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ÖgeEncapsulation and release of amino acids in double emulsions(Graduate School, 2021-03-18) Kocaman, Esra ; Van Der Meeren, Paul ; 506162503 ; Food Engineering ; Gıda MühendisliğiDouble emulsions have been studied for many years, given their potential as encapsulation systems. It is also possible to control the release of diverse bioactive components by means of double emulsions. As amino acids might be degraded to some extent due to environmental factors such as pH, temperature, light exposure as well as some reactions (i.e. oxidation, Maillard), their encapsulation may be advantageous to avoid these issues. Besides, encapsulation may enable to release of these compounds in a later stage of the gastro-intestinal tract. The main research question of our research project was to what extent the release of encapsulated components from double emulsions can be controlled by the emulsification method, emulsion composition and environmental factors. Moreover, it was evaluated whether the release kinetics were substantially influenced by the molecular properties of the encapsulated compounds. Hence, this thesis studies the influence of some parameters on double emulsion stability as well as amino acid encapsulation and release in double emulsions. The current study consist of the evaluation of these parameters: solute characteristics (i.e hydrophobicity, molar mass) and concentration, pH of the aqueous phases, hydrophobic and hydrophilic emulsifier, homogenization and thickener. For the investigation of the effect of these parameters, the emulsion droplet size, and the entrapped water volume fraction were evaluated to characterize the double emulsions. Moreover, the release of amino acids was observed during storage using spectrophotometric and Nuclear Magnetic Resonance (NMR) techniques. A modification of the original method was performed to enable the optimum conditions for amino acid quantification (section 4.1). Due to the high background absorbance of the reagent 2,4,6-trinitrobenzenesulfonic acid (TNBS) which was the case for many of the measured concentrations, different TNBS concentrations were evaluated in order to determine the optimum concentration. Hence, the solution containing 0.6 mM TNBS was choosen as it demonstrated the lowest absorbance among the studied concentrations as a blank and the TNBS solution reacted with leucine. As the absorbance was not substantially changed after 3 hours, it was used as the reaction time. In section 4.2, the effect of solute characteristics on double emulsion stability and release of encapsulated compounds were presented. Different amino acids (i.e. hydrophilic and hydrophobic) were used to investigate the hydrophobicity effect at different temperatures. Also, di-peptides were used as encapsulated compound in order to evaluate the influence of molecular mass. The results showed that an increase was observed from 50 up to 90 μm in the average droplet size for the samples homogenized with Ultra-turrax at 17500 rpm within the 32 days time frame. The double emulsions at 4 °C indicated a higher increase in average droplet size as compared to 37 °C. To investigate the main instability mechanism in the emulsion, double emulsions were diluted with sodium dodecyl sulfate (SDS) before laser diffraction measurement. The measurement of the droplet size in the presence of SDS showed that flocculation was the main instability mechanism, which caused an increase in droplet size. On the other hand, a constant enclosed water volume fraction was found in double emulsions during 16 days of storage, independent from the temperature and hydrophobicity studied in this thesis. The encapsulation efficiency of amino acids in the inner water droplets was found to be higher than 80% in all cases. From the release results, amino acid hydrophobicity and storage temperature were found to largely influence the release rate of the encapsulated amino acids. The amino acid release rates were fastest at 37 °C, which was the highest temperature examined in this section of the thesis. This can be explained by the higher solubility as well as increased diffusion rate of amino acids in the intermediate phase. Also, an increase was observed in the release rates of amino acids as a result of higher hydrophobicity. The significant effects of hydrophobicity and temperature, as well as the constant enclosed water volume revealed that the release of amino acids from the inner to the outer water phase was mainly governed by a direct diffusion mechanism. As the di-peptides released faster than the amino acids, it follows that the increased solubility overruled the effect from the decreased diffusion coefficient of the dissolved compound in the oil phase. In section 4.3, the influence of solute concentration (i.e. 5, 10, 20 and 40 mM) on the release and double emulsion stability was investigated. The varying concentrations of amino acid did not cause a significant difference in the increase of volume weighed droplet size during 16 days. The entrapped water volume was stable for double emulsions that contained varying solute concentrations except from the double emulsion which contained 40 mM where a decrease was observed through 16 days of storage. This can be a result of the faster diffusion velocity of the amino acid across the oil phase to the external water phase as compared to the diffusion of potassium chloride (KCl) through the oil phase to the internal water phase. Hence, a fraction of the internal phase was expelled to the external water phase to equalize the osmotic pressure which resulted in a decrease in yield of entrapped water volume. Regarding the average residence time (ta) values, the double emulsion that contained the highest solute concentration studied (i.e. 40 mM) in this thesis indicated a faster release as compared to the other samples at 37°C, whereas there was no significant difference among the samples at 4°C. The pH effect of the aqueous phases on the release of amino acids and di-peptides was evaluated in section 4.4. Regarding the average droplet size, there was no significant difference between samples as a function of pH of the aqueous phases. Considering the release, the transport of the amino acids and di-peptides was faster at neutral pH as compared to acidic and basic pH values, which was thought to be due to the increased solute solubility in the oil phase for the zwitterionic (rather than ionic) form of the more hydrophobic molecules at neutral pH. The oil type effect on amino acid release and double emulsion stability was demonstrated in section 4.5 comparing long chain and middle chain triglycerides. The average droplet size of the long chain triglyceride (LCT) containing double emulsions were larger than of the medium chain triglyceride (MCT) containing samples. This can be due to the stronger