 (PEO13 –PPO30 –PEO13 ) were examined. The properties at the surface were determined by surface pressure measurements for a 1:1 molar mixture. Comparison of the measured with the calculated isotherms show attractive interactions at surface pressures above 9 mN/m. The miscibility gaps of solutions containing 0.004–0.2 mmol/l ␣s -casein and 0.02–0.1 mol/l polymer were examined. It was found that a one-phase region exists at distinct mixing ratios and temperatures. Comparison of the cloud points of mixtures of ␣s -casein and PE6400 with pure ␣s -casein showed that the presence of the triblock copolymer enhanced the solubility of the protein. The ␨-potential of the ␣s -casein solution decreased by addition of PE6400 to zero. Our results thus suggest that ␣s -casein and PE6400 are miscible. The results of the cloud point and ␨-potential measurements were explained by formation of a mixed aggregate where the PPO chains are anchored inside the hydrophobic part of the ␣s -casein while the PEO chains cover the charged hydrophilic part of the ␣s -casein thereby leading to an increase of the cloud point and a decrease in ␨-potential. This is in agreement with the attractive interactions between ␣s -casein and PE6400 as observed via surface pressure measurements at the surface. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Investigations focus increasingly on determining the properties of naturally occurring compounds which are surface active and self-assemble into micellar structures suitable for the delivery of hydrophobic functional ingredients. These structures are of interest to various industries, including the food, pharmaceutical and personal care industries. Examples of naturally occurring surface active compounds are the caseins which are the main protein source in milk. Caseins consist of several proteins with four principal ones being ␣s1 -, ␣s2 -, ␤and ␬-casein in bovine milk [1,2]. The structural units of proteins are amino acids having different hydrophobicities. When amino acids with similar hydrophobicities are sequentially arranged in the chain, distinct hydrophilic or hydrophobic blocks are formed. The

∗ Corresponding author. Tel.: +49 711 459 24415; fax: +49 711 459 24446. E-mail address: [email protected] (J. Weiss). http://dx.doi.org/10.1016/j.colsurfb.2014.03.030 0927-7765/© 2014 Elsevier B.V. All rights reserved.

location of such blocks depends on the conformation of the chain. Caseins are highly surface active due to a lack of a secondary structure. Hydrophobic and hydrophilic residues can therefore readily assume suitable configurations at interfaces [1]. The distribution of hydrophilic and hydrophobic dominated parts in the different caseins was identified by determining the regions of the casein sequence which adsorb at hydrophobic surfaces by self-consistent field theory [3]. Results of these calculations led to conclude that ␤-casein is a diblock polymer where the C-terminal region has hydrophobic and the N-terminal hydrophilic properties, while ␣s1 casein is a triblock polymer with two hydrophobic ends and a hydrophilic center [3,4]. Aside from such naturally occurring block copolymers, a huge number of synthesized block copolymers are commercially available. The PEOx –PPOy –PEOx surfactants (tradename Pluronic PEs (BASF) or Synperonic PE Series (Croda)) have, for example, structural characteristics that are similar to the ␣s1 -casein. They consist of a hydrophobic polypropylene oxide (PPO) block in the center being enclosed by two hydrophilic polyethylene oxide (PEO)

