Emulsions stabilised with pectin-based microgels: Investigations into the break-up of droplets in the presence of microgels

Journal of Food Engineering 294 (2021) 110421

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Emulsions stabilised with pectin-based microgels: Investigations into the break-up of droplets in the presence of microgels G.I. Saavedra Isusi *, N. Lohner, H.P. Karbstein, U.S. van der Schaaf Karlsruhe Institute of Technology, Institute of Process Engineering in Life Sciences – Chair of Food Process Engineering, Gotthard-Franz-Str. 3, Building 50.31, 76131, Karlsruhe, Germany

A R T I C L E I N F O

A B S T R A C T

Keywords: Microgel particles Pectin Emulsions Droplet size Emulsification process Microgel break-up

Pectin-based microgels (MGP) are a promising alternative for the stabilisation of emulsions in vegan foodapplications. Previous work has focused on the preparation of pectin-based MGP and on the effect of MGP concentration on the resulting emulsion properties. However, the effect of the emulsification process on microgel properties, such as size and integrity, still needs clarification. In this study, we emulsified oil in the presence of pectin MGP of different sizes. We found that throughout the emulsification process, microgel particles, ranging from 17 μm to 137 μm in size, were comminuted to a size that depended on the energy input of the emulsification parameters. Higher mechanical energy (Δp = 450 bar) resulted in microgels of mean diameters equal to 89 nm. This led to emulsions of a constant oil droplet size with a mean droplet size of 0.8 μm, regardless of the initial microgel size used for emulsion stabilisation. Lower energy inputs during the emulsification process resulted also in constant droplet sizes of around 2.8 μm for all initial microgel sizes. We also showed that the presence of oil enhances microgel breakage, leading to smaller microgels than in the absence of oil under the same constant process conditions.

1. Introduction Emulsion-based food products such as, drinks, salad dressings, des­ serts, and sauces, are part of our daily lives. These products are composed of two immiscible liquids (oil and water), and are thermo­ dynamically unstable, meaning, they require the use of emulsifying and thickening agents in order to guarantee stability and hinder phase sep­ aration (McClements, 2016). There is a broad range of emulsifying agents that can be used for this purpose, some typical examples are proteins and hydrocolloids, but also particles and low molecular ten­ sides (Dickinson, 2015). Low molecular tensides decrease the interfacial tension between the water and oil interface (McClements, 2016). Whereas particles stabilise Pickering emulsions by adsorbing onto the oil-water interface (Tavernier et al., 2016), they build a layer sur­ rounding the droplets, which protects the droplets against coalescence (Pickering, 1907). Pickering emulsions can be formed by means of “limited coales­ cence” in order to obtain narrow oil droplet distributions (Arditty et al., 2003). To achieve this, a certain concentration of particles is used to stabilise an emulsion. During the emulsification process, oil droplets are broken up and particles adsorb onto the oil droplet surface. However,

the amount of particles is not sufficient to cover all the newly formed droplets completely. The insufficiently covered droplets coalesce until a critical droplet size is reached at which the amount of particles in the emulsion is enough to cover and stabilise the decreased droplet surface. Although Pickering emulsions are stable and have narrow droplet size distributions, the use of Pickering particles (latex, clay, silica, etc.) as emulsifying agents in food products is limited due to their lack of biodegradability, poor biocompatibility and low consumer acceptance (Li et al., 2019). Microgel particles (MGP) based on biopolymers are a promising alternative to Pickering particles for food applications. Microgel parti­ cles are particulate polymer networks, whose properties are more complex than those of single polymer chains and particles (Dickinson, 2015). The combination of colloidal and gel properties in a single par­ ticle results in thermal and pH responsiveness, reversible swelling, deformability, and interfacial activity among other characteristics (Brugger and Richtering, 2008; Dickinson, 2015; Lyon and Fernandez-Nieves, 2012; Ngai et al., 2006). Microgels based on food biopolymer have been successfully produced (Dickinson, 2015) and there are many different methods for synthesising microgel particles (Freitas et al., 2005; Paques et al., 2014; Pravinata et al., 2016). Each

* Corresponding author. Karlsruhe Institute of Technology, BLT-LVT Gotthard-Franz-Str. 3, Building No. 50.31 D-76131, Karlsruhe, Germany. E-mail address: [email protected] (G.I. Saavedra Isusi). https://doi.org/10.1016/j.jfoodeng.2020.110421 Received 4 August 2020; Received in revised form 26 November 2020; Accepted 29 November 2020 Available online 1 December 2020 0260-8774/© 2020 Elsevier Ltd. All rights reserved.

G.I. Saavedra Isusi et al.

