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Remarks on emulsions 2 primary emulsions are made: Emulsion prepared by homogenizing using only the Ultraturrax denoted as UT. Emulsion prepared by using the Ultraturrax after which the microfluidizer is used to make the water droplets even smaller denoted as MF. Remarks on procedure Standard procedure: Beakers are filled with oil-phase at 60 °C after which the water-phase (60°C) is added drop wise. An Ultraturrax (type S50 N - G45 F, IKA®-Werke, Germany) is used to prepare the emulsion by homogenizing at 5200 rpm for 5 minutes. The emulsion is made in a beaker of 300 mL after which a sample (UT) is taken and the rest is use to make the MF-emulsion. Premix W/O emulsion is homogenized using a Microfluidizer (type M110S, Microfluidics) at a driving air pressure of 6 bar for 1.5 minutes. The homogenized W/O emulsion is collected in a beaker.

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Measurements were done in hoso and in dodecane (mixture of isomers). The refractive indices were set to the values found in literature first (1.33+0i for water and 1.472 for hoso and 1.421 for dodecane) and afterwards adjusted to get the right vol% of particles. Increasing the imaginary index leads to a decrease of the volume% calculated (further away from the actual one) thus the real part of the RI of the water phase itself was varied instead (normally 1,33).

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The vol% calculated is closest to the real one when a ri between 1.40 and 1.43 is used for measurements in Hoso while an ri between 1.39 and 1.40 is optimal for measurements in dodecane.

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Measurements were done in hexane, the refractive index was set to 1.372.

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For the measurement, Δ was set to 180 ms, δ was set to 2500 µs and the magnetic field gradient was varied. Ds of the W1-phase was measured to be 1.427E-09 m/s2.

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* PFG-NMR only sees particles of which the diameter is bigger than (8*D*δ)1/2

With δ the duration of the pulses and D the free diffusion coefficient (1.261E-9 m/s2 (for pure water)) References Fourel et al. (1994). Determination of water droplet size distribution by low resolution PFG-NMR. Journal of colloid and interface science, 164, 48-53.

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Remarks on composition All waterphases were composed of Milli-Q water. Remarks on procedure Standard procedure using either only the Ultraturrax (UT) or both ultraturrax and microfluidizer afterwards (MF).

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Increasing the PGPR-concentration clearly leads to a linear increase of the oil viscosity. A higher lipid phase viscosity is related to smaller internal water droplets (Weiss & Muschiolik, 2007). Thus with higher viscosity, smaller inner water droplets are obtained. High concentration of emulsifier (PGPR) thus has a double reducing effect on the droplet size of the primary emulsion: reduction due to higher viscosity and due to the presence of more emulsifier during emulsification. Reference Weiss J. & Muschiolik G. (2007). Factors affecting the droplet size of Water-in-Oil emulsions (W/O) and the oil globule size in Water-in-Oil-in-Water emulsions (W/O/W). Journal of Dispersion Science and Technology, 28, 703-716.

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Remarks on laser diffraction measurements Measurements were done in miglyol 812 (MCT) oil. The refractive indices were set to 1,33+0,0i and 1,472 thus for water in hoso. The refractive index of miglyol 812 is 1,450 using this index, the difference is very small compared to the used refractive indices (slightly smaller diameters obtained). In a subsequent test (vide supra), the optimal refractive index settings were determined. Here the original values were used but this does not really matter for this test because the relative position of the peaks remains the same with a different refractive index.

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The results obtained using laser diffraction are quite influenced by the presence of air. Not a lot of information can be obtained from the primary data indicating that the measurement was not too good. Though it can be seen that for higher concentrations the highest signal is obtained for higher detector numbers, indicating smaller droplets. Also the more multimodal distribution for lower concentrations can be distinguished.

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Decreasing the concentration PGPR leads to an increase of the size of the oil layer on top of the emulsion after centrifugation. This indicates that lower PGPR-concentrations lead to bigger droplets which sediment faster. Also the viscosity increases with increasing PGPR-concentration, magnifying this effect. Remarks on LUMiFuge analysis: A rectangular synthetic cell with 2,2 mm light beam trough the sample was used. Volume sample: 0,4 mL Light intensity: 200% Speed: ± 3000 rpm Duration: 60 min. Transmission was recorded every 30 seconds.