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blocks. The aggregation properties of PEOx –PPOy –PEOx surfactants are extensively examined and shown to be highly temperature sensitive [5]. At low temperatures and concentrations, the surfactant is present as a monomer, while an increase in temperature and concentration leads to micelle formation, the size of which is influenced by temperature. A further increase in temperature and concentration leads to the formation of liquid crystalline phases [5]. Self-assembled structures of caseinates and ␤-caseins [6] or PEOx –PPOy –PEOx [7] were shown to be useful carrier systems for physiologically active components such as nutraceuticals or pharmaceuticals. Self-assembly in mixtures of triblock copolymers and various surfactants were examined [8–11]. Properties of mixtures of caseins and classical surfactants were investigated at interfaces [12]. Moreover, their ability to stabilize foams [13,14] and emulsions [15,16] were thoroughly studied. To the best of our knowledge, only one study investigated the self-assembly of triblock copolymers and caseins [17], namely ␤-casein, a natural diblock copolymer, mixed with PEO101 –PPO56 –PEO101 (Lutrol). In this study, we examined the surface properties, miscibility and self-assembly of ␣s -casein and PEO13 –PPO30 –PEO13 . This polymer was chosen because it is easy to handle as it is liquid and water-soluble. Furthermore PEO13 –PPO30 –PEO13 seems to be a good starting point to examine the influence of the PEO resp. PPO content in the molecule systematically in further studies. The triblock copolymer-like structure of the ␣s -casein and the results of Portnaya et al. [17] led us to hypothesize that ␣s -casein forms mixed aggregates with other block copolymers due to structural similarities. There are however two important differences in comparison to the study of Portnaya et al. [17]. First, we examined ␣s -casein, a triblock copolymer rather than using ␤-casein, a diblock copolymer and second, the PEOx –PPOy –PEOx triblock copolymer used in our study is of much lower molecular weight and less hydrophilic than the one used by Portnaya et al. [17]. Hence, we will discuss our result in the context of the results obtained by Portnaya et al. [17] in terms of the different molecular architectures.

of 243.02 mm2 . The surface pressure  was measured by a Wilhelmy plate. Aliquots of 30 ␮l of the surfactant solutions having a concentration of c = 0.45 mmol/l were spread at the surface. After an equilibration time of 30 min, the monolayer was compressed by moving the barriers at a speed of 20 mm/min while the surface pressure was recorded as a function of the surface area A. Values of the calculated elasticity E were smoothed using a moving average filter with a span of 10 (Matlab, Ver. 6.0, Mathworks, Natick, USA). The Langmuir–Blodgett trough was cleaned with water, ethanol (Carl Roth GmbH, Karlsruhe, Germany) and chloroform (Sigma–Aldrich Chemie GmbH, Steinheim, Germany). The success of the cleaning process was determined by measuring the surface pressure of water as a reference. Water with a conductivity of 0.055 ␮S/cm (Purelab Classic, Elga) was used for the experiment.

2.3. Surface tension The surface tension of the samples was measured using the du Noüy ring method (STA-1 Sinterface Technologies, Berlin, Germany). Prior to each measurement, the surface tension of water was measured to ensure that the ring and the vessel were surfactant-free. Surface tension was recorded as a function of time. Corrections after Harkins and Jordan were used [20]. The surface tension of water was determined as 71.41 ± 0.05 × 10−3 N/m at 22 ◦ C, which is in agreement with values reported in the literature [21]. In order to calculate the critical micelle concentration (CMC), results of measurements of the equilibrium surface tension ¯ eq below the ¯ eq plateau were fitted with a second order polynomial and the CMC was determined at the concentration c at which the polynomial fit intersected the plateau. The surface concentration  was calculated using the Gibbs isotherm for ionic surfactants [22],  =−

1 2RT



deq d ln c

 ,

(1)

p,T

2. Materials and methods 2.1. Preparation of surfactant solutions Solutions were prepared in 20 mmol/l imidazole/HCl buffer (Carl Roth GmbH, Karlsruhe, Deutschland) at pH 6.6. PEO13 –PPO30 –PEO13 (Tradename PE6400, BASF, Ludwigshafen, Germany) with a molecular weight of 2900 g/mol. It was used as received. The ␣s -casein rich casein with molecular weight of 23,000 g/mol, a protein content of 0.689 ± 0.013 g protein/g powder and a composition of 0.539 ± 0.013 g/g ␣s -casein, 0.318 ± 0.011 g/g ␤-casein and 0.15 ± 0.017 g/g ␬-casein was fractionated and protein stock solutions were prepared as described previously [18,19]. It should be mentioned, that ␣s -casein is composed of ␣s1 and ␣s2 -casein, but an analysis of the ␣s1 - and ␣s2 -casein content is not possible with the method used in this study. Henceforth the ␣s -casein rich casein fraction is referred to ␣s -casein. Below, both PE6400 and ␣s -caseins are referred to as surfactants. Doubledistilled water was used throughout the entire study unless stated otherwise. All glassware was soaked in diluted Deconex 11 universal solution (Borer Chemie AG, Zuchwil, Switzerland) overnight and rinsed extensively with double-distilled water prior to experiments. 2.2. Surface pressure Monolayers were prepared and compressed by a Langmuir–Blodgett trough (KSV Minitrough, KSV NIMA, Espoo, Finland) made of Teflon with two moveable barriers and a surface

