Colloids and Surfaces B: Biointerfaces 117 (2014) 368–375

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Investigations into aggregate formation with oppositely charged oil-in-water emulsions at different pH values Christiane Maier, Benjamin Zeeb, Jochen Weiss ∗ Department of Food Physics and Meat Science, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany

a r t i c l e

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Article history: Received 24 January 2014 Received in revised form 21 February 2014 Accepted 4 March 2014 Available online 12 March 2014 Keywords: Oil-in-water emulsion Emulsifier Stability Aggregation Heteroaggregate Confocal laser scanning microscopy

a b s t r a c t The pH-dependent formation and stability of food-grade heteroaggregates from oppositely charged oilin-water (O/W) emulsions was investigated. After screening suitable emulsifiers, 10% (w/w) oil in-water emulsions (d32 ≈ 1 ␮m) were prepared at pH 3–7 using a positively charged emulsifier (Na-lauroyl-larginine ethyl ester; LAE) and four negatively charged ones (citric esters of mono- and diglycerides, soy lecithin, sugar beet pectin, and Quillaja saponin). The oppositely charged emulsions were then combined at constant pH values at a volume flow rate ratio of 1:1. Emulsions and heteroaggregates were characterized by their surface charge, particle size distribution and microstructure using dynamic and static light scattering as well as confocal laser scanning microscopy. The emulsifier type was found to greatly influence the type of heteroaggregates formed, as well as the pH value, specifically in combined LAE/Quillaja saponin emulsions. Larger aggregates particularly were formed with increasing pH values (2.71 ± 1.21 to 46.53 ± 4.30 ␮m from pH 3 to 7, respectively), while LAE/pectin aggregates appeared not to be affected by pH over the full pH range investigated (3.80 ± 2.89 to 3.94 ± 2.78 ␮m from pH 3 to 7, respectively). Our study thus provides valuable first insights into the mechanism of the formation of food-grade heteroaggregates for later use in food systems. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Rational design of food ingredient structures has led to the development of new applications, such as fat replacers, carrier systems for bioactives, and texture and aroma modifiers. A promising innovative approach to design such novel food structures is to combine oppositely charged particles, leading to the formation of so-called heteroaggregates [1]. These may be assembled from particles having varying physicochemical properties, such as interfacial composition, charge density and size [2,3]. A prominent application of colloidal heteroaggregation in the food industry is the clarification of fruit and vegetable juices, using positively charged gelatin to induce the flocculation of negatively charged pectin-stabilized haze particles [4]. However, applications to create innovative food products due to aggregation of oppositely charged O/W droplets are scarce to date, although its use for altering the sensory perception of food products was proposed recently. Different products from low viscous liquids to highly viscous, paste-like products were achieved by manipulating attractive and repulsive interactions between two oppositely charged droplets of O/W emulsions [1]. A major issue in

∗ 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.2014.03.012 0927-7765/© 2014 Elsevier B.V. All rights reserved.

bringing emulsion droplets into close proximity is a phenomenon known as coalescence. Thereby, de-emulsification is often induced due to the fusion of different droplets forming larger ones if no repulsive forces are present on the droplets’ surfaces [5]. Further information on droplet coalescence was described by the extended DLVO theory [6,7]. Hence, special attention was paid to the interfacial characteristics that can be controlled by the use of suitable surfactants to rationally induce the transformation from single food-grade emulsion droplets to large three-dimensional aggregates with intact individual droplets [8]. Biopolymers, such as proteins or polysaccharides, as well as low-molecular weight surfactants, are typically used in food emulsions. Lecithins, for instance, tend to form inter˚ corresponding to the thickness of two facial layers of about 80 A, double lipid layers [9]. By contrast, sugar beet pectins were shown to form thick layers of around 140 nm, which closely agrees with the hydrodynamic diameter of the pectin chains [10]. Besides the emulsifiers’ molecular geometry and, therefore, their interfacial layer thickness, the charge of their hydrophilic group (positive, negative and zwitterionic, respectively) and pH-dependent charge changes are of paramount importance for electrostatic attraction and repulsion. Successful aggregation of oppositely charged droplets of O/W emulsions, for instance, was recently induced by combining emulsions stabilized by the surface active proteins ␤-lactoglobulin and

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lactoferrin [11,12], and whey protein isolate and modified starch [13]. However, the use of proteins is limited due to their pHdependent instability at their isoelectric point and their charge shift from positive to negative above it. As outlined above, controlling interfacial properties to induce aggregation is intricate due to the limited number of food- or cosmetic-grade charged emulsifiers that can be utilized within a broad pH range. Therefore, our study aims at the formation of stable heteroaggregates produced from oppositely charged O/W emulsions after selection of appropriate emulsifiers (e.g. electrical charge, layer thickness) in an initial emulsifier screening. In addition to the emulsifier type, we sought to influence the aggregate size by varying the pH of the aqueous phase, leading to altered interfacial interactions. 2. Materials and methods