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When the microfluidizer is used for oil with less than or equal to 3.3 wt%, some of the water drops are unstable, a water layer is formed at the bottom after centrifugation. This effect is indicated by the increase in transmission at the bottom of the flask.

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Remarks on preparation procedure: Primary emulsions prepared using standard procedure but using only the ultraturrax. To make the double emulsion 45 g of the W2-phase is brought in a beaker of ~150 mL and 35 g prepared W1/O-emulsion is added with a pipette to this 45 g of W2-phase. This mixture was mixed with an Ultraturrax S25-10G (IKA®-Werke, Germany) at 24000 rpm for 48 seconds and directly afterwards further emulsified with a continuous Ultraturrax DK25 (IKA®-Werke, Germany) at 24000 rpm for 1 minute 36 seconds.

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Remarks on yield Yields were determined using PFG-NMR: Δ was set to 180 ms, δ was set to 2500 µs and the magnetic field gradient was varied. Ds of the W1-phase was measured to be 1.309E-09 m/s2.

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Remarks on laser diffraction measurements The refractive indices where set to 1,5295+0,1i and 1,33 (3OHD).

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Remarks on composition All waterphases were composed of Milli-Q water. Remarks on procedure Standard procedure using either only the Ultraturrax (UT) or both ultraturrax and microfluidizer afterwards (MF).

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Remarks on laser diffraction measurements Measurements were done in miglyol 812 (MCT) oil. The refractive indices were set to 1,33+0,0i and 1,472 thus for water in hoso. The refractive index of miglyol 812 is 1,450 using this index, the difference is very small compared to the used refractive indices (slightly smaller diameters obtained).

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Ultraturrax Presence of NaCl leads to a smaller oil layer on top of the emulsion after centrifugation indicating smaller drops. Microfluidizer Presence of NaCl leads to a smaller oil layer on top of the emulsion after centrifugation indicating smaller drops when NaCl is present. The stability of the water drops seems to be lowered by the presence of NaCl, indicated by the increase of transmission at the bottom of the flask after centrifugation caused by the coalescence of water drops. Remarks on LUMiFuge analysis: A rectangular synthetic cell with 2,2 mm light beam trough the sample was used. Volume sample: 0,4 mL Light intensity: 200% Speed: ± 3000 rpm Duration: 60 min. Transmission was recorded every 30 seconds.

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Remarks on emulsions 2 double emulsions are made: Emulsion in which the primary emulsion is prepared by homogenizing using only the Ultraturrax. Emulsion in which the primary emulsion is prepared by using the Ultraturrax after which the microfluidizer is used to make the water droplets even smaller. The expectation is that the yield of UT will be higher than the yield of MF. This because of the fact that microfluidisation seems to lead to smaller but also less stable droplets (previous results). Remarks on procedure Standard procedure.

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Remarks on laser diffraction measurements The refractive indices where set at 1,5295+0,1i and 1,33 (3OHD). Remarks on PFG-NMR measurements For the measurement, Δ was set to 180 ms, δ was set to 2500 µs and the magnetic field gradient was varied. Ds of the W1-phase was measured to be 1.309E-09 m/s2.

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Remarks on LUMiFuge measurements The conclusions obtained here are confirmed by visual observation: After storage at 4 °C for 20 hours, the cream layer of MF seemed a lot smaller than the one of UT. The serum layers were not clear in both cases (milky). A rectangular synthetic cell with 2,2 mm light beam trough the sample was used. Volume sample: 0,4 mL Light intensity: 200% Speed: ± 3000 rpm Duration: 60 min. Transmission was recorded every 30 seconds.