Journal of Food Engineering 294 (2021) 110421

method requires controlling the microgel particle size either before or after the gelation process. Microgels of biopolymers are mostly formed via controlled droplet gelation in an aqueous solution or in an oil based continuous phase, or by grinding macrogels into smaller units (Burey et al., 2008). Controlled droplet gelation or emulsion polymerisation can achieve droplets in the submicron and micron ranges. However, this method requires the use of surfactants and is followed by solvent removal (Fessi et al., 1989; Paques et al., 2014). Therefore, microgel preparation using an emulsion polymerisation approach can be time and energy consuming. In the top-down approach, larger gels are ground into smaller pieces. Gels break if stresses transmitted from the sur­ rounding medium exceed cohesive forces in the material (Kavanagh and Ross-Murphy, 1998). In order to achieve micron-sized or submicron-sized microgel particles, great amounts of energy must be applied to them (Saavedra Isusi et al., 2019). Although this method is also energy-consuming, the removal of solvent or surfactant from the microgel dispersion is not necessary. The biopolymer used for microgel formation can determine or in­ fluence the functionality and characteristics of the obtained microgel particle: aggregation, self-assembly, complexation or denaturation. Most importantly, the biopolymer chosen has to build stable networks to ensure the long-term integrity of microgel particles (Dickinson, 2009). A variety of polysaccharides or proteins can form microgel particles. Microgels produced from proteins such as whey are considered to have great potential in the stabilisation of foams (Schmitt et al., 2014) and emulsions (Destribats et al., 2014b). However, proteins from animal sources are an unthinkable alternative for vegan products. Moreover, protein-based MGP from plant or animal sources coacervate at their isoelectric point (Chen et al., 2020). Polysaccharide-based MGP, such as pectin MGP, thus are of interest. MGP from polysaccharides can be used in wider pH ranges than those from proteins. Alginate, carrageenan, gellan gum, agar, curdlan, and pectin, are some examples used for MGP formation. Alginate MGP have been successfully produced using high-pressure homogenisation and indirect complexation via emulsifi­ cation. These particles can be used as encapsulating devices in phar­ maceutical products and cosmetics for targeted or delayed release of substances of interest (Paques et al., 2014). Carrageenan forms micro­ gels upon cooling in the presences of salts (Burey et al., 2008). Ellis et al. (2009) have manufactured k-carrageenan microgel using a high-velocity mixer to control the texture and reduce fat in food products. Agar as well as curdlan and gellan gum-based MGP have also proven to stabilise emulsions (Ishii et al., 2018). Agar, alginate, and carrageenan, are gained from seaweeds. Curdlan and gellan gum are both obtained from microbes. Pectin, on the con­ trary, is found in higher plants, where it gives firmness and structure to plant tissues. Pectin is gained from side streams of the food industry, e.g. from juice or sugar production. This makes pectin a sustainable plant polysaccharide from natural sources (Thakur et al., 1997) and a well-accepted consumer food ingredient. Compared to pectin as an in­ dividual polymer, pectin-based microgels maintain the same emulsi­ fying properties regardless of possible fluctuations in pectin source and molecular structures (Saavedra Isusi et al., 2020a). Therefore, pectin-based microgels have the potential to overcome variations in the polymer raw material. Pectin is a hetero block-copolymer, composed of three different polysaccharide structures forming a single pectin mole­ cule: homogalacturonan (HG), rhamnogalacturonan I (RGI), and rhamnogalacturonan II (RGII). Low methyl-esterified pectin chains form gels in the presences of divalent cations and form physical gels (Morris et al., 1982; Thakur et al., 1997). Previous work has focused on the choice of pectin type for microgel formation and the effect of microgel concentration on the resulting emulsion properties (Saavedra Isusi et al., 2020a, 2020b). However, the underlying mechanisms responsible for pectin microgel emulsifying properties are still unclear. Moreover, the effect of the emulsification process on microgel properties, such as size, and integrity, still needs clarification. Hence, further research on this topic is necessary to be able to use pectin microgel particles as

emulsifying agents in vegan food-applications. A key parameter to obtain a stable microgel-stabilised emulsion is the microgel size. Destribats et al. (2014a) studied this for microgel particles of inorganic polymers. The authors applied the concept of limited coalescence to emulsions stabilised with poly (N-isopropylacrylamide) (pNIPAM) microgels and demonstrated that the oil droplet size depends on the surface coverage of the droplets by microgels and microgel size, amongst other parameters. Equation (1) was postulated in order to describe the dependency of oil droplet size on microgel size and surface coverage (Destribats et al., 2014a): 2 nmp *π*dmp 1 = D 24*C*Vd

(1)