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Thereby, minimal amounts of emulsifier necessary to obtain stable stock emulsions were determined in preliminary experiments (0.2% (w/w) Citrem, 0.5% (w/w) soy lecithin, 0.1% (w/w) LAE, calculated as the actual LAE amount (14.5%) within the Mirenat-LAE product, 0.5% (w/w) Quillaja saponin, and 0.5% (w/w) sugar beet pectin, respectively). After the addition of 10% (w/v) Miglyol to 90% (w/w) emulsifier solution, a pre-emulsion was formed by mixing oil and emulsifier solution at 24,000 min−1 for 3 min, using a high-shear blender (labworld-online, Germany). Subsequently, pre-emulsions were ultrasonicated at 15% amplitude for 1 min (HD2200 Bandelin Sonopuls, Bandelin electronic GmbH & Co.KG, Germany). Emulsions used for confocal laser scanning microscopy (CLSM) were stained using 0.005% (w/w) of the lipophilic Nile Red prior to homogenization, while the continuous phase was stained by 0.1% (w/w) Nile Blue A added after homogenization. All emulsions were stored at room temperature for 24 h prior to analysis and the pH values were adjusted if necessary.

2.1. Materials Distearyldimethylammonium chloride (Varisoft® TA 100) and C8-18 alkylamidopropyl betaines (TEGO® Betain C60 and TEGO® Betain ZF) were provided by Evonik Industries AG (Essen, Germany). Citric esters of mono- and diglycerides (MD/DG; Grindsted Citrem RO kosher) and diacetyl tartaric esters of MD/DG (Panodan M2020 kosher) were called Citrem and Datem in the following, respectively, and were supplied by Danisco AG (Grindsted, Denmark). Sodium stearoyl-2-lactylat (Cognis Prefera SSL 6000) was donated by BASF SE (Illertissen, Germany). Soy lecithin (Ultralec P) was supplied by Archer Daniels Midland Company (Illinois, USA). Quillaja saponin (Andean QDP Ultra Organic) was purchased from Desert King International (Quilpué, Chile), labeling a saponin content of 62.5% (w/w). Sodium caseinate (Rovita FN 5 S 0222) was provided by Rovita GmbH (Engelsberg, Germany) and whey protein isolate (BiPRO® ) was donated by Davisco Foods International, Inc. (Minnesota, USA). Sugar beet pectin (degree of esterification: 55%) was donated by Herbstreith & Fox KG (Neuenbürg, Germany). Sodium lauroyl-l-arginine ethyl ester (Mirenat® -LAE), abbreviated to LAE in the following, was purchased from Meat Cracks Technologie GmbH (Mühlen, Germany). The manufacturer stated the LAE amount to be 14.5% (w/w). The cold water fish-skin gelatin, the fluorescence dyes Nile Red and Nile Blue A and sodium azide were purchased from Sigma–Aldrich Co. (Steinheim, Germany). The gelatin’s average molecular weight and pI value were reported to be ca. 60 kDa and pH 6, respectively. Sodium dodecyl sulphate (SDS) of analytical grade (>99%), hydrochloric acid and sodium hydroxide were purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). The medium chained triglyceride Miglyol 812N was purchased from Cremer Olio GmbH & Co. KG (Hamburg, Germany). Ultrapure water was used for the preparation of all samples. 2.2. Preparation of emulsifier solutions for an initial screening Aqueous emulsifier solutions were prepared by dispersing 0.05% (w/w) SDS, 0.5% (w/w) betaines, soy lecithin, distearyldimethylammonium chloride or sodium stearoyl-2-lactylat, and 1% (w/w) of all other emulsifiers (Table 1) into ultrapure water and subsequent stirring overnight. 0.02% (w/w) sodium azide was added to the solutions to inhibit microbial growth. From each stock solution, samples were adjusted to different pH values (pH 3, 4, 5, 6, and 7), using HCl or NaOH. 2.3. Preparation of selected stock emulsions Based on the initial emulsifier screening, appropriate emulsifiers including Citrem, LAE, Quillaja saponin, soy lecithin, and sugar beet pectin were selected for the formation of stock emulsions.

