Emulsions Stabilized by Chitosan-Modified Silica ... - ACS Publications

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Emulsions stabilized by chitosan-modified silica nanoparticles: pH control of structure-property relations Lauriane Alison, Ahmet Faik Demirörs, Elena Tervoort, Alexandra Teleki, Jan Vermant, and André R. Studart Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00622 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 2, 2018

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Emulsions stabilized by chitosan-modified silica nanoparticles: pH control of structure-property relations Lauriane Alison†, Ahmet F. Demirörs†, Elena Tervoort†, Alexandra Teleki§, Jan Vermant‡, Andre R. Studart†* ‡ †Complex Materials, Department of Materials and Soft Materials, Department of Materials, ETH Zürich, 8093 Zürich, Switzerland § Nutritional R&D Center Formulation and Application, DSM Nutritional Products Ltd., P.O. Box 2676, 4002 Basel, Switzerland (current address: Drug delivery group, Department of Pharmacy, Uppsala University, 752 36 Uppsala, Sweden)

*E-mail: [email protected]

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Abstract In food-grade emulsions, particles with an appropriate surface modification can be used to replace surfactants and potentially enhance the stability of emulsions. During the life cycle of products based on such emulsions they can be exposed to a broad range of pH conditions and hence it is crucial to understand how pH changes affect stability of emulsions stabilized by particles. Here, we report on a comprehensive study of the stability, microstructure and macroscopic behaviour of pH-controlled oil-in-water emulsions containing silica nanoparticles modified with chitosan, a food-grade polycation. We found that the modified colloidal particles used as stabilizers behave differently depending on the pH, resulting in unique emulsion structures at multiple length scales. Our findings are rationalized in terms of the different emulsion stabilization mechanisms involved, which are determined by the pH-dependent charges and interactions between the colloidal building blocks of the system. At pH 4, the silica particles are partially hydrophobized through chitosan modification, favouring their adsorption at the oil–water interface and the formation of Pickering emulsions. At pH 5.5, the particles become attractive and the emulsion is stabilized by a network of agglomerated particles formed between the droplets. Finally, chitosan aggregates form at pH 9 and these act as the emulsion stabilizers under alkaline conditions. These insights have important implications for the processing and use of particle-stabilized emulsions. On the one hand, changes in pH can lead to undesired macroscopic phase separation or coalescence of oil droplets. On the other hand, the pH effect on emulsion behaviour can be harnessed in industrial processing, either to tune their flow response by altering the pH between processing stages or to produce pH-responsive emulsions that enhance the functionality of the emulsified end products.

Keywords Food-grade emulsions, emulsion stabilization, pH dependence, pickering emulsions, oil-inwater emulsions


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Introduction Particle-stabilized oil-in-water emulsions are used increasingly in the food and pharmaceutical industries, as they offer superior stability and potential safety advantages compared to surfactant-stabilized 1-2 1,3-6 emulsions. A number of food-grade particle emulsifiers have been reported in recent years. Among them are chitosan-modified silica nanoparticles, which have been shown to be good emulsion stabilizers at a pH of 7 5.5. Since the pH value of this emulsion system can be easily varied, either intentionally or through unavoidable changes during processing or use, understanding the effect of pH on emulsion structure and stability is of major practical importance. More specifically, the pH is likely to affect several relevant physicochemical characteristics, including the charge density of the particles, the ionization degree of the chitosan 8 molecules and the adsorption behaviour and associated conformation of chitosan on the particle surface. These variables should in turn change the hydrophobicity, wettability and surface homogeneity of the particles, and therefore ultimately dictate the stability of the emulsion. Unravelling the influence of pH on the microstructure and stability of emulsions containing chitosan-modified silica nanoparticles should therefore enhance the design space available for the formulation of biocompatible complex emulsions for the food, pharmaceutical and cosmetic industries. 9 Chitosan is a pH-responsive weak polyelectrolyte, whose charge density and tendency to adsorb on 10-11 solid surfaces can be tuned through protonation of its amino groups. Protonation of such groups at pHs 12 13 below 6.5, the pKa value of the amino group, makes chitosan soluble in water. In this state, chitosan 14 adsorbs on an oppositely charged surface. In contrast, high pH induces polymer-chain association through hydrophobic interactions, leading to the formation of chitosan aggregates, which do not adsorb on hydrophilic surfaces but can still adsorb at oil-water interfaces to produce stable Pickering emulsions.13,15 Although chitosan on its own has been considered in food applications,12,16 its combination with nanoparticles provides greater opportunities for designing stable systems due to the cooperative interactions between these building blocks. Insights into the possible interactions of chitosan molecules with silica nanoparticles can be inferred from previous work on planar oxide surfaces. In particular, the adsorption behavior of chitosan and its molecular conformation on a model silica surface was shown to depend strongly on the pH.14 The affinity between chitosan molecules and oppositely charged silica surfaces is expected to arise mainly from attractive electrostatic forces. However, the release of counterions and the hydrophobic interactions due to the chitosan backbone also play a role in the adsorption process.10,14 It has been shown that at low pH (4–5), small amounts of chitosan adsorb on silica.14 By increasing the pH of the chitosan solution towards the pKa (6-8), chitosan becomes progressively neutral and the decreased repulsion between adsorbed molecules increases the concentration of the polyelectrolyte on the surface. At high pH (9), chitosan adsorption via electrostatic attraction is not possible as the amino groups are no longer protonated. When combined with colloidal particles, chitosan is expected to affect the stability of emulsions in different ways, since multiple pH-dependent interactions may occur between molecules, particles and droplets. Although chitosan-modified silica particles have already been shown to be adequate emulsion stabilizers,7 the effect of pH on the multiple possible colloidal interactions and the resulting emulsion stability have not been studied. In the present work, we investigate how pH-dependent interactions between chitosan molecules, silica nanoparticles and oil droplets affect the structure-property relations and the stability of complex emulsions made from such building blocks. To this end, we first systematically studied the microstructure of emulsions stabilized by chitosan and silica nanoparticles at multiple length scales and different pHs. Using a wide range of advanced characterization tools, we demonstrate that at least three distinct mechanisms may control the stability of such complex emulsion systems. Zeta potential analysis, diffusing wave spectroscopy, interfacial and bulk rheology, confocal microscopy and cryo-SEM imaging are then utilized to probe the structure and rheological properties of these emulsions at three representative pHs across several length scales. Finally, we show that the stability of our emulsions can be changed in situ by exploiting the pH-


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dependent reversible adsorption of chitosan on colloidal silica particles.

