Nano-encapsulation of olive leaf phenolic compounds through WPC-pectin complexes and evaluating their release rate

In this study, W/O micro-emulsions as primary emulsions and a complex of whey protein concentrate (WPC) and pectin in the external aqueous phase were used to produce W/O/W emulsions. Average droplet size of primary W/O emulsion and multiple emulsions stabilized by WPC or WPC-pectin after one day of production was 6.16, 675.7 and 1443 nm, respectively, which achieved to 22.97, 347.7 and, 1992.4 nm after 20 days storage without any sedimentation. The encapsulation efficiency of phenolic compounds for stabilized W/O/W emulsions with WPC and WPC-pectin were 93.34 and 96.64, respectively, which was decreased to 72.73 and 88.81 at 20th storage day. The lowest release of phenolics observed in multiple emulsions of WPC-pectin. These results suggest that nano-encapsulation of olive leaf extract within inner aqueous phase of W/O/W emulsions was successful, and there could be a high potential for the application of olive leaf extract in fortification of food products. © 2015 Elsevier B.V

Influence of oxidized oils on digestibility of caseins in O/W emulsions

The impact of lipid oxidation on protein modifications in emulsions and the consequences on protein digestibility remains unclear. In this study, this impact is evaluated in casein (6 mg mL(-1)) based emulsions containing oxidized soybean or fish oil (3%) in presence (0.3%) or absence of the emulsifier Tween 20. Emulsions are prepared using oils at three oxidation levels and subsequently the impact on protein digestibility is evaluated after 24 h incubation at 4 degrees C. Remarkably, protein digestibility increases in emulsions containing medium and highly oxidized fish oil: 70 +/- 0.4% and 73 +/- 0.4% of the proteins are digested, respectively, whereas protein digestibility in emulsions containing low oxidized fish oil amounted to 63 +/- 0.4%. Protein digestibility in emulsions containing soybean oil stabilized by Tween 20 is not influenced by the oxidation level of the oil used. A remarkable tendency is observed for the malondialdehyde content of the emulsions depending on the presence of Tween 20. For soybean oil based emulsions, malondialdehyde concentrations are consistently higher in the presence of Tween 20. On the other hand, for the fish oil based emulsions an opposite trend is observed, except at the highest oxidation level evaluated, for which no significant differences can be detected. It is concluded that the composition of the interface in emulsions depends strongly upon the degree of oil oxidation and the presence of other emulsifiers. If the oil is more oxidized, less protein is present in the interface restricting the impact of lipid oxidation products on the proteins and hence their digestibility

Emulsifying properties of hemp proteins: Effect of isolation technique

peer-reviewedHemp protein was isolated from hemp seed meal using two different isolation procedures: alkali extraction/isoelectric precipitation (HPI) and micellization (HMI). The ability of these proteins to form and stabilize 10% (w/w) sunflower oil-in-water emulsions (at pH = 3.0) was studied at three different concentrations, 0.25, 0.75 and 1.5% (w/w), by monitoring emulsion droplet size distribution, microstructural and morphological properties, rheological behaviour and stability against flocculation, coalescence and creaming. In addition, hemp proteins were analysed for water solubility, denaturation degree and surface/interfacial activity. HMI protein, which was found to be less denatured after isolation, exhibited higher solubility and slightly higher surface/interfacial activity than HPI protein. HMI emulsions possessed a smaller volume mean droplet diameter (d4,3 = 1.92–3.42 μm in 2% SDS) than HPI emulsions (d4,3 = 2.25–15.77 μm in 2% SDS). While HMI stabilized emulsions were characterized with individual droplets covered by protein film, both confocal laser scanning microscopy and flocculation indices indicated occurrence of bridging flocculation in HPI stabilized emulsions. Protein aggregation, which induced flocculation of the droplets, contributed to higher apparent viscosity of HPI stabilized emulsions compared to HMI stabilized emulsions. Interestingly, emulsions stabilized with 1.5% (w/w) HPI exhibited much better creaming and coalescence stability than other emulsions due to the formation of a weak transient network of floccules and higher continuous phase viscosity which both suppressed the movement of the droplets

pH-triggered phase inversion and separation of hydrophobised bacterial cellulose stabilised Pickering emulsions