where R is the ideal gas constant, T is the absolute temperature and c is the concentration. The area A occupied by every molecule at the surface is given as [22] A=

1 ,  · NA

(2)

where NA is the Avogadro constant. The maximal surface concentration  max and the minimum surface area Amin respectively are found at the CMC. All calculations were carried out with Matlab (Ver. 6.0, Mathworks, Natick, USA).

2.4. Turbidity The absorbance was measured in a UV spectrophotometer (8435, Hewlett Packard Development Company LP, Palo Olta, USA) at 400 nm as a function of temperature to determine the turbidity of the solutions. To adjust the temperature, the measurement cells of the photometer were thermostatted with a water bath (Julabo EH, Julabo, Seelbach, Germany). Sample temperature was adjusted and recorded with a thermometer (GMH 3710 High Precision Digitalthermometer, Greisinger Electronics, Regenstauf, Germany) with a PT100 probe (GTF 401, 1/10 DIN, Greisinger Electronics, Regenstauf, Germany). Disposable polystyrene cuvettes (Brand GmbH + CO KG, Wertheim, Germany) were used. Samples were allowed to equilibrate for 30 min at each temperature prior to carrying out the measurements.

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The absorbance AA was measured as a function of temperature ϑ from which the cloud point ϑcp was calculated. The absorbance at the cloud point Aϑcp was defined as Aϑcp = AA,min + 0.05 for c > 0.004 mmol/l

(3)

and Aϑcp = AA,min + 0.01 for c = 0.004 mmol/l

(4)

with AA,min being the lowest absorbance observed. ϑcp was determined by linear interpolation. For samples with c > 0.004 mmol/l a change of 0.05 was considered to be significant, while for c = 0.004 mmol/l it was a change of 0.01 which was interpreted as the onset of phase separation. All calculations were carried out with Matlab (Ver. 6.0, Mathworks, Natick, USA). The cloud points measured were verified visually by heating up the sample under mild agitation in a transparent water bath (usual in trade basin) equipped with a heating unit (Julabo MA; Julabo, Seelbach, Germany) and a thermometer with a PT100 probe. The possible formation of liquid crystalline phases was assessed by use of cross polarizers (Polfilter linear, ES49, Heliopan LichtfilterTechnik Summer GmbH + CO KG, Gräfelfing, Germany). 2.5. ␨-Potential The ␨-potential of the samples was determined by a Zetasizer (Nano ZS, Malvern Instruments GmbH, Herrenberg, Germany). All samples were first filtered (0.02 ␮m regenerated cellulose (RC) syringe filters, Rotilabo, Carl Roth GmbH, Karlsruhe, Germany) and then poured in a cuvette (disposable folded capillary cell, Malvern Instruments GmbH, Herrenberg, Germany). Samples were allowed to equilibrate for at least 10 min in the sample cell prior to analysis to ensure a uniform temperature. Attenuator, voltage, measurement time and number of measurements used to determine the electrophoretic mobility were selected automatically by the software. The ␨-potential was calculated by the Henry equation and Smoluchowski approximation using the measured electrophoretic mobility [23]. The viscosity, refractive index and dielectric constant of water at the respective measurement temperature were used. 2.6. Statistical analysis All measurements were repeated at least three times using duplicate samples. Means and standard errors for a 95% confidence interval, P = 95%, were calculated. ␨-Potential measurements were analyzed by multiple analysis of variance (ANOVA) and the differences were determined by multiple comparison test performed with the statistics toolbox of Matlab (Ver. 6.0, Mathworks, Natick, USA). The level of statistical significance ˛ was set at 0.05.