Materials and methods Materials Chitosan oligosaccharide dispersible in water between pH 3 and 7 was supplied by Haide Bei (China). The ability to disperse and solubilize in water over this wide pH range arises from the use of chitosan in its chlorinated form, which contains protonated -NH2 groups. This improves the dispersibility in water over a broader pH range beyond the acidic conditions needed for the solubilization of common chitosans. For this chitosan grade, the degree of deacetylation is higher than 85 %, the molecular weight MW ≤3 kDa and the measured pKa is 6.5. Corn oil (specific gravity of 0.9 g.mL-1, Newtonian fluid with viscosity 4.45 mPa.s-1), silica Ludox TM50 suspensions and D-(+) gluconic acid delta-lactone (≥ 99 %) were purchased from Sigma-Aldrich (Germany) and used without any purification. The Ludox TM50 suspensions consisted of 50 wt% of 22 nm silica -1 nanoparticles in water at a pH of 9 (suspension density of 1.4 g.mL ). Milli-Q water with an electrical resistivity of 18.2 mΩ.cm was used to prepare chitosan solutions and to dilute the as received Ludox particle suspensions. Preparation of chitosan-modified silica suspensions Chitosan solutions were prepared at a concentration of 20 wtT% by adding small amounts of chitosan powder into Milli-Q water under stirring until it was entirely dissolved. wtT% denotes the weight percentage of chitosan relative to the total weight of solution. The pH value of the chitosan solution was about 6. Chitosanmodified suspensions were obtained by diluting the Ludox TM50 suspension with Milli-Q water and then adding the chitosan solution dropwise under magnetic stirring. Silica suspensions were prepared either at a concentration of 8.7 wtsusp% or 1 wtsusp%. wtsusp% denotes the silica concentration calculated with respect to the total weight of the suspensions. Unless specified otherwise, the concentration of chitosan was 5 wt%, as calculated with respect to the silica content (wtSiO2%). 5 wtSiO2% of chitosan corresponds to 0.435 wtT% relative to the total suspension. To improve readability, chitosan concentrations (wtSiO2%) will be denoted simply by wt% in the following. The mixture was stirred at 1000 rpm for 15 minutes. The pH was adjusted to 4 or 5.5 using a 1M HCl solution and to 9 using a 1M NaOH solution, both prepared from Titrisol concentrates of Merck Millipore. To facilitate identification of the investigated suspensions, we use a nomenclature in which “S#” stands for the silica concentration in # wtsusp% and “C#” stands for the chitosan concentration in # wtSiO2%. For example, S1C1 refers to a 1 wtsusp% of silica suspensions modified with 1 wt SiO2% of chitosan. Preparation of Pickering emulsions stabilized by chitosan-modified silica nanoparticles Emulsions were prepared with 10 wt% of corn oil and 90 wt% of aqueous chitosan-modified silica suspensions. This oil-to-water ratio was used throughout this work unless declared otherwise. The constituents were mixed by hand before the pre-emulsification step performed with an Ultra-Turrax rotor–stator mixer (disperser T25 digital, dispersing tool S 25 N – 18 G, IKA, Germany) using a dispersing head operating at 10,200 rpm for 1 minute. Emulsification was realized with a high-pressure homogenizer (Nano DeBEE Laboratory Homogenizer from BEE international, US) using a diamond nozzle (D5, orifice 130 µm). The emulsion was processed four times: three times at 1,380 bar and then once at 2,760 bar. To study the stability of the emulsions over time, they were stored in a fridge at 4°C for several months. Zeta-potential measurements The zeta potentials of the chitosan-modified silica suspensions were measured using the electroacoustic colloidal-vibration-current technique, using a DT300 device (Dispersion Technology, US) at 25°C. The measurements were either performed at a fixed pH or obtained by titrations when the zeta potential was recorded as a function of pH. The zeta potential values for chitosan concentrations from 0 to 30 wt% (0 to 2.61 7 wtT% relative to the total suspension) and for pHs 4, 5.5 and 9 were extracted from our previous work. The titrations during the zeta-potential measurements were performed from high to low pH using 1-M HCl solution. Adsorption experiments by total organic carbon (TOC) analysis


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Adsorption isotherm measurements were conducted using a total organic carbon analyser (TOC VCSN, Shimadzu, Japan), which provided a quantitative measure of the non-adsorbed fraction of chitosan in the modified suspensions (Laboratory of Génie Chimique, Toulouse). 8.7 wtsusp% silica suspensions coated with different concentrations of chitosan from 0 to 30 wt% (0 to 2.61 wtT% relative to the total suspension) were prepared at pHs 4, 5.5 and 9 (see Figure S1). The suspensions were centrifuged at 20,000 rpm for 2 h at 20 °C and the supernatant aliquots were analysed to estimate chitosan adsorption. Diffusing wave spectroscopy measurements Due to the high particle concentrations, the turbidity, and the polydispersity of the samples, diffusing wave spectroscopy (DWS) was used to characterize the suspensions. DWS measurements were performed at 25 °C in a DWS apparatus (LS instruments, Switzerland). The laser used as a light source had a wavelength of 683 nm and the backscattered light was collected on a photomultiplier. The samples were introduced in glass cuvettes with an optical path length of 1 cm. The intensity fluctuations were measured by a digital correlator and the electric-field autocorrelation function g2(τ)-1 was determined. Macroscopic stability and droplet size determination The droplet size of the non-diluted emulsions, the dynamics of the phase separation (migration rate) and the backscattering profiles of the emulsions were obtained using an optical scanning analyser at 25 °C (Turbiscan LAB, Formulaction, France). This device measures the stability of emulsions using a moving detection head composed of a pulsed near-infrared light source (λ = 880 nm) with a detector for the backscattered light (at 45°). The detection head scans the entire height of a sample by moving vertically along the analysis cell and acquiring backscattering data every 40 µm. These scans were performed at various pre-programmed times and the obtained profiles against ageing time were overlaid on one graph in order to show the nascent destabilisation processes. Over time, changes in the backscattering level due to sample instability are recorded. These changes indicate droplet migration in the sample driven by coalescence, flocculation, sedimentation or creaming, as well as droplet size change. The graphs are displayed in reference mode, whereby the first profile is subtracted from all other profiles to highlight variations. With the same optical analyser, it is also possible to measure the droplet size in the concentrated regime. Taking the volume fraction of the dispersed phase (10 wt%) and the refractive indices of the constituent phases, the mean size of the oil droplets could be calculated for elapsed time t = 0 using the backscattering signal of the whole homogeneous sample. Refractive indices of 1.33, 1.47 and 1.5 were assumed for the continuous aqueous phase, the dispersed oil phase and for the modified particles, respectively. Confocal microscopy analysis Emulsion droplets were observed with a Leica SP2 laser scanning confocal microscope (Leica Microsystems, Germany). The oil phase was labelled with Nile Red (abcr, Germany) or Bodipy 493/503 (Invitrogen, US). The stained emulsions were placed inside a glass capillary (0.1 x 1 mm, Vitrocom, US) and sealed with epoxy adhesive on a microscope slide (76 x 26 mm). Samples were observed with a 63x oil-immersion objective (oil refractive index of 1.515) at an excitation wavelength of 561 nm. Bulk and interfacial rheological measurements The bulk rheological properties of the suspensions and emulsions were measured using a rheometer (Physica MCR 502, Anton Paar, Austria) equipped with a double gap Couette geometry (DG 26.7) using a Peltier element to keep temperature at 25.0 °C. The strain amplitude sweeps combined with in situ pH change were carried out adding 1.5 wt% of δ-gluconolactone (GDL). To measure the properties of the interfacial adsorption layer, interfacial shear rheology measurements were performed at 25 °C with a rheometer (Physica MCR 501, Anton Paar, Austria) using a biconal geometry (BI-C68-5), which was placed at the oil–water interface. The Boussinesq number Bo was calculated to be greater than 1, and interfacial flow can hence be treated as being decoupled from the bulk phase flow. The structure of the adsorption layer formed between the lower aqueous phase (chitosanmodified silica suspensions) and the oil phase (corn oil) was probed with a time sweep. The measurement with in situ pH change was performed at 25°C with a rheometer (HR 3 Discovery Hybrid Rheometer, TA instruments,