D is the droplet diameter, C is the surface coverage coefficient, dmp, is the microgel diameter, nmp is the microgel number concentration, and Vd is the oil volume. Pectin-based microgels stabilise emulsions in the same manner, as previous works have demonstrated (Saavedra Isusi et al., 2020b). Because microgel particle’s size determines the obtained oil droplet size, controlling microgel sizes should be guaranteed throughout the emulsification process. In this study, we aim to understand the effect of the emulsification process on the size of pectin-based microgels pre­ pared by a top-down approach. We focus on possible changes in microgel size during the mechanical emulsification as they may deter­ mine the resulting oil droplet size. Microgels have already been reduced in size during their preparation. However, it remains unclear whether the emulsification forces also affect microgel sizes. A possible size reduction could be dependent on the initial microgel size. Large microgel particles might break up due to harsh emulsification parame­ ters, thus affecting the resulting oil droplet size. Smaller microgel par­ ticles, in contrast, could remain unaffected by the same emulsification conditions. This study can help in optimising the emulsification process when using microgels as emulsion stabilizers. 2. Materials and methods 2.1. Materials Apple pomace pectinic acid (with a molecular weight of 34 kDa, a degree of esterification of 2% and a protein content of 3.1%) was a gift from Herbstreith & Fox (Neuenbürg, Germany). Calcium chloride dihydrate was obtained from Merck KGaA (Darmstadt, Germany). Me­ dium chain triglyceride (MCT) oil with C8 and C10 chains with 60:40 ratios was bought from IOI Oleo GmbH (Hamburg, Germany). Ester gum from Symrise AG (Holzminden, Germany), was used to increase the density of the MCT oil and retard creaming of emulsions. 8 wt% of ester gum was added to the oil. The mixture was then stirred at 50 ◦ C until dissolution. The MCT oil thereafter had a density of 0.96 kg/L at room temperature. 2.2. Preparations of pectin solution Pectinic acid solutions, with 2 wt% pectin in water, were prepared by dissolving 4 g pectin in 196 g demineralised water in a 600 mL beaker at 60 ◦ C using a high-shear mixer Ultraturrax T-25 digital (IKA® Werke GmbH & Co. KG, Staufen, Germany) at a rotational speed of 10.000 rpm for 30 s. Then solutions were left to cool down to room temperature. The pH was not adjusted and had a value of 4.7 ± 0.1 at room temperature. 2.3. Preparation of pectin microgel suspensions Pectinic acid solution, prepared as described in 4.2, was mixed with a 40 mM CaCl2 solution (volume ratio 1:1) at room temperature and under constant stirring. Once the pectin solution came in contact with the calcium solution, it gelled immediately, as described in previous own works. At 2 wt% pectin concentration, the gelled pectin solution does 2

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Journal of Food Engineering 294 (2021) 110421

not undergo volume changes during and after the gelation. Therefore, it is assumed that the mixture of pectin solution and calcium solutions contains 50 wt% microgels. As soon as the pectinic acid solution was completely poured into the CaCl2 solution, the gel suspension was mixed with a high-shear mixer (Ultraturrax T-25 digital) at a rotational speed of 13.000 rpm for 3 min in a 600 mL beaker, in order to reduce the microgel size. The obtained microgel suspension was diluted with demineralised water to obtain microgel suspensions with a mass con­ centration of 1 wt% microgel particles in water. These standard micro­ gels, referred to as MGP L, were used for further experiments. They either were used without any additional homogenising steps as emul­ sifying agents or were dispersed additionally to obtain smaller microgel particles. Additional homogenising steps were dispersing with a colloid mill (IKA MagicLab, IKA® Werke GmbH & Co. KG, Staufen, Germany) or a homogenising step using a high-pressure homogeniser (HPH). The exact processing parameters and processing devices for each formula­ tion are found in Table 1. Milled microgels are referred to as MGP M, and homogenised microgels as MGP S. The used HPH was a Microfluidizer MF 110 EH (Microfluidics Corporation, Newton, MA, USA), equipped with a Y-type interaction chamber with a microchannel diameter of 75 μm and an auxiliary processing module (APM) with a diameter of 200 μm. All samples were prepared in triplicate.

(IKA MagicLab, IKA® Werke GmbH & Co. KG, Staufen, Germany). Table 2 shows an overview of all formulations and process parameters used for these experiments. Each emulsion type was prepared in tripli­ cate if not stated otherwise. 2.6. Measurement of oil droplet size distribution The droplet size distribution (DSD) of the prepared emulsions was determined by static laser light scattering using a HORIBA LA-950 Particle analyser (Retsch Technology, Haan, Germany). The results were depicted as the cumulative volume distribution Q3. The charac­ teristic Sauter mean diameter d3,2 was chosen for comparison of the emulsifying results. The refractive indices were set at n = 1.4494 for MCT oil and n = 1.333 for water for all emulsions. The determination of the droplet sizes was made according to the Fraunhofer theory. All measurements were conducted in triplicate at room temperature. Emulsions were observed under a Zeiss Axiolab Microscope (Carl Zeiss AG, Oberkochen, Germany). Micrograhps were taken with an Axiocolor 105 camera. Micrographs of the samples were taken with 10, 20, or 40-fold magnification lenses. 2.7. Statistical analysis

2.4. Determination of microgel size

Each sample preparation was made in triplicate. If not specified otherwise, all analyses were conducted at least three times per inde­ pendent test. All data was assessed by a multifactorial analysis of vari­ ance (ANOVA) and a Sheff´ e test as post-hoc test. Dissimilarities in samples were considered statistically relevant at a level of p ≤ 0.05. The software OriginPro 2019 (OriginLab Corp., Northampton, MA, USA) was used for the statistical analysis, calculation of averages, and standard deviations.

The particle size distribution (PSD) of the prepared microgel sus­ pensions was measured by means of static laser light scattering, using a HORIBA LA-950 Particle Analyser (Retsch Technology GmbH, Haan, Germany). The distributions are given as the cumulative volume dis­ tribution Q3. The mean Sauter particle diameter d3,2 was chosen to compare the samples. The refractive index of pectin was set to n = 1.5470 + 0.01i and the index of water to n = 1.333 for all measurements. The scattering patterns were analysed according to the Fraunhofer theory, since the size of the produced microgel particles was well over 1 μm. However, the obtained sizes are explained as equivalent diameters, as pectin-based microgel particles are not perfect spheres. Numerical values do not correspond to absolute sizes and are used for comparison purposes only. The hydrodynamic diameter of MGP S was determined by dynamic light scattering with a particle size analyser Horiba Nanopartica SZ-100 (Horiba Scientific, Kyoto, Japan). Samples were measured at least 12 times. Measurements were conducted at a scattering angle of 173◦ and at 22. ±1 ◦ C.