2.4. Combination of oppositely charged stock emulsions to induce aggregation After pH and -potential verification, oppositely charged stock emulsions with appropriate pH values were combined under controlled flow conditions in a Y-mixing chamber at a volume flow rate ratio of 1:1 (4.1 ml/min). The combined emulsions were allowed to stand for 30 min prior to a repeated pH verification and subsequent storage at room temperature for 24 h before further analysis. 2.5. Characterization of emulsifier solutions, stock emulsions and combined emulsions 2.5.1. -potential measurements Emulsifier solutions were used without dilution, whereas emulsions and combined emulsions were diluted to a droplet concentration of approximately 0.005% (w/v) with ultrapure water having an appropriate pH value. Subsequently, a particle electrophoresis instrument (Nano ZS, Malvern Instruments, Malvern, UK) was used for the determination of the -potential. The Smolouchowski equation was used to calculate the -potential. The values reported represent means ± standard deviation of each three readings of two independently prepared samples. 2.5.2. Particle size analysis The particle size distributions of the stock and combined emulsions were determined by static light scattering (Horiba LA-950, Retsch Technology GmbH, Haan, Germany). Samples were diluted with pH-adjusted ultrapure water to a droplet concentration of approximately 0.005% (w/w) to prevent multiple scattering effects. The instrument measures the angular dependence of the intensity of the laser beam scattered by the stirred diluted emulsions and then uses the Mie theory to calculate the droplet size distributions that give the best fit between theoretical predictions and empirical measurements. A refractive index of 1.42 was used. The particle size measurements were reported  3 as mean volume-surface diameter d32 , which is defined as di ni / di ni 2 , where ni is the number of droplets of diameter di . Droplet diameters were determined from at least two freshly and independently prepared samples each with four measurements. As already stated by Mao and McClements [12], particle size measurements on highly aggregated combined emulsions should be treated with caution, since the Mie theory assumes spherical particles with well-defined refractive indices for the calculation of particle size distributions. However, heteroaggregated emulsions might be non-spherical and non-homogeneous with a broad refractive index range. Thus, the particle sizes calculated should only be

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Table 1 Selected food- and cosmetic-grade emulsifiers and their potential suitability (yes/no) to initiate aggregation of oppositely charged O/W emulsions. Emulsifier type

Criteria for emulsifier choice for a pH-dependent screening to initiate aggregation of oppositely charged O/W emulsions

Suitability

Mirenat LAE Distearyldimethyl-ammonium chloride Fish gelatin* Sodium caseinate* Whey protein isolate* Betaine C60# Betaine ZF# Sodium stearoyl-2-lactylat

Highly positive charged over broad pH-range, food-grade Positive charge, approved for cosmetics

Yes No No

Citric esters of MG/DG (Citrem) Diacetyl tartaric esters of MG/DG Quillaja saponins Soy lecithin Sodium dodecyl sulphate Sugar beet pectin

* : Not suitable for pH-shift experiment due to charge shift from positive to negative at IEP # : Only barely charged, furthermore charge shift from positive to negative due to zwitterionic character High negative charge, no stable O/W emulsions were obtained Negative charge highly pH-dependent, food-grade Similar to Citrem, weaker charged and only weakly pH-dependent Weakly negative charged, contain sugar side groups that increase steric repulsion, food-grade Negative charge highly pH-dependent, food-grade Negative charge, approved for cosmetics Negative charge highly pH-dependent, contains sugar side groups that increase steric repulsion, food-grade

treated as a rough estimation of the cluster size, rather than as accurate and precise representations.

No No Yes No Yes Yes No Yes

2.5.3. Microstructure analysis Three-dimensional structures of the combined emulsions were visualized by a confocal laser scanning microscope (CLSM; EclipseC1, Nikon GmbH, Düsseldorf, Germany). The microscope was equipped with a 20× objective and settings were controlled with the Nikon-ECZ1 software. Dispersed oil phases were stained with Nile Red, exciting at 488 nm and detected at 590/50 nm. The undiluted combined emulsion pairs were gently shaken and put into the specimen holders. For the contrasting of oppositely charged droplets of heteroaggregates, the dispersed phase of the Quillaja saponin-stabilized pre-emulsion was stained using Nile Red, whereas the continuous phase of the LAE-stabilized pre-emulsion was stained using Nile Blue A (excitation: 637 nm; detection: 650LP nm). Pre-emulsions were used for this experiment since only droplets of a larger size could be visually differentiated. Adobe Photoshop CS4 was used to adapt the contrast and brightness of the microscopic images obtained.

Due to the reasons mentioned above, only Mirenat LAE was selected as the most promising positively charged emulsifier. Regarding negatively charged emulsifiers (Fig. 1d–f), a broader variety of emulsifiers were found to maintain a substantial negative -potential, while shifting the pH from 3 to 7. The -potential of the soy lecithin solutions (Fig. 1e), for instance, decreased from ca. −35 mV to ca. −64 mV when the pH was increased from 3 to 7. In addition, citric esters of mono- and diglycerides (Fig. 1d), Quillaja saponin (Fig. 1e) and sugar beet pectin (Fig. 1f) revealed strong pHdependent charge magnitudes and were, therefore, selected for the preparation of negatively charged emulsions. Based on that screening, five emulsifiers were selected to investigate the aggregation behavior of combined emulsions within a broad pH range due to their high positive or negative electrical charges. Hereby, the LAE solution was the only positively charged one. Quillaja saponin, soy lecithin, sugar beet pectin, and Citrem solutions remained negatively charged, but differed from each other within their molecular geometry. The selection criteria for potential emulsifiers that might be used to initiate the aggregation of oppositely charged stock O/W emulsions at various pH values are summarized in Table 1.