Fig. 1. Surface pressure  (black) and elasticity E (gray) of (a) ␣s -casein (b) PE6400 and (c) mixtures thereof. The (A)-curves of the mixtures were measured (straight) and calculated (dotted). Different structures and phase transitions are labeled. An average of three measurements is given.

3. Results and discussion

interactions between the molecules in mixed monolayers may be obtained by calculating the ideal isotherm by

3.1. Surface pressure and elasticity

Aideal () = ACas () · xCas + APE () · xPE

The surface pressure isotherms for the ␣s -casein, PE6400 and an equimolar mixture of both are shown in Fig. 1. The surface pressure increases gradually with decreasing surface area. The surface pressure isotherm of the polymer is shifted to lower areas compared to those of the ␣s -casein which is most likely due to the lower molecular weight of the polymer. The equimolar mixture of ␣s -casein and PE6400 has a compression behavior that is in between that of the protein and the polymer. Polymer and ␣s -casein isotherms determined here are in agreement with results reported in the literature for ␤-casein [24] and PE6400 films [25]. Information about

where Aideal is the idealized area occupied per molecule at the surface at a specific surface pressure  [26]. Aideal () was calculated for the mixed monolayer by multiplying the area occupied by the ␣s casein ACas () and the polymer APE () with the mole fraction x of the respective component. The additive approach of Eq. (5) assumes that there are no differences in the interactions between different and similar molecules. In general, an equal progression for ideal and measured isotherms indicates that there are no differences in the interactions between different and similar molecules which is interpreted as ideal miscibility or no miscibility. In contrast a

(5)

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negative/positive deviation suggests attractive/repulsive interactions between the surfactants meaning synergistic/antagonistic mixing [26]. Therefore we conclude that for  ≤ 9 mN/m the surfactants forming the monolayer are ideally mixed or immiscible as the calculated and measured isotherm are equal while for  ≥ 9 mN/m attractive interactions between the molecules at the surface are taking place and a mixed monolayer was likely formed as the measured isotherm deviates slightly from the calculated ones. It should be mentioned, that a “squeezing-out” of protein from the film into the bulk phase, as described in the literature for casein systems [24], was not observed in this study. If this had been the case, one would have observed a parallel alignment of the isotherms of the polymer and the mixture. The determined E(A)-curve of the ␣s -casein film (Fig. 1a) has several extrema. In general, extrema of the E(A)-curves [28] are related to phase transitions and can be associated to the corresponding . For ␤-casein, a molecule similar to ␣s -casein, the orientation and arrangement behavior of the molecules in the monolayer at the surface during compression are explained in literature as followed [29]: • At low , amino acids segments are arranged like trains at the interface (structure 1) • At higher  and below film collapse, amino acid segments are “pushed” into the underlying aqueous solution forming loops and tails (structure 2). Applying this explanation to the data obtained in this study, one can conclude that at  ≤ 5 mN/m the protein molecules were arranged in structure 1, while with higher compression ( ≥ 12 mN/m) the proteins were arranged in structure 2 (Fig. 1a). Thus, a transition range is located at 5 ≤  ≥ 12 mN/m. The transition range determined agrees reasonably well with the transition point of ␤-casein at  = 12 mN/m as reported in literature [24]. The E(A)-curve of the PE6400 film (Fig. 1b) also has several extrema The shape observed for the E(A)-curve agrees well with the shape determined for other Pluronics, such as for example Pluronic P84 (PEO19 –PPO43 –PEO19 ) [25]. For P84, three different conformations of PEO–PPO–PEO molecules depending on the extent of the surface compression are described [25], • At low , the polymer chains are oriented horizontally to the surface (structure a) • At intermediate , the PEO parts extend into the aqueous phase (structure b)