3. Results and discussion 3.1. Microgel particles of different sizes In order to investigate the influence of emulsification process on the microgel particle size and on the stabilised oil droplet size, microgel particles of different sizes were prepared. For this reason, the standard microgel suspension (MGP L), obtained by high-shear mixing, was subjected to further homogenising steps to procure microgels of smaller sizes. MGP were either homogenised in a colloid mill (MGP M) or in a high-pressure homogeniser (MGP S). Each homogenising step increased the energy input applied to the particle suspension. Therefore, microgels were subjected to greater deformation forces, which ultimately led to particle break-up and to smaller sizes. Hence, smaller particles were produced with increasing energy input. The PSD of the microgel sus­ pensions was measured by means of static light scattering, as microgels are assumed to be well above 1 μm. The measured distributions are found in Fig. 1. As seen in Fig. 1, the obtained microgel sizes decrease with increasing mechanical energy input. The standard microgel suspension (MGP L), which was only dispersed once, had a mean Sauter diameter d3,2 of 137.5 ± 12.3 μm. This sample possessed by far the largest par­ ticles. Microgel suspensions, which were further homogenised, revealed

2.5. Preparation of pectin microgel-stabilised emulsions Emulsions were prepared by dispersing 5 vol% MCT oil (disperse phase) into a 1 wt% microgel suspension (continuous phase). Microgel suspensions were prepared as described in 4.3. MCT oil was dispersed into the continuous phase under constant mixing with a high-shear mixer Ultraturrax at a rotational speed of 15.000 rpm over 30 s in a 600 mL beaker. The coarse emulsions were mixed for another minute at the same rotational speed. In order to obtain fine emulsions, the coarse emulsions were homogenised using either an HPH Microfluidizer MF 110 EH (Microfluidics Corporation, Newton, MA, USA), or a colloid mill Table 1 Process parameter used for microgel preparation. Microgel name

Dispersing step

Process parameters

Standard (MGP L)

No additional, only high-shear mixer Additional dispersing step with colloid mill Additional dispersing step with high-pressure homogeniser

13.000 rpm, 3 min

Milled (MGP M) Homogenised (MGP S)

Table 2 Emulsification process parameters and formulations.

15.000 rpm, 2.5 min, gap width 0.3 mm 600 bar

*MGP = microgel particle.

Microgel type

Emulsification device

Process parameters for fine emulsion

MGP L, M, S

Colloid mill

MGP L, M, S

High-pressure homogeniser

15.000 rpm; 2.5 min, gap width 0.3 mm 450 bar

*MGP = microgel particle. 3

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Journal of Food Engineering 294 (2021) 110421

distributions with mean Sauter diameters of 43.5 ± 5.9 μm (MGP M) and 17.1 ± 1.7 μm (MGP S) for samples prepared with a colloid mill machine or a high-pressure homogeniser (HPH), respectively. The investigated suspensions are statistically different at a significance level of 0.05. As discussed above, the MGP size data presented are for comparative purposes only. In order to confirm that the measured sizes are repre­ sentative for the actual microgel particles, microscope images were taken. Exemplary microscope pictures are found in Fig. 2. As seen from Fig. 2, the microgel sizes concur with the measured distributions depicted in Fig. 1. Standard MGP L, which did not undergo additional homogenising steps (C), have no clear shape. The constant shearing during microgel preparation could be responsible for the elongated shape displayed in the image. Moreover, MGP L are the largest of the microgels shown in Fig. 2. Further grinding of MGP L decreased the microgel sizes. This can be seen in the upper left (A) and right (B) pictures. Microgel particles additionally ground in a colloid mill (MGP M) still do not display clear shapes. It can be seen that particles are not round but show an oval, elongated shape. This can also be due to shearing inside the colloid mill. Compared to particles prepared with a colloid mill, particles homo­ genised with an HPH are significantly smaller (MGP S). This corresponds to the elevated mechanical energy input inside an HPH (Schuchmann and Danner, 2004). Samples homogenised at 600 bar display a flake-like appearance. It appears that the flakes are composed of individual microgel particles. The flakes themselves appear to have a diameter of around 20 μm. This size is in good agreement with the measured data (Fig. 1). However, it indicates that the static laser scattering method

Fig. 1. Volumetric particle size distributions of pectinic acid microgel sus­ pensions (1 wt% microgels in water). Suspensions were prepared with different dispersing machines: Large microgels (MGP L): no additional dispersing step; Medium microgels (MGP M): additionally ground with colloid mill; Small microgels (MGP S): additionally high pressure homogenised at 600 bar.