3. Results and discussion

3.2. Characterization of stock emulsions

3.1. Selection of food- and cosmetic-grade emulsifiers—a screening

The minimum amount of emulsifier (Citrem, LAE, soy lecithin, sugar beet pectin, and Quillaja saponin) that is required to form stable emulsions without depletion flocculation was determined in preliminary experiments (data not shown). Subsequently, 10% (w/w) oil-in-water stock emulsions were prepared by mixing and subsequent ultrasonication, obtaining a mean emulsion droplet diameter of ca. 1 ␮m at all tested pH values (Table 2). Moreover, Fig. 2 illustrates the pH-dependent behavior of the emulsions surface charges (-potential). Soy lecithin stock emulsions revealed a negative ␨-potential from −44.75 ± 4.45 mV at pH 3 to −81.78 ± 2.05 mV at pH 7, which is in agreement with the results of the emulsifier screening mentioned above, and previously shown by Ogawa et al. [15]. The charge characteristics of all other emulsions showed similar pH-dependent behavior, i.e. the -potential of sugar beet pectin-stabilized stock emulsions ranged from −18.60 ± 3.82 mV to −50.68 ± 1.96 at pH 3 to pH 7, respectively. The -potentials of Citrem-stabilized oil droplets were between −26.85 ± 15.82 mV at pH 3 to −92.58 ± 9.22 mV at pH 7. -potentials for the Quillaja saponin-stabilized emulsions ranged from −14.90 ± 5.75 (pH 3) to −58.10 ± 12.96 mV (pH 7), while the

Only widely available food- and cosmetic-grade emulsifiers were included in the screening (Table 1), aiming at such emulsifiers that would maintain substantial positive or negative -potentials while varying the pH from 3 to 7. Representing a further selection criterion, food-grade emulsifiers were given higher priority than cosmetic-grade emulsifiers. Mirenat LAE maintained a constant positive -potential of ca. +85 mV over the full pH range investigated, while the -potential of the cosmetic-grade distearyldimethylammonium chloride increased from approximately +45 mV at pH 3 to +70 mV at pH 7, as shown in Fig. 1a. By contrast, the positive -potential (+20 and +30 mV at pH 3) of the proteins used (sodium caseinate and whey protein isolate, respectively) changed as expected to negative values at pH > pI (ca. pH 4.5, Fig. 1b)—a fact that was previously described in the literature [14]. Fish gelatin, betaine C60 and ZF similarly showed shifting -potentials at generally low magnitudes of charge (Fig. 1c) and were thus excluded.

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Fig. 1. Influence of pH on electrical charge (-potential) of various food- or cosmetic-grade emulsifiers, dissolved in ultrapure water that has been adjusted to pH 3–7: (a) positively charge emulsifiers, (b) and (c) zwitterionic emulsifiers, (d), (e), and (f) negatively charged emulsifiers. The number in brackets indicates the emulsifier amount (% w/w).

-potentials of the only emulsion providing positively charged lipid droplets (LAE-stabilized) varied from +93.72 ± 1.30 mV at pH 3 to +65.73 ± 17.06 mV at pH 7. The stock emulsions were stable against self-aggregation, due to sufficiently small particle sizes and high electrostatic (and steric) repulsive interactions—a fact that is important for the transformation of single droplets into heteroaggregation.

3.3. Characterization and classification of the combined emulsions The positively charged LAE-stabilized emulsion was combined with each of the negatively charged Citrem-, soy lecithin-, sugar beet pectin-, and Quillaja saponin-stabilized stock emulsions, using a Y-mixing chamber at a controlled flow rate ratio of 1:1. Each emulsion pair was produced and combined at five different pH levels (pH

3, 4, 5, 6, and 7). Combinations were analyzed in terms of surface charge, particle size and visual appearance (Figs. 3–5). After storage at room temperature for 24 h, visual observations showed three clearly different types of combined systems, as illustrated by a mechanistic model in Fig. 6: emulsion breakdown (type 1), stable combinations (type 2), and heteroaggregates (type 3). As a first type (type 1), rapidly occurring thick oil layers on top of the combined emulsions’ surface indicated emulsion breakup—a phenomenon which was observed for all LAE/Citrem combinations, irrespective of pH. A second class of combinations remained visually stable, having a uniform milky appearance throughout all pH values. LAE/sugar beet pectin combinations at all pH values fell into this category (type 2). The third type (type 3) of combined emulsions was represented by the formation of visually noticeable macroscopic aggregates, as observed for the LAE/Quillaja saponin combinations. For these combinations, aggregate size apparently increased with increasing pH, since a larger gravitational separated

Table 2 Mean particle diameter (d32 ) of 10% (w/w) O/W emulsions as a function of pH (3–7). Emulsions were prepared by mixing oil and emulsifier solution for 3 min at 24,000 min−1 , using a high shear blender and subsequent ultrasonication for 1 min at 15%. Emulsifier type