• At high , the monomers form thick brushes with overlapping polymer chains which are stretched in normal direction to the surface (structure c). Our results indicate formation of similar structures for the monomers in PE6400 films (Fig. 1b). The E(A)-curve of the mixture (Fig. 1c) also has a maximum which we attribute to the following arrangement of the monomers: • At  ≤ 9 mN/m, the structure is similar to the structure 1 and the structure a (henceforth referred to as structure I). • At  ≥ 9 mN/m, the structure is similar to structures 2, b and c (henceforth referred to as structure II) The arrangement of structure I is characterized by relatively long distances between the molecules and consequently the molecular interactions are small which is also reflected by the similarities between the ideal and the measured isotherm (Fig. 1c). At the phase transition ( = 9 mN/m) the measured isotherm deviated from the ideal one, meaning that attractive interactions between protein and polymer may take place since the molecules are more close together. 3.2. Surface tension and related parameters The equilibrium surface tension ¯ eq of PE6400 as a function of the concentration c at 20 ◦ C is shown in Fig. 2a. It should be noted that when pluronics are investigated, the surface tension isotherms usually do not reach the value of pure water (72 mN/m) since even very low concentrations of surfactants lead to a marked decrease in surface tension [30–32]. A decrease of the ¯ eq with increasing c was observed until the CMC at 84 mM was reached. Above the CMC, the solution had a concentration independent ¯ eq,min of 34 mN/m. The maximum surface concentration  max was calculated as 8.12 × 10−7 mol/m2 and the minimal area occupied per molecule at the surface Amin was found to be 2.1 nm2 (Table 1), which is in agreement with values in the literature ( max = 6.27 × 10−7 mol/m2 , Amin = 2.65 nm2 ) [33]. CMC values reported in the literature are lower (40 mmol/l [33] or 16 mmol/l [34] at 25 ◦ C, and 40 mmol/l [35] at 30 ◦ C) compared to the value measured here, namely 84 mmol/l. However, previous measurements were carried out at higher temperatures than those used in the study at hand (22 ◦ C). The CMC of PEO–PPO–PEO polymers was found to increase with decreasing temperature [5], explaining our higher CMC values.

Fig. 2. (a) Equilibrium surface tension  eq and (b) miscibility gap of PE6400 as a function of PE6400 concentration cPE ; values measured are marked with symbols. Fits are shown as lines in case of the surface tension and lines are guides to the eyes miscibility gap, average and standard error (P = 95%) are given.

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Table 1 Characteristic parameters of PE6400 at the surface determined by surface tension measurement. Parameter

Unit

Value

CMCa  eq,min b  max c Amin d

mmol/l mN/m 10−7 mol/m2 nm2

84 33.8 8.12 2.1

a b c d

CMC, critical micelle concentration.  eq,min , averaged equilibrium surface tension at concentrations above the CMC.  max , maximum surface concentration, determined at the CMC. Amin , minimal area occupied per molecule at the surface, determined at the CMC.