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Outline of the test Objective: 2 different double emulsions, one with Tween 60 (model for low MW) and one with Na-Caseinate (model for high MW) as external emulsifiers (table 1) were prepared and their swelling/shrinking behavior in hypo-, iso- and hypertonic solution during storage was followed. Therefore 25/25/50 (shrinking) and 10/40/50 (swelling) (v/v/v) W1/O/W2 emulsions with optimal concentrations of external emulsifier and optimal concentration PGPR (from previous tests) are made. In practice these emulsions were prepared and the initial droplet size and enclosed water fraction was measured after 12 hours. After this measurement these emulsions were diluted with hypo- (for 10/40/50) or hypertonic (for 25/25/50) solution. The hypotonic storage solution consists of deionized water, 2 wt% emulsifier and 0.02 wt% NaN3 while for isotonic storage the samples as such are used. The hypertonic storage solution consists of 0.2M KCl , 2 wt% emulsifier and 0.02 wt% NaN3. Measurements will be performed after 1, 2, 4, 8, 12, 24 hours and 2, 4, 8, 16, 32 days storage using laser diffraction. Remarks on procedure Standard procedure except that only the Ultraturrax S25-10G (IKA®-Werke, Germany) at 24000 rpm for 3 minutes was used to make the W/O/W emulsions (no continuous step).

Remarks

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Because the osmotic effects went faster than expected (results composition 1 and 3), it was decided to do the measurement at the right osmotic pressure for composition 2 and 4.

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Remarks on measurements The refractive indices where set to 1,5295+0,1i and 1,33 (3OHD). Composition 1 Emulsions were stored at 4 °C without stirring. Each measurement was repeated 5 times. Composition 2 Emulsions were stored at room temperature without stirring. Each measurement was repeated 5 times.

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Composition 3 Emulsions were stored at 4 °C without stirring. Each measurement was repeated 5 times. Composition 4 Emulsions were stored at room temperature without stirring. Each measurement was repeated 5 times.

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Osmotic effects are a lot bigger for the low MW surfactant: composition 2 shrinks with about 20% while composition 1 only shrinks with about 7%. Composition 4 swells by about 30% while composition 3 swells by about 20%.

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% obtained using 31P-NMR for Lecico and liquid chromatography for VAV life sciences

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Remarks on emulsions: All emulsions are prepared using distilled water. With unstable , phase separation occuring after less than one hour at room temperature is indicated. Remarks on preparation procedures: 1. Both phases heated to 60 °C after which the water is added dropwise to the oil.

The Ultraturrax S25-10G is used to mix 3 minutes at 24.000 rpm. 2. Following Akhtar and Dickinson (2001), both phases heated to 65 °C after

which the water is added dropwise to the oil while stirring magnetically, afterwards the Ultraturrax S25-10G is used to mix 3 minutes at 24.000 rpm.

3. Lecithin is dissolved in hoso by sonication. Water at room temperature is added drop wise to the oil phase (50 °C) while stirring with the Ultraturrax S25-10G for 5 minutes at 6.500 rpm (of which 4 minutes water addition). Afterwards the Ultraturrax S25-10G is used to mix 3 minutes at 24.000 rpm.

4. Lecithin is dissolved in distilled water by leaving the solution at room temperature for 12 hours. Water at room temperature is added drop wise to the oil phase (rt) while stirring with the Ultraturrax S25-10G for 5 minutes at 6.500 rpm (of which 4 minutes water addition). Afterwards the Ultraturrax S25-10G is used to mix 3 minutes at 24.000 rpm.

5. Lecithin is dissolved in distilled water by stirring for 30 minutes at 50 °C. Water at 50 °C is added drop wise to the oil phase (50 °C) while stirring with the

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Ultraturrax S25-10G for 3 minutes at 6.500 rpm (of which 1.5 minutes water addition). Afterwards the Ultraturrax S25-10G is used to mix 3 minutes at 24.000 rpm.

References Akhtar M. & Dickinson E. (2001). Water-in-oil-in-water multiple emulsions stabilized by polymeric and natural emulsifiers. In Dickinson E. and Miller R., Food colloids fundamentals of formulation. Special publication 258 (pp 133-143). Cambridge: Royal Society of Chemistry.