Emulsifier amount (% w/w)

Citric esters of MG/DG LAE Quillaja saponins Soy lecithin Sugar beet pectin

0.2 0.1 0.5 0.5 0.5

Mean particle diameter d32 (␮m) pH 3

pH 4

± ± ± ± ±

1.23 0.88 0.74 0.80 0.92

1.78 0.92 1.11 0.79 1.02

0.23 0.22 0.36 0.01 0.07

pH 5 ± ± ± ± ±

0.07 0.21 0.00 0.02 0.01

0.93 0.87 0.76 0.86 1.05

pH 6 ± ± ± ± ±

0.21 0.22 0.07 0.02 0.06

0.87 0.92 0.70 0.82 1.16

pH 7 ± ± ± ± ±

0.11 0.28 0.03 0.12 0.04

0.85 0.87 0.71 0.91 1.15

± ± ± ± ±

0.09 0.24 0.02 0.03 0.06

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might be highly prone to coalescence when oppositely charged droplets are forced together, while the rigid layers formed by polysaccharide-coated fat droplets might reveal superior stability during aggregation [16].

Fig. 2. -potentials of 10% (w/w) oil-in-water emulsions at pH 3–7, stabilized by 0.1% (w/w) LAE, 0.2% (w/w) citric esters of mono- and diglycerides (MG/DG), 0.5% (w/w) sugar beet pectin, 0.5% (w/w) soy lecithin, and 0.5% (w/w) Quillaja saponin, respectively.

opaque layer on top of a clearer serum layer at the bottom was observed at higher pH values by visual inspection. In contrast to the uniform behavior of the LAE/Citrem (Fig. 6, type 1 only), LAE/sugar beet pectin (Fig. 6, type 2 only) and the LAE/Quillaja saponin combinations (Fig. 6, type 3 only), the LAE/soy lecithin combination remained stable at pH 3 and 4 (type 2), but thick oil layers on top of the combinations at pH 5–7 were clearly visible (type 1). 3.3.1. Behavior of unstable combined emulsions (type 1) Emulsion breakup after combination was only observed for emulsion combinations stabilized by small molecular surfactants, i.e. for LAE/Citrem (pH 3–7) and LAE/soy lecithin (pH 5–7). Small surfactants may lead to more mobile interfacial layers and, thus,

3.3.2. Behavior of stable, combined emulsions (type 2 and 3) The combined emulsions that did not show oiling-off behavior (Fig. 6, type 2 and 3) were examined more intensively by -potential and particle size measurements and CLSM visualization. The -potentials of respective emulsion combinations decreased with increasing pH, i.e. the combinations LAE/sugar beet pectin and LAE/Quillaja saponin decreased from −15.68 ± 5.68 mV (pH 3) to −43.90 ± 0.57 mV (pH 7) and from −6.02 ± 6.80 (pH 3) to −48.10 ± 1.18 mV (pH 7), respectively (Fig. 3b). It is noteworthy that the pH-dependent behavior of the -potentials of both combined emulsions were highly similar to the respective negatively charged stock emulsions and not the approximated mean value of the positive and negative charge, as observed previously by several groups [2,13,17]. Despite their -potential being similar to the respective negatively charged emulsion, mean droplet diameter distributions of the two combinations (LAE/Quillaja saponin, LAE/sugar beet pectin) differed considerably from each other, specifically with increasing pH values (Fig. 3a). Along with increasing absolute potential values of the LAE/Quillaja saponin combinations from pH 3 to 7, mean particle diameters (d32 ) increased from 2.71 ± 1.21 to 46.53 ± 4.30 ␮m, respectively. By contrast, the particle size distribution of the LAE/sugar beet pectin combination did not appear to be affected over the full pH range investigated (3.80 ± 2.89 to 3.94 ± 2.78 ␮m from pH 3 to 7, respectively (Fig. 3a). Additionally, CLSM was conducted to visualize the threedimensional structures of these emulsion combinations, staining the lipid phases with Nile Red. The pH-dependent microstructures of the combined emulsions LAE/sugar beet pectin and LAE/Quillaja saponin are shown in Fig. 4. Supporting the particle size measurements by static light scattering (Fig. 3a), CLSM micrographs confirmed a slightly larger particle size distribution for the LAE/pectin combinations, being congruent over the entire pH range. Increased particle sizes were also observed by Mao and McClements [12] for oppositely charged protein-coated droplets. They postulated that droplet coalescence within the microclusters may occur due to the strong attraction of the opposite charges.

Fig. 3. pH-dependent mean particle diameter (d32 ) (a) and -potential (b) of combined (comb.) 10% (w/w) O/W emulsions LAE/sugar beet pectin, LAE/Quillaja saponin and LAE/soy lecithin (emulsion breakup at pH > 4), volume flow mixing ratio: 1:1.