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United States) equipped with a subphase exchange cell combined with a titanium double-wall ring (DWR with Rinner = 3.45 cm and Router = 3.55 cm). The double-wall ring was placed at the oil-water interface in a cup (top subphase: Rinner = 3 cm and Router = 4.05 cm; bottom subphase: Rinner = 3.1 cm and Router = 3.95 cm) as explained by Schroyen et al.17 Dimensions of the trough and of the ring are the same as for the original DWR setup 18 developed by Vandebril et al. A total of 3.15 mL HCl solution at 0.02 M was flushed in through the two -1 syringes attached to cell inlets by PFA tubings at 0.5 mL.min and the same volume was flushed out simultaneously, thanks to two syringes attached to cell outlets by PFA tubings to keep the volume of the subphase and the height of the interface constant. Cryo-scanning electron microscopy To determine the arrangement of particles in the bulk and at the droplet surface, the freeze-fracture technique coupled with cryo-SEM was used. In short, 4 µL of the desired Pickering emulsions were sandwiched between two pre-cleaned and hydrophilized 6-mm aluminium planchettes. The samples were frozen in a highpressure freezer HPM 100 (Bal-Tec/Leica, Vienna, Austria) and stored in liquid nitrogen. The high pressure prevented water crystallization and fixed the position of the emulsion components as they were in the liquid state. The frozen samples were subsequently fractured under high vacuum conditions in a pre-cooled freezefracture device at -130 °C (Bal-Tec/Leica BAF060 device). Samples were freeze-dried for 5 or 10 minutes at -7 100 °C and 6x10 mbar and then coated with 2.5 nm tungsten at a deposition angle of 45° followed by additional 2.5 nm at 90°. In this way, charging of the samples during imaging was supressed. Freeze-fractured metal-coated samples were then transferred to a pre-cooled SEM (-120 °C) (Zeiss Gemini 1530, Oberkochen, Germany) for imaging with inlens and SE2-detectors.

Results and discussion The structure of emulsions stabilized by chitosan-modified silica particles is controlled by the distinctive interactions between the building blocks at different pHs, affecting the structural features at different length scales. At the molecular scale, the charge of the chitosan molecules depends on pH; decreasing pH from 9 to 4 gradually increases the positive charges due to the protonation of amine functional groups. We chose to work at three pH values: pH 4, where chitosan is heavily charged; pH 5.5, close to its pKa value of 6.5, where chitosan is slightly charged; and pH 9, where chitosan is uncharged (Figure 1A). The silica nanoparticles exhibit negative electrical charges over the pH range 4–9, as a result of deprotonation of surface hydroxyl groups. Because of the attractive electrostatic interactions arising from these opposite charges, adsorption of chitosan on silica nanoparticles is possible at pHs 4 and 5.5 (Figure 1A,B). The amount of adsorbed chitosan gives rise to different degrees of hydrophobization for the modified silica particles. This degree of hydrophobization regulates the inter-particle interactions and the adsorption of the nanoparticles at the oil–water interface (Figure 1C). The adsorption of the particles at the interface and their interactions in the continuous phase dictate the emulsion microstructure. The resulting microstructure may induce gelation of the colloidal system and affect the density of the droplets (Figure 1D). The possible gelation of the system and the density of the particle-coated droplets determine the macroscopic stability, in particular the response of the emulsion to gravity. As a consequence, the pH also influences the stability and emulsion behaviour at the macroscopic scale (Figure 1E). Next, we investigate the effect of pH at progressively larger length scales to gain new insights into the mechanisms underlying the macroscopic colloidal behaviour of emulsions containing chitosan and particles as stabilizers.


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Figure 1: Schematic illustration of the formation of emulsions prepared with the same composition at different pHs. (A) pHdependent behaviour of chitosan, a weak polyelectrolyte with a pKa value of 6.5. At pH 4, chitosan is heavily charged; around its pKa value at pH 5.5 chitosan is slightly positively charged; and at pH 9 chitosan is poorly charged and insoluble in water. (B) Electrostatic modification of negatively charged silica by positively charged chitosan. (C) Chitosan is used as a modifier to partially hydrophobize the silica particles and influence their wettability at the oil–water interface. (D) After emulsification, the obtained droplets are stabilized through distinct mechanisms as a function of pH leading to different microstructures. (E) Different macroscopic behaviors of the emulsions obtained at pH 4, 5.5 and 9.

Chitosan adsorption on silica nanoparticles At the length scale of the individual particles, the amount of adsorbed chitosan directly influences the hydrophobicity of the silica nanoparticles. To directly quantify the amount of chitosan adsorbed on the silica particles, we performed a TOC analysis of the supernatant of chitosan-modified silica suspensions at different pHs after ultracentrifugation (Figure 2A). The analysis assumes that the precipitate contains the chitosanmodified silica nanoparticles and the supernatant is left with the non-adsorbed chitosan (free chitosan). In fact, as the total chitosan concentration in the colloidal suspensions is high, chitosan is expected to adsorb on 10 silica until a saturated layer is formed, while the excess remains in solution as free chitosan. From the initial chitosan concentration added to the suspension and the carbon content measured in the supernatant, the amount of carbon in the precipitate and therefore the fraction of adsorbed chitosan can be determined. At pH 9, the amount of precipitated carbon is the highest, but this does not correspond to the highest adsorption as the precipitate contains mostly insoluble chitosan. We corrected the amount of chitosan adsorbed on silica at this pH by subtracting the amount of chitosan that precipitates in a pure chitosan suspension containing no silica (see Figure S2). Overall, only a small amount of chitosan adsorbs at pH 9. At pH 4, a larger amount of chitosan adsorbs on silica, about 35% of the initial chitosan added in the suspension. Finally, the amount of chitosan adsorbed on silica is maximum at pH 5.5, which is slightly below the pKa of chitosan (6.5).19 Since the protonation degree of the chitosan molecules changes between pHs 4 and 9, our results suggest that the surface charges controls the chitosan adsorption process. We used zeta-potential measurements to study the surface charges of the bare and modified particles (Figure 2B). As shown previously, higher chitosan concentrations increase the zeta potential of the particles and this rise in potential depends on the pH.7 Irrespective of the initial chitosan concentration, the zeta potential value is maximal at pH 5.5, which corresponds to the maximum adsorption measured in the TOC analysis. Comparing the zeta potential of the particles in the absence or presence of chitosan allows us to indirectly infer the amount of electrostatically adsorbed molecules and thus gain further insights into the driving force of the adsorption process. Since the zeta potential varies with pH, we consider the difference between the zeta potentials of the modified and non-modified silica at pH 4, 5.5 and 9 as a function of the initial chitosan concentration (Figure 2C). Such zeta potential differences depend on the charges of the bare particles and the charges of free chitosan. At pH 4 and 5.5, chitosan is fully and partially positively charged, respectively, and thus soluble in water (Figure 1A). In contrast, at pH 9 chitosan is insoluble and not charged; its possible adsorption on the