We determined the surface properties of the second surfactant, ␣s -casein, previously [18]. 3.3. Cloud point Fig. 2b shows the cloud point temperatures ϑcp of PE6400 solutions having different concentrations. Below ϑcp the polymer is miscible with the solvent on a macroscopic scale while above ϑcp a phase separation takes place. An emulsifier-rich and an emulsifierpoor phase are formed in solution with one phase being finely dispersed in the other. The dispersed phase scatters light causing a turbid appearance of the solution above ϑcp . ϑcp of the PE6400 solution was a function of the surfactant concentration. ϑcp had a maximum with 62.5 ◦ C at 0.1 mol/l. Similar results are reported in literature [33,36]. The turbidity was not due to the formation of a liquid crystalline phase, a fact that became apparent when samples were observed visually through cross polarizers. Clouding is generally caused by an increase in polymer–polymer interactions while polymer–water interactions become less favorable with increasing temperature [37]. Almgren et al. [37] reviewed three possible explanations for this change in the interactions. First, the PEO fits well into the water structure at lower temperatures. With increasing temperature the water structure is increasingly perturbed and the compatibility vanishes. Second, hydrogen bonds between water and ether oxygen are present. The strength of these hydrogen bonds decreases with increasing temperature. Third, conformation changes take place in the PEO chain rendering it less water soluble. The concentration dependence of ϑcp may be explained by the fact that at low concentrations individual solvent depleted polymer molecules assemble into a separate phase while at moderate concentrations micelles remain assembled, and consequently more energy is required to deplete water from them causing an increase in ϑcp , a fact that was previously described for PEO10 –PPO23 –PEO10 systems [38]. The miscibility gap of ␣s -casein was determined previously [18]. Miscibility gaps were then determined for mixtures containing 0.02, 0.06 and 0.1 mol/l and 2 × 10−3 to 2 × 10−1 mmol/l ␣s -casein (Fig. 3). In comparison to ϑcp of PE6400 (Fig. 2b), addition of low amounts of protein (ccas = 0.004 mmol/l) did not altered ϑcp . A further increase in the ␣s -casein concentration however caused a noticeable decrease of ϑcp compared to the ϑcp of the pure polymer, independent of the polymer concentration (Fig. 3). With increasing PE6400 concentration from cPE = 0.02 to cPE = 0.1 mol/l, the phase boundary is moved to higher temperatures and a second lower phase boundary appeared which is moved to higher temperatures and lower ccas as the cPE increased from cPE = 0.06 to cPE = 0.1 mol/l. Compared to the phase diagram of ␣s -casein [18], the presence of cPE = 0.02 mol/l polymer decreased ϑcp (ϑcp = −24 ◦ C), while the presence of cPE = 0.06 and cPE = 0.1 mol/l increased ϑcp (ϑcp = +5 ◦ C). As mentioned above, the reasons for clouding of PEOx –PPOy –PEOx are attributed to an increase in polymer–polymer attraction and a decrease in polymer–water interactions with

Fig. 3. Miscibility gaps of mixtures containing (a) 0.02, (b) 0.06 and (c) 0.1 mol/l PE6400 as a function of ␣s -casein concentration ccas and temperature ϑ. Lines are guides for the eye. Average and standard error (P = 95%) are given.

increasing temperature. As the upper phase boundary moved to lower temperature with increasing temperature, the presence of protein alters the interactions between polymer and water in such a way that the solubility of PE6400 is decreased. A decrease of cloud points in PEOx –PPOy –PEOx systems is known to be induced by addition of salts. The salt is embedded in the polymer coil and increases the polarity difference between the polymer

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Fig. 4. ␨-Potential of aggregates in solutions containing 0.004 mmol/l ␣s -casein and 0.02 mol/l (black), 0.06 mol/l (gray) or 0.1 mol/l (white) PE6400. Average and standard error (P = 95%) are given.