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Fig. 4. Three-dimensional CLSM images of combined emulsions (10% (w/w) O/W) stabilized by LAE and sugar beet pectin (LAE/sugar beet pectin), respect, and LAE/Quillaja saponin (volume flow mixing ratio: 1:1) as a function of pH. Emulsions were prepared by mixing oil and emulsifier solution at 24,000 min−1 for 3 min, using a high-shear blender and subsequent ultrasonication at 15% for 1 min. The scale bars are 100 ␮m in length.

While CLSM micrographs of the LAE/Quillaja saponin aggregated emulsions also show some coalescence at pH 3 and 4, an outstanding pH-dependent formation of aggregates was observed, yielding three-dimensional cluster structures from pH 5 to 7. Moreover, aggregate size within the CLSM images seemed to be even greater (>100 ␮m) than the values measured using static light scattering (ca. 30–50 ␮m, Fig. 3a). However, since the samples were diluted and slightly sheared to avoid multiple scattering effects prior to particle size measurements by static light scattering, big clusters might be reduced to fractured smaller pieces. In order to visualize the aggregated structures formed by LAE/Quillaja saponin in-depth, combined pre-emulsions containing larger droplets were observed by CLSM at pH 7, since the largest aggregates were observed at this pH. Therefore, the continuous phase of the LAE-stabilized emulsion was stained using Nile Blue A, whereas the dispersed phase of the Quillaja saponin-stabilized emulsion was stained with Nile Red. Two-dimensional CLSM pictures of various droplets are depicted in Fig. 5a and b. According to

our staining technique, the black spots illustrate the LAE-stabilized droplets and the red dots represent the Quillaja saponin-stabilized ones. Two-dimensional cross-sections of heteroaggregates were observed immediately after emulsion combination. Pictures were taken directly after combination and observed to be stable within at least 1 h. Within the examination time, the oppositely charged droplets stuck to each other as individual entities, not showing any fusion of the two kinds of droplets emphasizing the formation of stable heteroaggregates, classified as type 3. 3.4. Theoretical considerations 3.4.1. Calculation of adsorption times Evaluating the substantial differences between the three performances observed after emulsion combination, namely emulsion breakup (type 1), increase in droplet diameter (type 2) and aggregate formation (type 3) (Fig. 6), approximated adsorption times for the different emulsifiers were calculated. Adsorption dynamics of

Fig. 5. CLSM images of combined pre-emulsions (10% (w/w) O/W) stabilized by LAE and Quillaja saponin at pH 7, respectively. The pre-emulsions were prepared by mixing oil and emulsifier solution at 24,000 min−1 for 3 min, using a high-shear blender. The red spots illustrate the Quillaja saponin-stabilized one, whereas the black spots illustrate the LAE-stabilized droplets. Images (a) and (b) depict two different aggregates. Pictures were taken directly after combination.

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Fig. 6. Illustration of a mechanistic model demonstrating proposed behavior of combined emulsions, containing oil droplets of opposite charge, with either emulsion breakup (type 1), increase of particle size (type 2), or formation of heteroaggregates (type 3), respectively.

emulsifiers provide important knowledge, e.g. when the stability of emulsions has to be explained. Hereby, instability phenomena greatly depend on the  (t) value, the surface concentration of emulsifiers at any instant in time, which, in turn, depends on the diffusion coefficient D of the emulsifiers. For short adsorption times, they depend on each other in the following way, with c0 being the emulsifier bulk concentration (Eq. (1)).



 (t) = 2c0

Dt 

(1)

In equilibrium, the adsorption and desorption fluxes of emulsifiers are in balance; for instance, if the actual surface concentration at the interface is smaller than the equilibrium one, then the adsorption flux to the interface dominates and vice versa [18]. Consequently, the time needed to obtain almost complete adsorption ( / ∞ ≈ 0.97) with  ∞ as the final surface concentration of emulsifier, can roughly be approximated by Eq. (2) [19]. tabs ≈ 10

2 ∞

c02 D

(2)

According to Ferri and Stebe [20], diffusion coefficients vary only weakly for surfactants of similar size. Most surfactants have D ≈ 5 × 10−6 cm2 /s and ∞ ≈ 3 mg/m2 , whereas the diffusion coefficients for macromolecular surfactants are generally smaller, by around a factor of 4 [19].  for sugar beet pectin is around 20 mg/m2 [10]. It should be noted that the assumptions may not be entirely correct, nevertheless, they allow a comparison of the data obtained. Representing the group of small surfactants, the diffusion time tabs calculated for citric esters of MG/DG is around 45 ms and for the macromolecule sugar beet pectin around 1.28 s, respectively. Although these adsorption times are drastically different, emulsifier exchange between non-adsorbed emulsifiers in the surrounding aqueous phase and the coated oil droplets and, most likely, also between the aggregated droplets happens