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silica particles will therefore not provide extra positive charges to the silica surface and will not change the zeta potential of the particles. The dependence of the charge of the chitosan molecules on the pH explains its interactions with the silica nanoparticles and thus the measured zeta potential difference. At pH 5.5, the zetapotential difference between the modified and non-modified silica is the highest at all chitosan concentrations. Because chitosan molecules and silica particles are oppositely charged at this pH, the significant zeta-potential difference and the high adsorption measured at this pH are likely to result from the 7 strong electrostatic attraction between molecules and particles. At pH 4, chitosan is strongly positively charged and silica is less charged, therefore less chitosan is adsorbed. In addition, electrostatic repulsion between strongly charged chitosan molecules can prevent adsorption of chitosan onto the silica surface before monolayer coverage is reached. At pH 9, chitosan is uncharged, therefore the zeta potential is expected to be close to that of the non-modified silica. However, due to the protocol used for suspension preparation, some adsorption occurs on the silica surface before we change the pH of the suspension to 9. The initially adsorbed molecules can remain on the surface of the particles after the pH change, preventing some of the SiOH groups to fully deprotonate and to reach the zeta potential value of bare silica particles. These protocol-dependent effect might also explain the similar zeta potential differences obtained for suspensions prepared at pHs of 4 and 9 (Figure 2C), despite the higher amount of chitosan adsorbed at pH 4 (Figure 2A). Overall, our TOC analysis combined with surface charge measurements indicate that the maximum amount of adsorbed chitosan is found at pH 5.5, due to the favourable electrostatic attractions between oppositely charged species. This is in reasonable agreement with previous work by Tiraferri et al.,14 who showed that chitosan adsorption on silica substrate is optimal around its pKa value and results in the formation of thick layers that are viscoelastic and dissipative.14 In our study, we use a water-soluble chitosan with a different acetylation degree and molecular weight, which explains the fact that the optimal adsorption of chitosan occurs at a slightly lower pH of 5.5 (Figure 2) compared to Tiraferri et al.14 At this pH, there is minimal intermolecular electrostatic repulsion between the chitosan molecules, favouring the accumulation of thick layers of chitosan on the silica surface, as schematically shown in the inset of Figure 2A. In addition, the surface charge density of the silica is sufficiently high, leading to a high concentration of counterions in the diffuse layer that screen the electrostatic potential between the already adsorbed chitosan molecules and result in an optimal chitosan adsorption.14 At pH 4, it was also previously shown that less chitosan is adsorbed compared to a pH close to the pKa value, leading to the formation of rigid and thin monolayers on silica particles (Figure 2A).14 At pH 9, we assume that the fraction of chitosan adsorbed on silica will arrange into flocs (as shown in the inset of Figure 2A) due to its insolubility at this pH (see Figure S6A). Because it directly affects the amount of adsorbed chitosan, the pH should also influence the hydrophobicity of the silica particles and thus the colloidal stability and rheology of suspensions prepared with these two types of building blocks.

Figure 2: Characterization of chitosan adsorption on silica nanoparticles in suspensions containing 8.7 wtsusp% modified silica at different pH values. (A) Fraction of chitosan adsorbed on silica particles in suspensions with 5 wt% of chitosan (S8.7C5) at pH 4, 5.5 and 9 determined by total organic carbon (TOC) analysis. The percentages correspond to the amount of adsorbed chitosan relative to the initial chitosan concentrations. The inset shows schematics of the expected configuration of the chitosan layer on a silica nanoparticle. (B) Zeta-potential measurements of silica particles modified with 5 wt% of chitosan as compared to bare silica. (C) Quantification of the particle modification through the zeta potential


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difference between the chitosan-modified silica particles and the non-modified silica particles at pH 4, pH 5.5 and pH 9 at different chitosan concentrations.

Colloidal behaviour and rheology We studied the colloidal stability of suspensions containing chitosan and silica particles at different pHs using diffusing wave spectroscopy, electron microscopy and rheological measurements. Cryo-SEM imaging (Figure 3A) shows that silica nanoparticles coated with chitosan agglomerate extensively at pH 5.5. The zeta potential of modified particles at this pH is not sufficiently high to prevent agglomeration of the particles against van der Waals and hydrophobic attractive forces (Figure 3B).7 The presence of agglomerates is confirmed by the high turbidity of the modified suspensions prepared at pHs 5.2, 6.3 and 7.3 (Figure 3B), which is directly reflected in the diffusing wave spectroscopy (DWS) measurements (Figure 3C). For pHs around the pKa of chitosan, there is no decay of the auto-correlation function, which means that diffusion of the particles is restricted (long lag time). This provides further evidence of the presence of agglomerates that move slower than individual particles in the modified suspensions or may even form a percolating network (Figure 3 C). In contrast, the cryo-SEM images obtained at pHs 4 and 9 (Figure 3A) indicate no visible agglomeration, since the nanoparticles in these cases are observed mostly as individual units (Figure 2). As no agglomerates are formed at pH 4 and 9, less turbid suspensions are obtained, as illustrated in Figure 3B. The lack of agglomeration at pH 4 is rather surprising, since the zeta potential of the modified particles at this condition is close to zero (Figure 2B). This reflects the fact that the positively adsorbed chitosan molecules neutralize and partly reverse the initially negative charges on the silica surface at this pH. Such a low zeta 14 potential and the expected flat confirmation of the chitosan on the oxide surface would be expected to favour particle agglomeration. However, less chitosan molecules are adsorbed on silica compared to the situation for pH 5.5 (Figure 2A), which reduces potential attractive forces arising from van der Waals and hydrophobic interactions between chitosan molecules adsorbed on different particle surfaces. Despite the low amount adsorbed and low overall zeta potential, the high charges of chitosan molecules at pH 4 are also expected to result in local electrostatic repulsion between surface-anchored chitosan molecules. As opposed to pH 4, the absence of agglomerates at pH 9 can be easily explained by the high zeta potential of the silica particles. At this pH, chitosan is uncharged and only a low amount of chitosan molecules adsorbs on the silica surface due to the suspension preparation protocol, as explained previously (Figure 2). Thus, the high absolute zeta potential values at this pH result mostly from the bare silica particles. The absence of agglomerates in the cryo-SEM is consistent with the macroscopic stability of the suspensions. The slight turbidity of the suspensions prepared around pH 4 and 9 is confirmed by the short decay time of the auto-correlation function of the DWS measurements (Figure 3C). The short lag times mean that the particles are not in an arrested state and do not agglomerate. Nonetheless, particles at pH 9 move slower than those at pH 4. This is presumably caused by the presence of insoluble chitosan in the suspensions, which slows down the diffusion of the silica particles. Insoluble chitosan was indeed observed by cryo-SEM images of an aqueous solution prepared at this pH (see Figure S3). The agglomeration process observed at pH close to 5.5 affects the rheological properties of the resulting modified suspension. The rheological properties of suspensions at different pHs was first monitored over a long time period of 20 hours (see Figure S4). With time, the modified suspension prepared at pH 5.5 forms a gel, which eventually becomes quite strong. The fact that the particles are not agglomerated at pH 4 and 9 explains the non-gelling behaviour of these modified suspensions. Oscillatory rheological measurements after 20 hours show that all the modified suspensions show viscoelastic behaviour (Figure 3D). Agglomeration at pH 5.5 increases the storage modulus of the suspension by orders of magnitude in comparison to the suspensions prepared at pHs 4 and 9. The formation of a network of agglomerated modified particles, possibly linked through interactions between chitosan multilayers and free molecules, is responsible for the strong elastic gel-like behaviour at pH 5.5.7,20 Breakdown of such gel at increasing strain amplitudes is indicated by the slight increase in loss modulus as the storage modulus starts to decay (Figure 3D).20 When suspensions


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prepared at pH 4 and pH 9 are compared, we find higher storage and loss moduli for the suspension displaying pH 4. This suggests that the low zeta potential of particles modified at this pH (Figure 2B) might lead to weak attractive interactions between particles, despite the absence of large agglomerates (Figure 3A,C).

Figure 3: Colloidal behaviour of 8.7 wtsusp% silica suspensions modified with 5 wt% of chitosan as a function of pH. (A) CryoSEM images of chitosan-modified silica suspensions at pH 4, 5.5 and 9. (B) Photograph of chitosan-modified suspensions prepared at different pHs measured after their preparation; zeta potential values are shown in the labels. Note that the turbidity of the suspensions is reflected in the DWS analysis shown in panel C. (C) Diffusing wave spectroscopy and (D) oscillatory amplitude sweeps measured after a time sweep of 20h for chitosan-modified suspensions prepared at different -1 pH values. The strain amplitude sweep was performed at a constant angular frequency of ω = 1 rad.s .