Fig. 2. Microgels synthesised from pectinic acid. A: Small microgels (MGP S), homogenised with a HPH at 600 bar. B: Medium microgel (MGP M), milled with a RSM machine at 15.000 rpm for 2.5 s. C: Large microgels (MGP L), no additional homogenising step. Scale bar equals 20 μm. 4

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Journal of Food Engineering 294 (2021) 110421

measured particle agglomerates instead of individual particles. For this reason, we also measured the hydrodynamic diameters of the individual microgels composing the agglomerates of MGP S by means of dynamic light scattering. Large agglomerates were not taken into consideration during the data processing, as a result only individual particles could be analysed. The individual microgels that form MGP S have a mean hy­ drodynamic diameter of 89.7 ± 0.7 nm. From Fig. 2, it is clear that the sizes of the individual microgels that compose the shown aggregates of MGP S are below 1 μm, otherwise we could see them clearly under the microscope. The aggregation of MGP S to flakes indicates that the par­ ticles are small enough so that van-der-Waals forces or other attractive forces can lead to aggregation (Kobayashi et al., 2005). 3.2. Influence of microgel particle size on droplet size distribution Oil-in-water emulsions were prepared using pectin-based microgel particles as emulsifying agents. The microgel mass concentration was kept constant, but the microgel size was varied. As microgels had sizes ranging from 17 μm to 137 μm, the number of microgel particles per mass unit differed in the emulsions from sample to sample. The number of microgels nmp can be calculated using Equation (2), which was adapted from an equation presented by Destribats et al. (2014a). The authors developed this relation for pNiPAM microgel particles. It as­ sumes round particles, which does not apply to this study. Nevertheless, this equation was used for means of comparison. nmp =

6*mpolymer

3 *ρ π*dmp mp

Fig. 3. Volumetric droplet size distributions of o/w emulsions (5 vol% MCT in water), stabilised with 1 wt% microgels (pectinic acid). Emulsions were pre­ pared with HPH (450 bar) and microgels of various diameter: Large micro­ gels (MGP L): d3,2 = 137.5 μm; Medium microgels (MGP M): d3,2 = 43.5 μm; Small microgels (MGP S): d3,2 = 17.1 μm.

(2)

mpolymer is the pectin mass concentration, ρmp is the microgel density and dmp is the microgel diameter. MGP L, MGP M and MGP S have 1.4*107, 4.5*108, 7.5*109 counts per mass unit, respectively. Even though the numbers are not exact, they show a difference of one order of magnitude in microgel number concentration. However, as the smallest microgels also have the largest microgel number per mass unit, the resulting oil droplet diameter with these microgels should be the smallest. Our underlying hypothesis states that microgels that were previously broken up under processing conditions harsher than the conditions of emulsification should not be affected by the additional processing step. The microgel size difference, however, might affect the obtained oil droplet sizes, even though constant emulsification process parameters were chosen. In order to test this hypothesis, we produced emulsions of same formulation (5 vol% MCT oil and 1 wt% MGP L, M or S) either using a high-pressure homogeniser HPH (high energy input) or a colloid mill (reduced energy input) (Karbstein and Schubert, 1995). Reduced energy conditions represent milder stresses. The conditions chosen (15.000 rpm for 2.5 min at a gap width of 0.3 mm) are the same pro­ cessing conditions used for microgel MGP M preparation (see Table 1). As lower stresses also affect the resulting oil droplet sizes, we expected to obtain larger oil droplets in the colloid mill. High energy input corre­ sponds to high stress conditions, which were achieved by using a HPH at 450 bar. The homogenising pressure difference is lower than the one used for MGP S preparation (600 bar). Therefore, we did not expect further breakage of MGP S in the emulsification step. The measured oil droplet size distributions are found in Fig. 3 and Fig. 4. The determined mean Sauter diameters for all formulations are found in Table 3. As seen in Table 3, the obtained Sauter mean diameter for all emulsions prepared with HPH or a colloid did not differ within the statistical deviation of the measurement. Figs. 3 and 4 show that irre­ spective of the initial microgel size, MGP S, M, and L all stabilised oil droplets of about 1 μm (HPH, 450 bar) or 2.8 μm (colloid mill, 15.000 rpm, 2.5 min, 0.3 mm gap width). Even though the microgel sizes were significantly different prior to the emulsification step, the obtained DSDs only depended on the energy input applied during emulsification. As expected, milder stress conditions during emulsification (colloid mill)

Fig. 4. Volumetric droplet size distributions of o/w emulsions (5 vol% MCT in water), stabilised with 1 wt% microgels (pectinic acid). Emulsions were pre­ pared with a colloid mill and microgels of various diameter: Large microgels (MGP L): d3,2 = 137.5 μm; Medium microgels (MGP M): d3,2 = 43.5 μm; Small microgels (MGP S): d3,2 = 17.1 μm. Table 3 Resulting mean Sauter diameters of emulsions prepared with a colloid mill or HPH, with MGP L, M or S. Microgel type MGP L MGP M MGP S

Oil droplet d3,2 HPD [μm] a

Oil droplet d3,2 colloid mill [μm]

0.80 ± 0.05

2.94 ± 0.67b

0.81 ± 0.03a

2.72 ± 0.63b

0.83 ± 0.05a

2.75 ± 0.87b

*MGP = microgel particle. Initial microgel mean Sauter diameter: MGP L 137.5 ± 12.3 μm; MGP M 43.5 ± 5.9 μm; MGP S 17.1 ± 1.7 μm.