and the water which facilitates the phase separation [37]. At pH 6.6 the ␣s -casein is negatively charged [18]. Taken together, we thus propose that the addition of the charged protein has a similar effect as the addition of salt: charges are introduced in the PE6400 micelles that increase the polarity difference between solvent and PE6400 which facilitates phase separation. Results can therefore be taken as a further confirmation that interactions between the polymer and the ␣s -casein take place. 3.4. ␨-Potential The ␨-potential of aggregates containing 0.004 mmol/l ␣s -casein in the one-phase region is shown in Fig. 4. Results for other ␣s casein concentrations are not shown since a further increase in ␣s -casein concentration did not alter the ␨-potential significantly (˛ = 0.05). The ␨-potential increased significantly (˛ = 0.05) with increasing polymer concentration, for example from −18 ± 4 mV to −1.0 ± 0.4 mV as the polymer concentration increased from 0.02 mol/l to 0.1 mol/l with 0.004 mmol/l ␣s -casein at 10 ◦ C. The ␨-potential of the solution containing 0.02 mol/l is similar to the ␨potential of the pure ␣s -casein being −18 mV [18]. The ␨-potential appears to possess a maximum at 30 ◦ C at all examined polymer concentrations. Portnaya et al. [17] similarly reported for mixtures of PEO101 –PPO56 –PEO101 (Lutrol) with ␤-casein also decreasing ␨potentials with increasing polymer concentration. They observed a decrease to a ␨-potential of approximately zero at a molar mixing ratio of 0.5 Lutrol/␤-casein. Examination of the structure of both molecules showed that the hydrophilic segments have the same molecular weight while the hydrophilic ␤-casein group is less than half of the length of the PEO chain. At a zero ␨-potential, ␤-casein and Lutrol have the same amount of hydrophilic groups. Therefore the authors proposed a model in which a long uncharged PEO segments extended out of the protein corona, “coating” the micellar surface and “masking” the micelle charge. In our study the ␨-potential was −3 ± 1 mV for a PE6400 to ␣s -casein ratio of 300 at 38 ◦ C, i.e. a mixture containing 0.06 mol/l PE6400 and 0.2 mmol/l ␣s -casein. This value is assumed to be zero in the publication of Portnaya et al. [17] We also examined the architecture of the molecules using the same approach as Portnaya et al. [17]. The hydrophobic part of the polymer has approximately a molecular weight of 1300 and a length of 30 units while the hydrophilic part consists of two chains

with a molecular weight of 570 for each chain with 13 units. The ␣s1 -casein, the main compound in our ␣s -casein consists of two hydrophilic groups, one between AS 45 and 90 while AS 40 and 80 is negatively charged and one between AS 113 and 132 which is uncharged [39]. Assuming an averaged molecular weight of 100 per amino acid, the first hydrophilic group has approximately a molecular weight of 4500 and the second one of 1900. Therefore the charged region of the ␣s1 -casein has approximately a ten time higher molecular weight as one of the PEO chains. Indeed not the molecular weight as assumed by Portnaya et al. [17] is characteristic for a successful shielding. It is in our opinion the length of the hydrophilic parts. One can estimate the length of the protein by assuming the length of the C C binding with 1.51 A˚ and of the C N binding with 1.32 A˚ for a peptide bound leading to a length of 2.83 A˚ of a bound amino acid. Since the molecules assumes a non-liner Ufolded configuration to enable micelle formation, the length of the hydrophilic region is roughly half of the chain length, namely 23 amino acids meaning 65 A˚ resp. 10 amino acids meaning 28.3 A˚ [40]. A similar approach can be used for the PEO, assuming a C C binding ˚ a C O binding length of 1.43 A˚ and a O H binding length of 1.51 A, length of 0.96 A˚ [41], leading to a length of PEO chain with 13 units ˚ Therefore the PEO chain is compared with of approximately 40 A. the longer hydrophilic ␣s1 -casein segment a bit shorter and for the smaller hydrophilic ␣s1 -casein segment a bit longer meaning that a shielding seems to be possible from a geometrical point of view. Although the molecular characteristics of the micellar units differ from those in the study of Portnaya et al. [17], we come to the similar structural conclusion, namely that PPO is anchored to the hydrophobic parts of the protein and the hydrophilic PEO units are extending out of the protein structure thereby shielding charges of the hydrophilic protein moieties. The proposed model agrees with the fact that the ␨-potential remains constant even if less protein was added and is in agreement with the results of Portnaya et al. [17]. Differences are found in the polymer–protein ratio, where the ␨-potential becomes zero: Portnaya et al. [17] found a value of 1 while we found a value of 300. There are two reasons why more polymer molecules are needed in this study. First, the hydrophilic group of the ␣s1 -casein have to be folded due to their location in the center of the amino acid chain. It is therefore more bulky and more thin PEO chains are needed for shielding. Second the CMC of the two polymers differ by a factor 60, PE6400 has a CMC of 5 mmol/l while Lutrol has a CMC of 0.08 mmol/l at 30 ◦ C. This means that the amount of polymer which is not part of the micelle is around 60 times higher for the PE6400 in comparison with Lutrol. It is therefore not possible to assume that the composition of the molecules in the micelle are the same as in solution. Taken together, our results let us conclude that the PE6400 and ␣s -casein form aggregates composed of both compounds. This is in agreement with results of an other study published by our group, where the results of fluorescence measurements also hinted at the presence of mixed aggregates [42].