Microstructure of the emulsions The colloidal behaviour of the modified particles in aqueous medium significantly influences the microstructure of emulsions prepared by incorporating an oil phase into the suspensions of chitosan-modified particles. The microstructure of the emulsions reflects the different stabilization mechanisms that may occur depending on initial pH of the suspensions of modified particles. Electron and optical microscopy images of compositions prepared at different pHs reveal the effect of particle hydrophobicity and agglomeration state on the final stabilization mechanism and emulsion microstructure. Next, we discuss the insights obtained by imaging emulsions obtained from suspensions prepared at distinct pHs. First, cryo-SEM images show that partially hydrophobized particles reside at the oil–water interface and thus form an armour around droplets in emulsions produced at pH 4. Since the overall particle concentration is relatively high, the excess of modified particles stays in bulk (Figure 4A,I). The dynamics of such emulsion was studied by watching the motion of the droplets (video V2 in SI) using confocal microscopy. Droplets were found to form aggregates with an average size of 2.2 µm, which move constantly driven by thermal energy (Figure 4A,II). Neither electrostatic nor steric repulsive interactions between the nanoparticles are high enough to keep the individual droplets separate. The formation of droplet aggregates is most likely caused by particle bridging effects21-22 and by the high shear forces imposed during the high-energy emulsification process. High shear forces promote hydrophobic interactions between particles23 and particle


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adsorption at fluid interfaces. The resulting aggregated droplets are surrounded by a layer of nanoparticles at the oil–water interface, indicating that Pickering stabilization is the dominant mechanism responsible for the microstructure of emulsions prepared at pH 4. Second, the chitosan-coated silica particles form relatively large agglomerates at pH 5.5, which prevents the formation of a close-packed armour of particles around individual droplets (Figure 2A). This suggests that the high particle hydrophobicity achieved at this pH makes them agglomerate in the aqueous medium before they can diffuse and adsorb at the oil–water interface. The cryo-SEM images shown in Figure 4B, I indicate that most of the particles are in the bulk and their colloidal behaviour is dominated by attractive interactions. The particle agglomeration observed throughout the bulk continuous phase shows that the emulsification process does not completely destroy the aggregates formed in the initial suspensions (Figure 3 A). In fact, we expect these agglomerates to partially break down during high-energy emulsification and 24 eventually re-form in the continuous phase after shearing ceases. The formation of a percolating network of 24 agglomerates around the droplets result in a gel-like emulsion. The gelation of the emulsion at pH 5.5 is also evident from the immobility of the droplets (video V1 in SI). These droplets have an average size of 1.8 µm and are well distributed throughout the emulsion (Figure 4B,II). Third, emulsions prepared at pH 9 feature particles that remain mostly in the continuous phase in a non-agglomerated state (Figure 4C,I and Figure 3A). Moreover, 2-µm-sized aggregates of droplets are produced at this pH, which eventually form a network of slow wiggling droplets (video V3 in SI). Although chitosan is not detected at the length scale of our microscopy images, we expect the surface of the droplets to be covered with insoluble hydrophobic chitosan molecules. The formation of aggregated percolating droplets (Figure 4C,II) most likely results from hydrophobic interactions between insoluble chitosan molecules adsorbed at the liquid interface and left in the continuous phase.

Figure 4: (I) Cryo-SEM images of emulsions prepared from 8.7 wtsusp% silica suspensions modified with 5 wt% of chitosan at (A) pH 4, (B) pH 5.5 and (C) pH 9. (II) Confocal images of emulsions taken three days after preparation. The oil phase was


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labelled with fluorescent Nile red and the average droplet size (ADS) measured with an optical scanning analyser using multiple light scattering at 4°C.

Interfacial and bulk rheology of emulsions Microscopy analyses were complemented by interfacial and bulk rheology measurements to gain more insights into the stabilization mechanisms arising from the different emulsion microstructures. Adsorption of the particles at the oil–water interface was examined by interfacial rheology, whereas the particle behaviour in the continuous phase was assessed by bulk rheological measurements. At pH 4, the interfacial storage (Gi’) and loss (Gi”) moduli of the interface formed between a suspension of chitosan-modified silica particles and corn oil increase by several orders of magnitude over a period of several hours (Figure 5A,I). This indicates the formation of a strong layer with significant linear viscoelastic moduli at the oil-water interface. These interfacial properties develop due to the spontaneous adsorption of the particles at the oil–water interface, confirming that emulsions at pH 4 are stabilized by a Pickering mechanism. The amount of chitosan covering the silica particles at this pH leads to a hydrophobization level that is sufficiently high to enable interfacial adsorption without extensive agglomeration of the silica particles throughout the aqueous medium. Control experiments performed with pure water at pH 4, pure chitosan solution or bare silica suspension instead of the suspension of chitosanmodified silica did not indicate any interfacial adsorption over the same time period of 24 h (data not shown). This highlights the fact that chitosan modification is important to enable particle adsorption at the oil-water interface. Interestingly, the strong viscoelastic moduli of the particle-laden interface contrasts with the weak viscoelastic behaviour of the bulk emulsion (Figure 5A,II). We expect this weak bulk viscoelasticity to arise from the network of attractive droplet clusters formed throughout the continuous phase (Figure 4A,II). The results obtained from interfacial rheology and the cryo-SEM images confirm that droplets are particle-stabilized at pH 4, as schematically depicted in Figure 5A,III. At pH 5.5, no interfacial viscoelasticity is detected (data are below the measurement sensitivity), indicating that no particles are adsorbed at the oil–water interface after one day (Figure 5B,I). Instead, most of the modified particles agglomerate extensively in between droplets at this pH, leading to an emulsion with strong elastic behaviour (Figure 5B,II). The strong bulk elasticity is a hallmark of emulsions stabilized at pH 5.5. Similar to the suspensions (Figure 3D), cooperative interactions between chitosan and silica particles are responsible for the strong gel-like properties of emulsions at this pH. If these constituents are used individually, the emulsions do not gel. The viscoelastic properties of the 3D network of agglomerated nanoparticles formed throughout the continuous phase keep the droplets isolated, thus ensuring efficient emulsion stabilization. Cryo-SEM images of aqueous solutions show that the chitosan molecules form long threads at pH 5.5 (see Figure S3B), which are likely to connect droplets7 and nanoparticles throughout the emulsion (Figure 3A). Such an aggregated fibrillar structure suggests that chitosan interacts through attractive hydrophobic interactions close to its pKa. Based on these results, we propose that the stabilization of emulsions at pH 5.5 relies on the formation of a strong network of agglomerated particles, as sketched in Figure 5B,III. This interpretation is in agreement with the cryo-SEM images presented in our previous work.7 Although interfacial rheology measurements do not suggest the presence of a strong adsorbed layer at pH 5.5 (Figure 5B,I), the cryo-SEM pictures indicate that a few particles are anchored at the oil–water interface, without forming an interfacial network (Figure 4B,I). This is probably due to the application of high shear rates, 24 which increase the frequency of collisions of particles with the interface, leading to irreversible adsorption during the emulsification process. At pH 9, insoluble chitosan molecules alone adsorb at the oil–water interface to form a viscoelastic layer, as indicated by the interfacial rheology measurements (Figure 5C,I). Comparison of interfacial rheology data measured for the chitosan-silica suspension and a chitosan aqueous solution shows that the presence of silica particles delays the adsorption of chitosan to the oil-water interface (Figure 5C,I). This probably results