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Remarks on emulsions: All emulsions are prepared using distilled water. Remarks on preparation procedures: Beakers are filled with oil-phase at room temperature after which the water-phase (room temperature) is added drop wise while stirring magnetically. After this, the Ultraturrax S25-10G is used for 3 minutes at 24.000 rpm. Observations A lot of foam can be seen while the lecithin is mixed in the oil so I left the mixture to stand until almost all foam settled. The oil phase is very viscous leading to the fact that water sinks difficultly after adding so difficult emulsification. W/O 10/90 : No good emulsion, 2 cream layers (top and bottom) after about 30 minutes. 20/80 : Quite solid, an oil layer can be seen on top after about 30 minutes. 30/70 : Cream like structure, small layer of oil after 2 hours. O/W 40/60 and 50/50 Foam formed during emulsifying.

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Used emulsifying setup (beakers, stir speed) might have a very big influence : water is sinking slowly into the very viscous oil layer while adding drop wise. This slow sinking might be partially responsible for the low maximal volume of water in W/O before inversion to O/W. It is better to heat both phases so the viscosity of the oil phase goes down (done in following tests).

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Remarks on emulsions: Oil-phase: -8 wt% To 20 g Hoso, 1.6 g lecithin is added and these solutions are stirred for 1 hour at room temperature. Following were the assumptions: - ½ of the lecithin are phospholipids - Average MW of 779 g/mol (calculated) - 50 A2/molecule PL - Specific surface silica is 500 m2/g Thus following amounts of silica were added to the oil solution: 0% : 0g 100%: 0.619g 150% : 0.929g 200%: 1.238g These solutions were stirred for 4 hours at room temperature after which these solutions were centrifuged for 10 minutes at 3392g. 14 g of the supernatants was used to make the emulsions, this solution was heated to 65 °C before emulsification.

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Water-phase: 6 g deionized water heated to 65 °C. Preparation procedure Beakers are filled with oil-phase at 65 °C after which the water-phase (65°C) is added drop wise. An Ultraturrax (type S25-10G, IKA®-Werke, Germany) is used to prepare the emulsion by homogenizing at 24.000 rpm for 3 minutes. Observations Also for the solution with 0% removal (no silica added), a subnatans can be seen after centrifugation. The more silica used (phospholipids removed), the less the emulsion is viscous. Also the more silica is used, the lighter the color of the oil-solution/emulsion. An oil layer can be seen on top of the emulsions after centrifugation. This layer is similar for 9 and 10 but increases for 11 and even further for 12. The viscosity goes down from 12 to 9 which might be the cause of this effect (faster sedimentation) or it might be that bigger drops are present in 12>11>10>9 or a combination of both effects. No destabilization of water drops is seen (no increase transmission at bottom of the tube). Remarks on LUMiFuge analysis: A rectangular synthetic cell with 2,2 mm light beam trough the sample was used. Volume sample: 0,4 mL Light intensity: 200% Speed: ± 3000 rpm Duration: 60 min. Transmission was recorded every 30 seconds.

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Remarks on emulsifiers: Ryoto sugar ester series type S-170, S-270, S-370, P-170, L-195, ER-190, O-170 and POS-135 were tested. 2 wt% S-270 and S-370 could not be dissolved in the oil phase by heating and stirring. L-195 gave an unstable W/O emulsion, structure breaks after less then 48 hours at room temperature and LUMiFuge indicates that water droplets are coalescing. Remarks on emulsions: All emulsions are prepared using distilled water. Remarks on preparation procedures: Beakers are filled with oil-phase at room temperature after which the water-phase (room temperature) is added drop wise while stirring at 6500 rpm for 3 minutes (of which 2 minutes addition) with the Ultraturrax S25-10G. After this the same Ultraturrax is used and the mixture is further emulsified for 3 minutes at 24.000 rpm. Observations S-170 gives a creamy emulsion which is stable (due to cream structure). The emulsion made using O-170 is more viscous than the one made using PGPR, this emulsion is flocky but still more fluid than S-170. The emulsion made using ER-190 is about as viscous as O-170 but the flocks are smaller. The emulsion obtained with

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P-170 is solid with very big flocks. POS135 is liquid with some flocks. After the first stirring step at 6500 rpm, POS gives a smooth emulsion.

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Remarks on LUMiFuge analysis: A rectangular synthetic cell with 2,2 mm light beam trough the sample was used. Volume sample: 0,4 mL Light intensity: 200% Speed: ± 3000 rpm Duration: 60 min. Transmission was recorded every 30 seconds.

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