4. Conclusion By considering all experimental data one may develop a mechanistic model that could explain the formation of structures in mixtures of ␣s -casein and PEO13 –PPO30 –PEO13 (PE6400) both at the surface and in solution (Fig. 5). At the surface, ␣s -casein and PEO13 –PPO30 –PEO13 (PE6400) form mixed monolayers. A comparison of the surface area needed by each molecule assuming an ideal behavior with the measured surface area shows that attractive interactions between the molecules at the surface occur above  = 9 mN/m. In solution, cloud point measurements showed that the two compounds are miscible at distinct mixing ratios and temperatures. The cloud point decreases with increasing protein

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Fig. 5. Schematic diagrams of the behavior of the surfactants (a) at the surface and (b) in solution including an inset which describes the arrangement of the surfactants in the mixed aggregates. The diagrams are based on surface pressure measurements, determination of cloud points and ␨-potential measurements. The hydrophobic regions of the ␣s -casein are taken from Swaisgood [39] (cPE , PE6400 concentration, Aideal , Areal , surfaces needed per molecule in the calculated ideal case or in the measured real case). Presumable, the composition of the mixed self-assembled structure are not the same as in the solution due to CMC differences.

concentrations, while a second miscibility gap appears at low temperature with increasing polymer concentrations. We think, that ␣s -casein and PE6400 form mixed self-assembled micelles which causes a polarity difference between the solvent and the aggregates facilitating phase separation. Measurements of the ␨-potential let also to suppose that mixed aggregates are present. The ␨-potential decreases to zero when PE6400 is added to the protein solution. We propose that the PPO unit is anchored in the self-assembled ␣s -casein micelle while the PEO units are shielding the charges of the hydrophilic protein part leading to the zero ␨-potential as shown schematically in Fig. 5. This is in line with the results of the surface pressure measurements, where attractive interactions between PE6400 and ␣s -casein are observed. Additional information about the size and morphology of the formed structures may be obtained in a subsequent study by small-angle X-ray scattering (SAXS) and freeze-fracture electron microscopy (FFEM). Comparison of our results with the results of Portnaya et al. [17] showed that aside from diblock–triblock combinations such as ␤-casein and Lutrol, triblock copolymers such as ␣s -casein and PE6400 are also able to form mixed structures, albeit these structures possess different properties. Presumable, mixing of synthesized and natural block copolymers could lead to a variety of micellar encapsulation systems whose properties could be adjusted easily by changing the molecular structure of the used synthesized block copolymers. Moreover, solubilization of pyrene in such mixed aggregates is possible [42], which emphasizes their use in various industries as carrier system of functional compounds.

Acknowledgements We would like to thank Aline Holder and Antonie Post at the Department of Dairy Science and Technology, University of Hohenheim, for fractionating the protein and performing the HPLC analysis. We also thank Julia Boos and Enda Carey, Institut für Physikalische Chemie, Universität Stuttgart, for assistance in the surface tension measurements and the generation of phase diagrams; Izabella Brand, Physikalische Chemie, Universität Oldenburg for her assistance in the surface pressure measurements and

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