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from the longer and more tortuous diffusion path that the chitosan molecules need to take towards the interface in the presence of silica particles. Since silica nanoparticles are only slightly modified with chitosan at pH 9 (Figure 2A), they remain hydrophilic and do not adsorb at the oil–water interface under the conditions of the interfacial rheology experiments (no imposed shear). Hydrophobic interactions between the insoluble chitosan molecules adsorbed at the interface and free in the suspension leads to emulsions with mild bulk elastic behaviour, which is much weaker compared to the emulsion at pH 5.5 but stronger compared to the system at pH 4 (Figure 5C,II). Our results suggest that chitosan suspended in an aqueous solution at pH 9 is sufficiently surface active to stabilize droplets even in the absence of silica nanoparticles. This was confirmed in control experiments (see Figure S5) and is in agreement with previous work.13,15 In another series of control experiments, we found that the fatty acids present in corn oil can also act as emulsifiers in basic medium (see Figure S8). Moreover, the observation by cryo-SEM of a few silica nanoparticles at the oil–water interface at pH 9 (Figure 4C,I) indicates that particles also play a role in the stabilization of our emulsions. We attribute the presence of few nanoparticles at the interface to the effect of high shear rates that increase the collision frequency between particles and droplets during high-pressure emulsification.24 The high-pressure homogenization process also forces chitosan to adsorb on the silica particles, as confirmed by the change in turbidity of the chitosan-modified silica suspension and the decrease of zeta potential at pH 9 (see Figure S6B, C). As the amount of adsorbed chitosan on silica nanoparticles rises during the high-energy process, particle agglomerates are expected to form after this intense emulsification procedure. This is in agreement with the gelation of the chitosan-modified silica suspensions at pH 9 when passed through the high-pressure homogenizer and let rest for one month (see Figure S6D). The adsorption of chitosan on the silica particles makes them sufficiently hydrophobic to partially adsorb at the oil–water interface. Thus, we conclude that a combination of stabilizers, including the insoluble chitosan molecules, the chitosan-modified silica particles and the fatty acids present in the corn oil, is responsible for the stabilization of the emulsion prepared at pH 9, as illustrated in Figure 5C,III. The distinct arrangement of modified particles in the bulk and at the oil–water interface at the different studied pHs also influence the macroscopic behaviour of the emulsions. This arises from the fact that interfacially adsorbed particles change the density of individual droplets, whereas the agglomeration of particles in the bulk lead to a load-bearing elastic network around the droplets. These effects determine the susceptibility of the emulsion to gravity-induced phase separation at the macroscopic scale, as discussed next.


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Figure 5: Interfacial and bulk emulsion rheology performed at pH (A) 4, (B) 5.5 and (C) 9 to shed light on the emulsion stabilization mechanisms. Unless otherwise indicated, the aqueous continuous phase contains 8.7 wtsusp% silica particles modified with 5 wt% of chitosan. (I) Linear viscoelastic time sweep of the interfacial layer formed between corn oil and an underlying aqueous suspension performed at a constant strain amplitude of γ = 0.3 % and angular frequency of ω = 1 rad.s 1 , (II) Strain amplitude sweep (bulk) of the respective emulsions after three days performed at a constant angular frequency -1 of ω = 1 rad.s and (III) schematics of the proposed arrangement of chitosan-modified silica particles around the oil droplets for emulsions stabilized at different pHs. G’ and G” correspond to the storage and loss moduli, respectively.

Emulsion macroscopic behaviour The macroscopic behaviour of the emulsions at the studied pH values depends on the density of the droplets and on the emulsion microstructure. Macroscopically, the emulsions phase separate at pH 4 and 9 while staying gelled at pH 5.5. The phase separation of the emulsions leads to a phase containing a concentrated emulsion (E) and a clarified phase with water (W) and particles (P). The formation of these phases was followed by illuminating the samples with light and measuring the variation of the backscattered signal versus the height of the emulsion over 2.75 days with an optical analyser (Turbiscan LAB). This enables the identification of destabilization phenomena occurring in the samples as a function of time. The thickness of


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the clarified layer (W + P) was then extracted and normalized over the entire sample height (Figure 6A). For each of the three emulsions, the kinetics of the clarification — marked by double arrows on the photographs displayed in Figure 6B — is represented by the evolution of the thickness of the clarified layer over time. We quantified the clarification rates at the different pHs by measuring the slopes of each curve segment from 16 hours to two days. The obtained rates can be physically interpreted as the migration rate of droplets driven by gravity and is directly linked to the shelf stability of the emulsion. The slower is the migration, the more stable is the emulsion. The data clearly shows that the pH of the suspension used to prepare the emulsion significantly influences its macroscopic stability. The most stable emulsion is the one prepared at pH 5.5, as it has the slowest droplet migration rate. This migration rate increases by a factor of more than two for the emulsions at pH 4 and by a factor of twelve for pH 9, indicating that the emulsion prepared at pH 4 is more stable than the one prepared at pH 9. At pH 5.5, only a thin clarified layer is observed (see Figure S9A). The emulsion remains macroscopically homogeneous with droplets well distributed throughout the volume and surrounded by an elastic network of particles and chitosan (Figure 5B, III). This keeps droplets in a ‘frozen’ state, in which no migration takes place even after two months (Figure 6B). At pH 4, the thickness of the clarified layer increases with time as the droplets sediment to the bottom of the sample container (see Figure S9B). The sedimentation of the droplets is due to the higher density of particle-coated droplets as compared to water. A photograph taken two months after the emulsion preparation (Figure 6B) highlights that a thin particle layer is visible between the lower emulsion phase and the upper water phase. This corresponds to the excess of modified particles that sediment and gel in the clarified phase over time (Figure 5A, III). At pH 9, the formation of a large clarified layer at a high droplet migration rate demonstrates that the emulsion rapidly creams. Creaming is driven by the lower density of chitosan-stabilized droplets as compared to water (see Figure S9C and Figure 5C,III). With aging, the clarified phase gels while the concentrated emulsion phase separates into two parts: a water-rich top and a particle-rich bottom phase (Figure 6B). The large thickness of the particle-rich layer is consistent with the observation that the silica nanoparticles remain predominantly in the continuous phase of emulsions prepared at this pH (Figure 4C). These phase separation experiments show that the pH plays a key role on the long-term macroscopic behavior of the emulsions. By combining the influence of pH and chitosan concentrations on the microstructure and macroscopic behaviour of the emulsions, we can build stability maps that show the conditions required to achieve distinct stabilization mechanisms using chitosan and silica nanoparticles as stabilizers, as shown below.

Figure 6: (A) Dynamics of phase separation in fresh emulsions prepared at pH 4, 5.5 and 9 examined over 2.75 days at 25 °C. Phase separation is quantified by the relative thickness of the clarified layer. The slopes of every curve from 16 hours to two days are used to evaluate the speed of the phase separation process. (B) Photographs of the emulsions prepared at the different pHs taken after 2 months. The schematics show the expected microscopic behaviour of the droplets and the modified particles at each pH. W stands for water, P for particles and E for emulsion.