led to larger droplets (Fig. 4) compared to higher stresses (HPH) which resulted in smaller oil droplets (Fig. 3). It is noticeable that the resulting oil droplet sizes all were signifi­ cantly smaller than the sizes of the microgels added for their 5

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Journal of Food Engineering 294 (2021) 110421

stabilisation. An explanation for this could be MGP breakage during the emulsification process. A further break-up can be expected especially for large MGP. As the MGP used in this study were prepared with pectinic acid, which forms brittle gels, they are easily broken up (Saavedra Isusi et al., 2019) especially under tensile stress, which is found in both the high-pressure homogeniser and the colloid mill (Saavedra Isusi et al., 2019). Oil droplets stabilised by microgels are a microgel-oil complex, since microgels are adsorbed at the interface. Since the investigated emulsions were emulsified under the same conditions, we assume that the oil droplet break-up should be the same for all formulations prepared either with colloid mill or an HDH. The only difference between the investi­ gated emulsions is the initial microgel size used for stabilisation. The resulting oil droplet size distributions indicate that all types of MGP (L, M, and S) were ground to the same sizes in an emulsification process under given process conditions. Otherwise, the oil droplet sizes would be different for each microgel type. MGP S, which were homogenised by HPH at a corresponding energy input (600 bar) prior to the emulsifi­ cation process, already were at the required size, but agglomerated to flakes. We assume that the flakes were broken up during the emulsifi­ cation process and the individual microgel particles adsorbed onto the oil-water interface prior to being able to reform flakes. Microgels are assumed to behave like Pickering particles. Once microgels have adsorbed onto an interface, they remain there almost irreversibly and are no longer able to agglomerate (Aveyard et al., 2003). This assump­ tion is supported by the absence of bimodal size distributions in Figs. 3 and 4. Larger particles and agglomerates would be detected in the same way as larger droplets by the laser scattering methods. However, one cannot discard that microgels can remain in the continuous phase and are measured with the emulsion droplets. As a means to confirm that no large microgel particles were detected as oil droplets, we took micro­ scope images of all samples emulsified with a colloid mill. Fig. 5 displays oil droplets from emulsions prepared in a colloid mill with MGP L (A), MGP M (B), and MGP S (C). The depicted droplet sizes

are in good agreement with the measured values using static light scattering, shown in Fig. 4. As seen in Fig. 5, microgel particles are not found in the micrographs. Microgel particles would show up as inho­ mogeneously shaped particles, as seen in Fig. 2. The fact that larger microgels are absent from the micrographs indicates that the particles were most likely located at the interface or were much smaller than the oil droplets. Moreover, all investigated emulsion formulations were stable for at least one week. This indicates that microgels are adsorbed onto the interface and hinder oil droplet from coalescing. One should consider that the resolution of the employed microscope is around 1 μm. This corresponds to the smallest objects found in Fig. 5 (MGP S) that were identified as emulsion droplets. Therefore, free MGP in the continuous aqueous phase or MGP adsorbed onto the oil droplets must be smaller than 1 μm. 3.3. Influence of oil on microgel break-up The results described in Section 5.2 indicate that the extent of microgel break-up depends on whether microgels were homogenised in the presence of oil or not. In the following sections, the phenomena occurring during emulsification will be discussed separately for the different microgel particle types. 3.3.1. Influence of oil on MGP S Microgels MGP S were produced by HPH at 600 bar. They were then used as emulsifiers at 450 bar. Prior to the emulsification process, MGP S formed agglomerates due to particle interactions. Nevertheless, due to the homogenisation history of the particles, we expected the least amount of changes in the microgel size during the emulsification pro­ cess. However, the results shown in Section 5.2 indicate that the sizes of agglomerates formed by MGP S are also affected by the presence of oil when stresses acted on them. MGP S agglomerates were larger than the droplets depicted in Figs. 4 and 5. After high-pressure homogenisation, they formed aggregates of sizes around 17 μm. Fig. 5 shows no

Fig. 5. o/w emulsions (5 vol% MCT oil in water) prepared with a colloid mill and 1 wt% microgel particles (pectinic acid) of varying sizes: A: Medium microgel particles (MGP M). B: Large microgel particles (MGP L). C: Small microgel particles (MGP S). Scale bar equal 50 μm. 6

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Journal of Food Engineering 294 (2021) 110421

agglomerate after the emulsification process with a colloid mill (lower mechanical energy input compared to HPH). However, one would as­ sume that MGP S should stabilise smaller droplets than MGP L and MGP M due to their smaller size and higher microgel count per mass unit. Nevertheless, this is not the case (see Fig. 4). This indicates that the MGP S agglomerates are not completely disrupted and are broken up to the same sizes as MGP M and L. Fig. 6 shows a schematic representation of the events happening during the emulsification process with MGP S. Large microgel particles are homogenised and agglomerate due to their resulting small sizes. These agglomerates are used as emulsifying agents. During the emulsi­ fication process, agglomerates are broken up and individual microgels (or smaller agglomerates) are brought to the interface. There, they adsorb almost irreversibly, thus hindering re-agglomeration. The extent of agglomerate breakage and the resulting oil droplet size depends on the mechanical energy input.