The pH-triggered transitional phase behaviour of Pickering emulsions stabilised by hydrophobised bacterial cellulose (BC) is reported in this work. Neat BC was esterified with acetic (C2–), hexanoic (C6–) and dodecanoic (C12–) acids, respectively. We observed that C6– and C12–BC stabilised emulsions exhibited a pH-triggered reversible transitional phase separation. Water-in-toluene emulsions containing of 60 vol.% dispersed phase stabilised by C6– and C12–BC were produced at pH 5. Lowering the pH of the aqueous phase to 1 did not affect the emulsion type. Increasing the pH to 14, however, caused the emulsions to phase separate. This phase separation was caused by electrostatic repulsion between modified BC due to dissociable acidic surface groups at high pH, which lowered the surface coverage of the water droplets by modified BC. When the pH was re-adjusted to 1 again, w/o emulsions re-formed for C6– and C12–BC stabilised emulsions. C2–BC stabilised emulsions, on the other hand, underwent an irreversible pH-triggered transitional phase separation and inversion. This difference in phase behaviour between C2–BC and C6–/C12–BC was attributed to the hydrolysis of the ester bonds of C2–BC at high pH. This hypothesis is in good agreement with the measured degree of surface substitution (DSS) of modified BC after the pH-triggered experiments. The DSS of C2–BC decreased by 20% whilst the DSS remained constant for C6– and C12–BC

Influence of Mucilage Viscosity On The Globule Structure And Stability Of Certain Starch Emulsions

A study was carried out to determine the influence of mucilage viscosity on the globule structure (i.e. size and number) of certain starch emulsions. The starches investigated were cassava, potato and maize. The emulsions were prepared by mixing the starch mucilage of a predetermined concentration 4%w/v with arachis oil in the ratio 50:50, using a silverson mixer fitted with a dispersator head. The emulsions were stored at room temperature (28±20C) for 7 days. Changes in globule size were monitored by photomicroscopy. Viscosities of the mucilage and those of resulting emulsions were determined using a capillary flow method. The viscosities of the emulsions expressed as time of flow (seconds), were 680 (cassava starch), 369 (potato starch) and 270 (Maize starch), and for the mucilage 510 (cassava), 336 (potato) and 248 (maize). The corresponding mean globule sizes of the fresh emulsions were (µm) 28±6, 42±6 and 45±5 respectively. The increase in globule size during storage (measure of globule coalescence rate) was 1.8±0.2µm day -1 (cassava), 3.5±0.2µm day -1 (potato) and 4.6±0.3µm day -1 (maize). Thus, a higher viscosity of the dispersion medium is associated with the production of finer and more stable emulsions

Oil-in-oil emulsions stabilised solely by solid particles

A brief review of the stabilisation of emulsions of two immiscible oils is given. We then describe the use of fumed silica particles coated with either hydrocarbon or fluorocarbon groups in acting as sole stabilisers of emulsions of various vegetable oils with linear silicone oils (PDMS) of different viscosity. Transitional phase inversion of emulsions, containing equal volumes of the two oils, from silicone-invegetable (S/V) to vegetable-in-silicone (V/S) occurs upon increasing the hydrophobicity of the particles. Close to inversion, emulsions are stable to coalescence and gravity-induced separation for at least one year. Increasing the viscosity of the silicone oil enables stable S/V emulsions to be prepared even with relatively hydrophilic particles. Predictions of emulsion type from calculated contact angles of a silica particle at the oil–oil interface are in agreement with experiment provided a small polar contribution to the surface energy of the oils is included. We also show that stable multiple emulsions of V/S/V can be prepared in a two-step procedure using two particle types of different hydrophobicity. At fixed particle concentration, catastrophic phase inversion of emulsions from V/S to S/V can be effected by increasing the volume fraction of vegetable oil. Finally, in the case of sunflower oil + 20 cS PDMS, the study is extended to particles other than silica which differ in chemical type, particle size and particle shape. Consistent with the above findings, we find that only sufficiently hydrophobic particles (clay, zinc oxide, silicone, calcium carbonate) can act as efficient V/S emulsion stabilisers

Stability of water-in-oil-in-water emulsions formed by membrane emulsification : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology at Massey University, Palmerston North, New Zealand