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Stability maps In the experiments described so far, the composition of the modified suspensions was fixed to 8.7 wtsusp% silica and 5 wt% chitosan, respectively. When the chitosan concentration at the three studied pHs is 7 decreased, we observe different emulsion microstructures and stability, as reported in our earlier work. To identify the stabilization mechanisms at different pHs and chitosan concentrations, we combine all these experimental results in an emulsion stability map. In the first representation of this stability map, confocal images of the different emulsions are used to highlight the distinct emulsion microstructures as a function of chitosan concentration and pH of the modified suspensions (Figure 7A). In a second representation, results obtained by image correlation between two timelapse images are displayed to quantify the droplet kinetics at different sets of pHs and chitosan concentrations (Figure 7B). The droplet kinetics is quantified by the Pearson correlation coefficient, ρ. This parameter takes values between 1 and -1, with ρ = 1, 0 and -1 corresponding to fully correlated, uncorrelated and anti25 correlated images, respectively. Thus, a ρ value close to 1 indicates that the two images are well correlated and that the droplets have not moved significantly with the time window probed; instead, a gelled emulsion has formed. A low ρ value indicates that the two images are significantly different and that the droplets have moved through Brownian motion. As discussed earlier, three distinct pH-dependent mechanisms are at work for emulsions containing 5 wt% of chitosan. Pickering emulsions formed at pH 4 contain particle-stabilized droplets that are relatively mobile (ρ = 0.71, Figure 7B). By contrast, droplets stabilized at pH 5.5 and 9 are trapped within a gel-like continuous phase comprising a network of modified particles and chitosan molecules, respectively (high ρ values). Videos V1 to V3 in the SI show the real-time dynamics of the droplets, supporting this interpretation. Additional control experiments were performed to verify the efficacy of chitosan or silica alone in stabilizing the emulsions at the pH values studied (see Figure S7). With the exception of the emulsion prepared with pure chitosan at pH 9 (see Figure S5),13,15 none of the control emulsions exhibit stability as high as the one observed for the emulsions prepared with 8.7 wtsusp% silica and 5 wt% chitosan. Analysing the effect of chitosan concentration at one fixed pH provides further guidelines for the design and formulation of the investigated emulsion system. The minimum chitosan concentration required for emulsion stabilization depends on the pH. At pH 4, the stability map shown in Figure 7A indicates that aggregated droplets are obtained for the entire range of chitosan concentrations studied, that is between 0.2 and 5 wt% of chitosan. These droplet aggregates likely result from hydrophobic interactions between modified particles and from interdroplet particle bridges formed during the high-energy emulsification process. Despite the formation of droplet aggregates, the absence of a percolating gel throughout the continuous phase makes the droplets relatively mobile with comparatively low ρ values in the range 0.66 – 0.71 (Figure 7B). Interfacial rheology confirms that all compositions studied at pH 4 are Pickering emulsions, as a particle-laden film with strong viscoelastic response is formed at the oil–water interface (see Figure S10). As opposed to pH 4, the stabilization mechanism at pH 5.5 changes as the chitosan concentration is varied between 0.2 and 5 wt% (Figure 7A).7 For 0.2 wt% of chitosan, the droplets are unstable because the particles are not sufficiently hydrophobic to adsorb at the oil–water interface. This insufficient hydrophobicity might result from the fact that these molecules adsorb in a random-coil configuration on the oxide surface,14 leaving large uncovered areas of hydrophilic character on the particle surface. By contrast, fully charged chitosan molecules at pH 4 adsorb in a flat stretched conformation, ensuring sufficient hydrophobization of the particles modified with only 0.2 wt% of chitosan (see Figure S10). For 1 wt% of chitosan, the droplets become hydrophobic enough to adsorb at the oil-water interface and thus stabilize the emulsion through the Pickering mechanism. Although particle interfacial adsorption is confirmed by interfacial rheology (see Figure 7 S11), the resulting Pickering emulsion also displays a weakly gelled continuous phase. This is a noticeable difference relative to the emulsion prepared with the same chitosan concentration at pH 4. The particlecoated droplets of the latter emulsion sediments over time, whereas those prepared at pH 5.5 remain homogeneously distributed in a gelled emulsion (see Figure S12). This indicates that emulsions containing 1 wt% chitosan at pH 5.5 are stabilized through a combination of Pickering and network stabilization mechanisms. Further increase of the chitosan concentration to 5 wt% leads to emulsion stabilization through network formation. This transition from unstable (0.2 wt%) to mixed Pickering/network stabilization (1 wt%) to pure network stabilization (5 wt%) is nicely captured by the increase in the Pearson correlation coefficient (ρ) from 0.48 to 0.94 with increasing chitosan concentration at pH 5.5 (Figure 7B). Progressively higher ρ values indicate more immobile and gelled colloids, supporting our physical interpretation of the results.


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Finally, the stability of the emulsions at pH 9 increases with increasing chitosan concentration (Figure 7A). At this pH, chitosan takes the role of a Pickering stabilizer by adsorbing at the oil–water interface. When added in high concentrations, free chitosan molecules present in the continuous phase also work as a binder between droplets (see Figure S5). At the lowest concentration of 0.2 wt%, the amount of chitosan is not sufficient to fully stabilize the emulsion, resulting in the release of oil at the top of the emulsion after three days. At 1 wt% of chitosan, Pickering stabilization is possible but no droplet agglomeration is observed due to the low concentration of free molecules in the continuous phase. Further increase of the chitosan concentration to 5 wt% leads to an excess of free molecules, leading to the gelation of Pickering-stabilized oil droplets. This trend is captured by the increase in the Pearson correlation coefficient as the chitosan concentration is increased (Figure 7B).

Figure 7: Stability map illustrating the different stabilization mechanisms as a function of pH and chitosan concentration for emulsions prepared from suspensions containing 8.7 wtsusp% silica. (A) Confocal images of emulsions highlighting their different microstructures. Scale bars: 10 µm. (B) Results of image-correlation analyses between two confocal images with a time lag of 5 s and the corresponding Pearson correlation coefficient (ρ), which reflects the motion of the droplets and thus the degree of gelation.

Tuning emulsion stability through in situ pH change The rich phase behaviour of the emulsions enables control of the underlying stabilization mechanisms by changing a single parameter in the design space, either the pH value or the chitosan concentration. This opens up the possibility to create responsive emulsions or emulsions whose colloidal behaviour can be tuned to best fit desired processing conditions. According to the stability map of our emulsion, switching the pH of the continuous phase in situ from 5.5 to 4 for example allows us to transform a network-stabilized emulsion into a Pickering-stabilized emulsion. Such transformation is schematically indicated by an arrow on the stability map (Figure 7A). We illustrate this possibility by first investigating the behaviour of the oil–water interface as the pH is changed in situ through the controlled addition of an acid. Indeed, our results show that the in situ change in pH from 5.5 to 4 leads to the spontaneous adsorption of modified particles at the oil–water interface, which is in qualitative agreement with the experiments where the initial pH is adjusted before emulsification (Figure 5 & Figure 3). The in situ pH change during an interfacial rheology experiment was possible by using a sub-phase exchange cell combined with a double-wall ring (DWR) (Figure 8A). As demonstrated previously in Figure 5B, the modified particles do not adsorb spontaneously at the oil–water interface at pH 5.5; therefore no viscoelastic adsorption layer is detected at the beginning of the experiment (Figure 8A). By changing in situ the pH from 5.5 to 4 after 20 h using the appropriate amount of 0.02M HCl solution, a viscoelastic film is formed at the oil–water interface, as indicated by the steady increase in interfacial storage and loss moduli with time. Because the equilibrium amount of chitosan adsorbed at pH 5.5 is higher than at pH 4 (Figure 2), chitosan is