Fig. 7 displays emulsion droplets of the coarse emulsion, emulsified for 1 min. All three micrographs display droplets and microgels found in the same sample. The upper left image depicts a large microgel particle with droplets attached to its surface. The upper right image shows oil droplets with smaller microgels adsorbed onto the interface. The bottom image shows oil droplet agglomerates, as well. Besides, the droplet surfaces show a golf ball-like structure. In this case, the droplet surface is covered by even smaller microgel particles than those seen in the other two images. The sizes of the microgels seen at the droplet surface border the resolution limit of the microscope of 1 μm which is much smaller than the initial MGP size of 137.5 μm. Fig. 8 shows an exemplary view of an emulsion homogenised for 5 min in the high-shear mixer. Only droplets and droplet agglomerates were found (Fig. 8). Microgel-droplet aggregates comparable to those shown in Fig. 7 (A and B) were not detected anymore. Both emulsions were not stable, as an oil film was noticeable on top of the emulsion hours after preparation. This indicates an insufficient stabilisation of the interface. Moreover, droplet agglomerates seen in Figs. 7 and 8 are caused by microgel particle bridging, which is often seen when rela­ tively large microgels adsorb at the interface (Destribats et al., 2014b). From the discussed images, it is clear that microgels and oil droplets interact with each other and lead to an increased microgel breakage in the presence of oil compared to microgel homogenisation without oil. Fig. 9 displays a schematic representation of microgel and oil droplet breakage during a common emulsification step with large microgel particles. As is evident from Fig. 9, large microgels and oil droplets undergo the same stresses during the emulsification step. This leads to a parallel microgel and oil droplet break-up. Small microgels and microgel frag­ ments adsorb onto the droplet surfaces and stabilise them. Larger microgels can also attach themselves to oil droplets. Microgel segments attached to the interface are sheared from the rest of the microgel. At the interface, the deformation stresses applied to the microgel surface are evidently not the same as the ones a microgel is exposed to when being homogenised in the absence of oil. Microgels are known to modify the mechanical properties of an interface and increase interfacial stress (Truzzolillo et al., 2015). The interface can become more rigid due to the adsorbed particles. The reduced deformability of the interface may also influence microgel rupture. Furthermore, the anchoring of microgel segments to a droplet

3.3.2. Influence of oil on MGP L In order to obtain MGP M, MPG L (size 137 μm) were homogenised with a colloid mill at a rotational speed of 15.000 rpm for 2.5 min. The resulting MGP M have diameters of 43.5 μm. Emulsions prepared with a colloid mill were emulsified under the same process conditions as those used for the preparation of MGP M (15.000 rpm for 2.5 min). MGP L were used for the stabilisation of these emulsions prepared with the colloid mill. Hence, MGP L underwent the same mechanical stress dur­ ing the emulsification process as during the preparation of MGP M. Therefore, we assumed that MGP L used for emulsion stabilisation are broken up to sizes equal to MGP M after the emulsification process. Nevertheless, after emulsification, only droplets or microgel particles ranging from 1 to 10 μm, i.e. much smaller diameters than those of MGP M, are detected. This indicates that MGP L microgels were broken up more extensively in the presence of oil than in its absence under the same process conditions. To get a better understanding of the processes taking place during emulsification, coarse emulsions were prepared using an Ultra-Turrax high-shear mixer at 15.000 rpm for 1 min or 5 min. As stabilizers, MGP L were used because of the large observed size reduction during the emulsification process. The aim of this experimental setup was to make oil-MGP interactions visually accessible. Microscope images of the ob­ tained emulsions are displayed in Fig. 7 and Fig. 8.

Fig. 6. Schematic representation of the microgel homogenising step and the emulsification step with microgel agglomerates as emulsifying agents. 7

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Journal of Food Engineering 294 (2021) 110421

Fig. 7. Coarse o/w emulsions (5 vol% MCT in water) prepared with an Ultra-Turrax machine, 1 wt% microgel particles (pectinic acid) and MGP L (large microgels), at 15.000 rpm for 1 min. A: Large microgel particles with oil droplets attached to its surface. Microgel size around 70 μm. B: Oil droplets with microgel particles adsorbed onto the water-oil interface. Microgel diameter around 5 μm. C: Oil droplets with small microgel particles adsorbed onto the oil-water interface. Microgel diameter around 1 μm. Scale bar equals 10 μm.

interface may influence its deformation in continuous phase flow. We assume that at the microgel-oil interface, the no-slip boundary condition is valid and oil droplets act as walls. Moreover, Kinoshita et al. (2007) showed that oil droplets in a flowing aqueous phase possess an internal flow. This internal flow is oriented in the opposite direction of the flow direction of the outer phase. This should increment the stress applied to microgels, increase their surface deformation and improve their break-up. Chernyak et al. (1986) described wall-polymer interactions leading to polymer relaxation or even rupture. These first observations motivate a thorough investigation of microgel break-up in two-phase flow to clarify the underlying mechanisms in detail. 3.4. Calculation of microgel size after the emulsification process From the results discussed above, it is clear that the emulsification process affects both oil droplet and microgel sizes. On the one hand, the energy input during emulsification clearly determined the oil droplet and microgel size. On the other hand, according to theories postulated for microgels and Pickering particles (Binks and Lumsdon, 2001; Des­ tribats et al., 2014a), droplet sizes are determined by the microgel sizes. However, the initial microgel size was changed during the emulsifica­ tion and the resulting microgel size was not accessible by the employed measurement methods. Light microscopy methods are able to detect objects sized up to 1 μm, consequently small microgels at the interface are not visible. Only larger microgels are seen at the interface of the coarse emulsion. However, once the energy input is increased and