directly while combining the emulsions. This rapid exchange simultaneously leads to a rapid charge approximation between the two kinds of droplets [17]. Subsequently, insufficient electrostatic stabilization may result in emulsion breakup if other stabilizing principles are absent, as, for instance, for Citrem and soy lecithin. However, the combination LAE/soy lecithin remained stable against droplet coalescence at pH 3 and 4, possibly due to soy lecithin’s zwitterionic character. Its major phospholipid components (as stated by the manufacturer) were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acids, with primary pKa values (of the phosphate group) varying from ca. 1.0–3.9 [21]. If the pH drops below these values, the majority of phosphate groups are protonated, whereas the ammonium group remains unaffectedly positively charged, resulting in electrostatic repulsion of the likely charged LAE-stabilized droplets. At higher pH (pH > 3.9), the phosphate groups of the lecithin phospholipids will be deprotonated, thus initiating droplet attraction and subsequent coalescence of the combined emulsions, as observed in our study (Fig. 3a). As described above, further stabilizing principles beyond electrostatic stabilization are proposed to be a crucial prerequisite for the formation of stable heteroaggregates. In contrast to small molecular surfactants, Kirby et al. [22] stated that sugar beet pectin macromolecules adsorb with long dangling tails protruding out into the solution, leading to steric stabilization. By analogy, Quillaja saponin contains large sugar side groups that extend into the aqueous phase [23] and, therefore, also generate a substantial steric effect for droplet stabilization. Furthermore, as stated by the manufacturer, Quillaja saponin contains varying amounts of proteins (3.5–7%) and polyphenols besides 62.5% saponins. That mixture of different components might lead to increased droplet stability. Additionally, according to Chee et al. [10], sugar beet pectins also typically contain a small percentage of proteins (∼2%) which adsorb and, therefore, desorb very slowly [19,24]. Summarizing, steric effects for the LAE/sugar beet

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pectin and LAE/Quillaja saponin samples might have stabilized the combined emulsions against coalescence and subsequent emulsion breakup. Nevertheless, both systems showed a drastically different pH-dependent behavior as described above. 3.4.2. Proposed physicochemical properties of emulsifiers required for heteroaggregation In contrast to all other combinations, the LAE/Quillaja saponin combined emulsions showed considerable aggregate formation (Figs. 3a and 4) which cannot sufficiently be explained solely by adsorption dynamics and steric effects. The pH particularly was demonstrated to have a decisive effect on aggregate formation of the LAE/Quillaja saponin emulsions. Aggregate size increased with increasing pH, leading to large three-dimensional clusters. Ikedo et al. [23] described bovine serum albumin and soy saponin interactions. They concluded that interactions between proteins and saponins might have been induced due to electrostatic and hydrophobic effects. Their considerations might explain heteroaggregate growth with increasing pH, since the electrostatic effect particularly increases with increasing pH due to deprotonation of the carboxyl group contained in saponins. A further explanation for the formation of exceptional aggregates might be derived from the presence of polyphenols in the saponin-stabilized emulsions. Polyphenolic compounds have easily oxidizable structural elements, such as an ortho-diphenol structure, which are readily oxidized to an ortho-quinone by molecular oxygen, particularly when plant enzymes such as polyphenol oxidases are present. Subsequently, the highly reactive quinones may react with nucleophilic partners, such as amino or sulfhydryl side chains of polypeptides and proteins, to form covalent C–N or C–S bonds with the phenolic ring. Most polyphenols can undergo multiple reactions with nucleophilic compounds, leading to the formation of cross-linked polymers [25]. As for the LAE/Quillaja saponin combination, the Quillaja polyphenols could have reacted with Quillaja proteins (3–7% of the Quillaja saponin), stabilizing the membrane of the Quillaja saponin-coated emulsion. Furthermore, Quillaja polyphenols could have cross-linked with the nucleophilic ␣-amino and the guanidine group of the arginine moiety of LAE. In agreement with our observations, the reactivity of polyphenols and, hence, quinones increases with increasing pH [26,27]. Besides cross-linking, polyphenolic compounds may also react noncovalently with polypeptides via hydrogen-bonding at low pH values. At high pH values, they may also interact with hydrophobic patches, e.g. aliphatic chains or other aromatic groups, and with other ionized molecules [19,28]. The formation of stable heteroaggregates from LAE- and Quillaja saponin-stabilized emulsions is likely to be due to interaction of several of the considerations mentioned above. Therefore, a simplified mechanistic model is illustrated in Fig. 6 showing the requirements that are of utmost importance to at least inhibit coalescence and, furthermore, that might lead to the formation of heteroaggregates. 4. Conclusions This study has demonstrated that the formation of rationally designed food-grade heteroaggregates is feasible by choosing appropriate emulsifiers. The emulsifier type was of the highest importance for the type of heteroaggregates formed, i.e. generation of aggregates containing intact individual droplets versus formation of large coalesced oil droplets or emulsion breakup.