aggregation of LCT containing samples as a consequence of the higher viscosity of the LCT oil. From the release results, much faster transport of L-leucine was observed through MCT oil as compared to LCT oil due to its higher solubility. Also, the lower viscosity of MCT oil gives rise to a higher diffusivity of dissolved compounds, which may also fasten molecular transport. In section 4.6, the influence of the hydrophobic emulsifier concentration (from 1 to 5%) on the double emulsion stability and release of entrapped amino acids was demonstrated. The entrapped water volume fraction of the polyglycerol polyricinoleate (PGPR) stabilized samples remained around 100% during 32 days of storage, except from the one with only 1% PGPR which had a decreasing yield due to insufficient stabilisation of the internal water droplets. It follows that the use of higher concentrations of PGPR enabled the entrapped water volume to remain constant, whereas a PGPR concentration below the critical micelle concentration (CMC) caused a water flux from the internal to the external phase. The average residence time (ta) of enclosed L-leucine among the PGPR stabilized double emulsions was lowest at the highest PGPR concentration, which indicates the faster release of L-leucine in the presence of an excess of reverse PGPR micelles in the oil phase. The effect of partial replacement of PGPR by native and phosphatidylcholine (PC) depleted lecithin on double emulsion stability and amino acid release was shown in section 4.7. Although a droplet size increase was observed in the PGPR-stabilised double emulsions during storage, the use of 5% of a PGPR-native lecithin (1/1) mixture resulted in a constant droplet size during storage. The used PGPR and PC-depleted lecithin concentration influenced the droplet size of the double emulsions. The lowest droplet size was about 30 µm just after preparation and during storage in double emulsions containing 5% PC-depleted lecithin. This indicates that partial replacement of PGPR can be beneficial in terms of stability of the double emulsion droplet size. Considering the entrapped water volume, the inclusion of PC-depleted lecithin could not facilitate to overcome the instability at too low (i.e. less than 2% in this case) PGPR concentration. In fact, lecithin addition had a negative impact on the etrapped water volume fraction. The average residence time ta, on the other hand, was much lower in PC-depleted lecithin-containing double emulsions as compared to the emulsions with only PGPR. The effect of hydrophilic emulsifier concentration on amino acid release and double emulsion stability was investigated (section 4.8). It was found that the use of a higher Tween 80 concentration facilitated a less pronounced increase in average droplet size during storage. The use of less than 2% Tween 80 concentration seemed to be insufficient to cover the interface between oil and outer aqueous phase. A constant entrapped water volume fraction was obtained during storage regardless of the Tween 80 concentration. Differences in Tween 80 concentration, varying from 0.5 to 2.0%, did not change the release kinetics to a large extent. In section 4.9, the influence of microfluidization (at 0.75 and 1.00 bar of driving compressed air pressure) and rotor stator homogenization treatment (at 17500, 21500 and 24000 rpm of Ultra-turrax) and the presence of xanthan gum were investigated. Considering the particle size distribution, multimodal and monomodal particle size distributions were observed for microfluidized double emulsions and those prepared by rotor stator homogenization treatment, respectively. The inclusion of xanthan gum decreased the size of the oil droplets, which resulted from the decreased viscosity ratio between the oil and the aqueous phase. Also, an increased homogenization intensity induced a decreased droplet size, resulting from the higher shear stress applied to the fluid. The entrapped water volume fraction was about 90% for all double emulsions prepared with rotor stator homogenization treatment and without xanthan gum. As the cream and serum layers of the double emulsions stabilized with xanthan gum were not separated during 2 hours of analytical centrifugation, the reliable estimation of the enclosed water volume fraction was troublesome. The release rate of L-leucine in double emulsions prepared with rotor stator homogenization treatment was proportional with the homogenization level, which can be explained from the smaller droplet size: a faster release rate was observed at higher homogenization intensity as a result of a smaller droplet size. Xanthan gum addition remarkably increased the release rate of L-leucine, which was thought to be due to the smaller droplet size. Preliminary gastrointestinal tests indicated that double emulsion encapsulation provided a gradual release of amino acids in the gastrointestinal environment (section 4.10). The release of amino acids might be governed by diffusion in the gastric environment, whereas the oil digestion can change this mechanism as well as the relase rate. The smaller droplets obtained after intestinal digestion was likely due to the triglycerides hydrolysis which resulted in the disruption of the oil phase and hence release of encapsulated amino acid. In section 4.11, the release of L-phenylalanine was investigated by means of high resolution NMR diffusometry. As the first and last decay profile of water overlapped, it follows that the enclosed water volume fraction remained constant during incubation (at 30 and 50 °C). Moreover, a slower amino acid diffusion coefficient was obtained in the external water phase as compared to the internal water phase (i.e. before emulsification). This might be due to the presence of xanthan gum in the external (but not in the internal) water phase, which restricts the thermal motion of the amino acids, and hence the diffusion behaviour. The diffusion behaviour of L-phenylalanine in double emulsions exhibited a typical bi-exponential decay, which enabled to discriminate between encapsulated (slowly diffusing due to restriction in a spherical confinement) and released (fast diffusing due to the absence of confinements) amino acid. Whereas the main purpose of the experiment was to enable a more detailed investigation of the influence of the incubation temperature, a clear conclusion was hampered by the extensive release before the start of the NMR experiment. This research enables a better insight to understand the influence of molecular properties and double emulsion composition on the release kinetics. From a practical point of view, our results provide guidance in the design of colloidal systems for the encapsulation and controlled release for nutritional applications. In order to extend this study, the double emulsions containing amino acids can be incorporated in the food matrix or drugs.