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expected to desorb from the silica surface during acidification. However, this is only possible if the adsorption of chitosan on the silica particles is a reversible process within the pH range 5.5-4. The detection of a viscoelastic film by the interfacial rheology experiments confirms the reversibility of the adsorption process. This reversibility is attributed to the fact that the silica particles are only slightly charged at pH 4, reducing attractive electrostatic interactions between chitosan and silica. Moreover, chitosan is highly charged at pH 4, which should lead to repulsion between the already-adsorbed chitosan molecules when the pH is changed 26-27 from 5.5 to 4. Despite the reversibility of the chitosan adsorption process, we observed that the formation of the viscoelastic interface takes as much as several days, as opposed to tens of hours in the case of suspensions whose pH was initially adjusted to 4 (Figure 5A, I). The desorption of charged polyelectrolytes is known to be a very slow process, due to the great number of contacts between the macromolecular segments and the surface. This constitutes a significant kinetic barrier for desorption.28 The kinetics of desorption is also reduced by the slow convection and diffusion of ions upon exchange of the sub-phase in the double wall ring (DWR) 17 set-up. As expected, particles adsorbed at the oil–water interface at pH 4 no longer desorb even if the pH is changed back to 5.5 (data not shown). This suggests that the transformation of a Pickering emulsion into a network-stabilized emulsion through a pH change in the opposite direction is not feasible. To evaluate the effect of a pH shift from neutral to acidic values on the behaviour of the emulsion continuous phase, we also followed the bulk rheological properties of a suspension of modified particles whose pH is varied in situ via a time-delayed chemical reaction (Figure 8B). The in situ pH change was accomplished through the time-delayed hydrolysis of δ-gluconolactone (GDL) in water at a concentration of 29 1.5 wt%. Such a reaction has been exploited before for the direct coagulation of suspensions and would provide a practical means to formulate emulsions that are deliberately tuned to change structure and properties over time. Rheological properties of the bulk emulsion were measured while the GDL induced a pH change from 7.5 to 4.5 in a couple of hours. High storage and loss moduli were obtained at pHs in the range 7.5 - 5.5, when the suspension is gelled through the formation of a percolating network of chitosan-coated silica particles. As the pH decreases towards 4, the moduli drop by several orders of magnitude to values where only noise is recorded. During this pH reduction, the modified particles de-agglomerate from the initial network due to chitosan desorption from the silica surface, leading to a liquid-like behaviour of the modified suspensions. This in situ fluidization confirms that chitosan adsorbs reversibly on silica and demonstrates the reversibility of the agglomeration process. The reversibility of chitosan adsorption with pH is an interesting feature for industrial applications as it provides a flexible means to change the microstructure and rheological properties of a formulation at any time. In food and cosmetics applications, for example, a base emulsion formulation at pH 5.5 could be prepared at the production site and only later adapted through a simple pH change to the structure and rheological properties desired for the final product. The reversible feature also makes the stabilization of emulsions with chitosan-modified silica particles more resilient to possible variations in the emulsification procedure. For example, emulsions with identical properties can be prepared, regardless of whether the pH of the modified suspensions was adjusted before or after emulsification (see Figure S14). Indeed, we found that emulsions at pH 4 and 9 can be obtained either by adjusting the initial pH of the modified suspensions (as for all the results discussed so far) or by modifying the pH of an emulsion initially prepared at pH 5.5 to either pH 4 or 9 (see Figure S14). This reflects the kinetically trapped state of the network-stabilized emulsion prepared at pH 5.5. The removal of this kinetic constraint enabled by a pH change to 4 or 9 eventually set particles or chitosan molecules free from agglomerates, thus enabling their adsorption at the oil–water interface. 30 Since GDL is a food-grade chemical used for milk acidification, its addition to the formulation does not compromise the edibility and biocompatibility of our emulsion system. Moreover, the dependence of the hydrolysis rate on temperature31 makes this variable another control parameter for tuning on-demand the structure and rheological properties of the emulsions.


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Figure 8: (A) Effect of the in situ pH change on the interfacial shear rheology of an interface formed between corn oil and a 1 wtsusp% silica suspension modified with 5 wt% of chitosan. The silica suspension was prepared at pH 5.5 and acidified after 20 h with a 0.02-M HCI solution to reach pH 4. The time sweep was performed at a constant strain amplitude of γ = 0.3 % -1 and angular frequency of ω = 1 rad.s . (B) In situ pH change and associated bulk shear rheology properties of a chitosan modified suspension initially prepared in basic conditions (pH 10.5) and acidified with 1.5 wt% of GDL (δ-gluconolactone) until a pH of 4 is reached. The time sweep was performed at a constant strain amplitude of γ = 0.001 % and angular -1 frequency of ω = 1 rad.s . All G’ and G’’ values below 0.01 were found to be within the noise level and were therefore omitted from the plot.

Conclusions Changing the pH of chitosan-modified silica suspensions mixed with an oil phase affects the stability, microstructure and rheological properties of the resulting food-grade oil-in-water emulsions. The pH influences the surface charges of silica particles and chitosan molecules, and therefore their interactions in the continuous phase and with the oil droplets. When attractive interactions are present, chitosan adsorbs on the silica particles. The amount of adsorbed chitosan changes the particle hydrophobicity, thus affecting the affinity of these particles to the oil–water interface and their colloidal behaviour in the continuous aqueous medium. Because the amount of chitosan adsorbed on the silica particles is significantly affected by the pH, three distinct emulsion stabilization mechanisms may occur depending on the acidity level of the system. Pickering emulsions stabilized by silica particles are obtained at pH 4, whereas a network-stabilized emulsion is achieved at pH 5.5. At pH 9, stable emulsions result predominantly from the adsorption of insoluble chitosan at the oil-water interface. The reversible adsorption of chitosan on silica at pH 5.5 allows for switching the microstructure and properties of the network-stabilized emulsion on-demand by simply changing the pH. This provides the convenience of adjusting the pH of either the suspension or the emulsion without affecting the properties of the final product. The ability of the chitosan-modified silica particles to stabilize emulsion droplets at various pHs also offers a broad design space to address specific functional requirements in the food industry. For instance, emulsion droplets stabilized by nanoparticles modified at low pH can be spray dried and used for acidic beverages while the emulsion droplets prepared at 5.5 could stabilize edible emulsions like milk or ice cream. Besides food industry, this adaptable system can potentially be used for the preparation of oil-inwater emulsions with tunable stability in cosmetics, pharmaceutical or manufacturing products.


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Supporting Information Videos showing real-time dynamics and structure of emulsions prepared at pH 4, 5.5, and 9 Adsorption isotherm of chitosan on silica nanoparticles; behavior of chitosan at pH 9; cryo-SEM images of chitosan at pH 4, 5.5, and 9; bulk rheology measurements of chitosan-modified silica suspensions; experiments showing that chitosan molecules act as emulsifiers at pH 9; behavior of chitosan-modified silica suspensions at pH 9; control emulsions with only chitosan molecules, only silica nanoparticles, or only fatty acids as stabilizers; backscattering profiles of emulsions prepared at different pH; additional interfacial rheology measurements; macro and microscopic behavior of emulsions prepared at pH 4 and 5.5; compositional diagrams; reversibility of chitosan adsorption after high-pressure homogenization.

Acknowledgements This research was supported by DSM (Grant 3831/175101). The work also benefited from support from the Swiss National Science Foundation through the National Center of Competence in Research Bio-Inspired Materials. The authors would like to thank Dr. Bruno Leuenberger and Dr. Szilvia Mesaros for discussions, Bram Schroyen for the help with the interfacial rheology measurements, Dr. Michele Zanini for the cryo-SEM images of the modified suspensions, Sandrine Desclaux for the TOC analysis, Dr. Sreenath Bolisetty and Prof. Raffaele Mezzenga for providing access to the DWS apparatus, and Andreas Trabesinger for his contributions in revising this manuscript. We also thank Formulaction, particularly Dr. Christelle Tisserand, for the measurements carried out with the Turbiscan LAB. We are also grateful to ScopeM microscopy center at ETH, in particular to Stephan Handschin and Falk Lucas for the cryo-SEM images of the emulsions.


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TOC figure


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Emulsions Stabilized by Chitosan-Modified Silica ... - ACS Publications

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