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Emulsions, for instance, immediately coalesced when combining LAE-stabilized droplets with mono- and diglyceride-stabilized ones, while stable aggregates were formed when combining Quillaja saponin- and pectin-stabilized emulsions with LAE ones. The pH was found to have a decisive effect on aggregate formation, specifically in combined LAE/Quillaja saponin emulsions. Larger aggregates were formed with increasing pH values for LAE/Quillaja, while LAE/pectin aggregates did not appear to be affected by pH over the full pH range. Our study thus provides valuable first insights into the mechanism of the formation of food-grade heteroaggregates for later use in food systems. However, further studies investigating the interaction mechanisms of high purity samples of LAE and Quillaja saponin and other components contained should be carried out. This might result to an in-depth understanding of the heteroaggregation mechanism leading to a structure-function related approach to induce and modify those heteroaggregated structures. Acknowledgments We would like to thank Archer Daniels Midland Company (Illinois, USA), BASF SE (Illertissen, Germany), Danisco AG (Grindsted, Denmark), Davisco Foods International, Inc. (Minnesota, USA), Evonik Industries AG (Essen, Germany), Herbstreith & Fox KG (Neuenbürg, Germany), and Rovita GmbH (Engelsberg, Germany) for generously providing us with emulsifier samples. Furthermore, the authors would like to thank Martin Sramek (University of Hohenheim) for his kind assistance during the CLSM microscopy and Ralf Martin Schweiggert (University of Hohenheim) for his kind introduction to Adobe Illustrator and numerous fruitful discussions. References [1] Y. Mao, D.J. McClements, Food Chem. 134 (2012) 872. [2] S. Rollié, K. Sundmacher, Langmuir 24 (2008) 13348. [3] J.M. López-López, A. Schmitt, A. Moncho-Jordá, R. Hidalgo-Álvarez, Soft Matter 2 (2006) 1025. [4] U. Schobinger, Frucht- und Gemüsesäfte: Technologie, Chemie, Mikrobiologie, Analytik, Bedeutung, Recht. 2., neubearb. und erweit. Aufl. ed., Ulmer, Stuttgart (Hohenheim), 1987. [5] D.J. McClements, Food Emulsions: Principles, Practice, and Techniques, CRC Press Inc, 2005. [6] A.M. Islam, B.Z. Chowdhry, M.J. Snowden, Adv. Colloid Interface Sci. 62 (1995) 109. [7] Y. Mao, D.J. McClements, J. Appl. Polym. Sci. 130 (2013) 3833. [8] D.J. McClements, E.A. Decker, J. Weiss, J. Food Sci. 72 (2007) R109. [9] L. Rydhag, I. Wilton, J. Am. Oil Chem. Soc. 58 (1981) 830. [10] K.S. Chee, P.A. Williams, S.W. Cui, Q. Wang, J. Agric. Food Chem. 56 (2008) 8111. [11] Y. Mao, D.J. McClements, J. Colloid Interface Sci. 380 (2012) 60. [12] Y. Mao, D.J. McClements, Food Hydrocoll. 27 (2012) 80. [13] Y. Mao, D.J. McClements, Food Hydrocoll. 33 (2013) 320. [14] D.J. McClements, E.A. Decker, Y. Park, J. Weiss, Crit. Rev. Food Sci. Nutr. 49 (2009) 577. [15] S. Ogawa, E.A. Decker, D.J. McClements, J. Agric. Food Chem. 52 (2004) 3595. [16] C. Chung, B. Degner, D.J. McClements, Food Res. Int. 48 (2012) 641. [17] Y. Mao, D.J. McClements, Food Hydrocoll. 25 (2011) 1201. [18] S.S. Dukhin, G. Kretzschmar, R. Miller, Dynamics of Adsorption at Liquid Interfaces: Theory, Experiment, Application, Elsevier Science, 1995. [19] P. Walstra, Physical Chemistry of Foods, Dekker, New York, 2003. [20] J.K. Ferri, K.J. Stebe, Adv. Colloid Interface Sci. 85 (2000) 61. [21] D. Marsh, CRC Handbook of Lipid Bilayers, CRC Press, 1990. [22] A.R. Kirby, A.J. MacDougall, V.J. Morris, Food Biophys. 1 (2006) 51. [23] S. Ikedo, M. Shimoyamada, K. Watanabe, J. Agric. Food Chem. 44 (1996) 792. [24] V.B. Fainerman, R. Miller, J.K. Ferri, H. Watzke, M.E. Leser, M. Michel, Adv. Colloid Interface Sci. 123–126 (2006) 163. [25] G. Strauss, S.M. Gibson, Food Hydrocoll. 18 (2004) 81. ´ C.E. Banks, R.G. Compton, Electroanalysis 17 (2005) 1025. [26] B. S˘ljukic, [27] G. Jürmann, D.J. Schiffrin, K. Tammeveski, Electrochim. Acta 53 (2007) 390. [28] M. Saeed, M. Cheryan, J. Agric. Food Chem. 37 (1989) 1270.