Fig. 8. Coarse o/w emulsions (5 vol% MCT in water) prepared with an UltraTurrax machine, 1 wt% microgel particles (pectinic acid) and MGP L (large microgels), at 15.000 rpm for 5 min. Scale bar equals 10 μm.

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Journal of Food Engineering 294 (2021) 110421

Fig. 9. Schematic representation of the simultaneous microgel homogenising and emulsification step with large microgels as emulsifying agents.

droplet sizes are reduced, microgels are not detectable via light micro­ scopy. Additionally, light scattering methods are not useful either for this purpose, since microgels are adsorbed at the interface and the analysis methods only detect the microgel-oil complex as one object. Therefore, we estimated the resulting microgel size starting from the obtained oil droplet size. For this, we used the limited coalescence approach (Eq. (1)) described by Arditty et al. (2003) for solid particles at oil-water interfaces as well as by Destribats et al. (2014a) for microgels at interfaces. We used Equation (2) to substitute nmp in Equation (1) and have a term, which depends on the microgel diameter dmp. In our cal­ culations we used the mean Sauter diameter d3,2 as D. Assuming a full surface coverage and no changes in microgel size due to thermal fluctuations, the microgel size required to stabilise emulsion droplets of 2.9 μm (emulsion prepared with colloid mill) is 286 nm. Droplets with diameters of 2.7 μm should be stabilised with microgels of a size of 267 nm. For droplets of 0.8 μm (emulsion prepared with HPH), microgels should be 79 nm in size. According to the dynamic light scattering measurements, individual MGP S already had a mean hy­ drodynamic diameter of 89.7 ± 0.7 nm after preparation (see Section 5.1). This value is in good agreement with the calculated size of 79 nm. The calculations took a full surface coverage for granted, which prob­ ably does not represent reality. Previous studies reported surface coverage coefficients ranging from 0.36 to 0.71 for pNIPAM microgels (Destribats et al., 2014a) and 0.9 for a monolayer of hexagonally packed hard spheres (Destribats et al., 2013). Thus, a surface coverage coeffi­ cient less than 1 is plausible for our system. This would lead to larger calculated microgel diameters, which would agree even better with our measured particle sizes.

emulsion stabilisation. Lower energy input during the emulsification process resulted also in constant mean droplet sizes of around 2.8 μm for all initial microgel sizes. From the oil droplet sizes stabilised by microgel particles, we calculated the size of the MGP after the emulsification step. Comparing this value to the size of the MGP treated at the same energy input but without oil droplets, we derived that microgel break-up occurred in a different manner in the presence or in the absence of oil droplets. The presence of oil led to a greater extent of microgel breakage. We hypothesise that the non-slip boundary condition is valid for the microgel parts anchored to the oil interface. This might lead to a dif­ ference in velocity between anchored microgel parts and the rest of the particle resulting in an increased shear stress acting on the microgel particles anchored to oil droplets. This hypothesis must be investigated thoroughly in the future. The obtained data might help in understanding the behaviour of microgel particles during the emulsification process. The stresses applied for oil droplet break-up also significantly reduced MGP in size. The resulting small MGP apparently adsorbed directly at the oil droplet interfaces, preventing them from forming new agglomerates, as found in pure MGP dispersions treated with comparable energy input. With the gained information one could aim to combine the microgel preparation and the emulsification process. Thus, microgels, regardless of their size, can be used as emulsifying agents if the emulsification process is adequate, thereby saving an energy-intensive microgel preparation step. Credit author statement Gabriela I. Saavedra Isusi: Conceptualization, Methodology, Vali­ dation, Formal analysis, Investigation, Writing – original draft, Visual­ ization, Nils Lohner: Validation, Formal analysis, Investigation, Heike P. Karbstein: Funding acquisition, Writing Review & Editing, Resources, Supervision, Ulrike S. van der Schaaf: Conceptualization, Project administration, Funding acquisition, Supervision, Writing: Review & Editing

4. Conclusions In this study we emulsified oil in the presence of MGP particles of different sizes. We found that both, oil droplets and microgel particles, undergo deformation and break-up during the emulsification process. Microgel particles, ranging from 17 nm to 137 μm in size, were comminuted to a size that depended on the energy input of the emul­ sification process. Higher mechanical energy (450 bar homogenising pressure difference) resulted in small microgels of mean Sauter diameter equal to 89 nm. Even the largest MGP of 137 μm were reduced to this size. This led to emulsions of a constant oil droplet size with a mean droplet size of 0.8 μm, regardless of the initial microgel size used for

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 9

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Journal of Food Engineering 294 (2021) 110421

Acknowledgements

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