The main objectives of this study were to determine i. The effectiveness of encapsulating whey protein concentrates (WPC) within water-in-oil-in-water multiple emulsions produced by membrane emulsification. ii. The effect of the primary and secondary emulsification conditions and membrane operating parameters on the multiple emulsion properties; of particular concern were the yield and physical stability of the emulsions. The multiple emulsions were prepared by a two-stage emulsification process. The emulsification conditions were varied widely to determine the optimum conditions for the production of multiple emulsions. Ultra-Turrax, ultrasound and valve homogenisation were tried for the preparation of the primary emulsion; an Ultra-Turrax and Shirazu porous glass (SPG) membrane emulsification were used for secondary emulsification. The standard primary emulsions (water-in-oil) were prepared with 10% of the WPC and 6.4% glucose (as a marker) in the water phase with a water-in-oil volume fraction of 0.25. The oil phase (soybean oil) consisted of 10% hydrophobic emulsifier (Polyglycerol polyricinoleate [PGPR] or Span 80). Typically pre-emulsification was carried out in an Ultra-Turrax at 20500 rpm and the primary emulsification was done in a homogeniser at a pressure of 500/100 bars in the two stages; secondary emulsions were prepared with an SPG membrane of pore size 2μm or 3.8µm with the dispersed phase (water-in-oil) being pushed through the pores of the membrane at a specific transmembrane pressure (125-150 kPa) and dispersed phase flux into a continuous phase (water with a hydrophilic emulsifier concentration, usually Tween 80, of 1%) flowing through the inside of the membrane at a particular velocity (standard of 1 m/s). The yield of the multiple emulsions formed was estimated by measuring glucose release using an Advantage Glucose meter. Unlike Span 80, PGPR was able to form stable o/w emulsions and hence the initial yield of the multiple emulsions varied from 80% at 2.5% to 100% at 10% PGPR. The higher the concentration of water in the inner phase the lower the yield of the multiple emulsions and the higher the droplet size of the primary emulsions. A valve homogeniser gave the best results for primary emulsification. Of the 3 homogenisation pressures (250 bar, 500 bar, 1000 bar) tried, the w/o emulsion produced with 500 bar and 10% PGPR was taken as the standard as this was found to be stable for 6 months without physical damage. A 30% maximum loading of the WPC in the inner water phase was also determined. A further increase may destabilise the process by causing blockage to the membrane pores. The yield as well as the droplet size of the multiple emulsions was found to increase as the membrane pore-size was increased from 1.4 µm to 3.8 µm. Transmembrane pressure and continuous phase velocity did not have much influence on the yield of the multiple emulsions. However an increase in continuous phase velocity increased the opacity of the serum layer formed indicating that an increased amount of smaller droplets were formed. The dispersed phase flux was increased by increases in any of the transmembrane pressure, PGPR concentration and membrane pore-size. Hydrophilic emulsifiers (whey protein isolate, soy protein isolate and sodium caseinate) did not influence the yield; however the Tween 80 stabilised multiple emulsions showed a smaller droplet size. An increase in temperature from 20 - 50°C resulted in a lower yield as well as a higher droplet size. The osmotic gradient set up by glucose and WPC in the inner phase of the emulsion resulting in an influx of water from the outer phase causing bulging of the droplets. Sorbitol added at 1.7% in the outer phase gave a high initial yield (100%) as well as a low droplet size (2-3 µm). The cream layer formed as a result of storage was found to decrease with increase in sorbitol concentration (to 5.9%) due to the lower size ot the droplets formed. The key issues identified were to find an alternative to PGPR with lower Accepted Daily Intake (ADI) value without compromising the emulsification properties and to standardise ways to analyse droplet size of w/o emulsions. Overall the study proved that functional ingredients can be encapsulated using stable w/o/w multiple emulsions prepared using SPG membranes under standardised conditions and hence appears to offer promise for manufacture of commercial products

Investigations on the emulsifying properties of egg white protein : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Auckland, New Zealand

Figures 2.2 & 2.3 were removed for copyright reasons, but Figure 2.1 remains for ease of access.Egg white proteins (EWP) have excellent foaming and gelling functional properties. However, their emulsifying properties are considered poor when compared to soy proteins or milk proteins. Some studies have attributed the poor emulsifying properties to the hydrophobic amino acid groups buried deeply in the interior of the protein conformational structure which is crucial for emulsification. Several methods, such as heat treatment, acid/acid-heat treatment, Maillard reaction, phosphorylation and enzymatic hydrolysis, have been used by some researchers to improve the emulsifying properties of EWP. Preliminary experiments carried out in this study showed that oil-in-water (O/W) emulsions prepared with egg white liquid (EWL) generated lots of visible large aggregates, which no other study has reported. Therefore, it was important to investigate the factors responsible for the formation of these aggregates. Investigations into improving EWP's emulsifying properties could offer opportunities in developing unique and well-defined egg white-based emulsions. The objective of this research project was to produce egg white emulsions with little or no aggregates. This thesis comprises three main parts. The first part focused on the effects of pH and heat treatment on protein aggregation and partial denaturation of proteins in EWL. The second part investigated the effects of heat treatment, oil concentration and protein concentration on the reduction of large visible aggregates in emulsions prepared with EWL. The third part studied the effect of enzymatic hydrolysis on the degree of hydrolysis and emulsifying properties of EWP hydrolysates. The emulsifying properties of original EWP and EWP hydrolysates were characterised in terms of size and zeta (ζ)-potential of emulsion droplets and emulsion stability (e.g. turbidity, microscopic examination and phase separation). Firstly, an experimental study was carried out to evaluate the effect of pH on protein aggregation and precipitation in EWL containing different protein concentrations (0.5, 1, 2, 3, 4, 5 and 10% w/w). It was found that at all the protein concentrations used and at pH less than around 5, ζ-potential values were all positive but decreased as pH increased from 2 to 5. At pH 5, ζ-potential was close to zero (this is the pI of most egg white proteins), while, at pH levels above 5, ζ-potential became negative and increased as pH increased from pH 5 to 11. The spectral absorbance (turbidity) of emulsion samples was also measured at 600 nm which revealed that for all protein concentrations, turbidity was observed to be higher at acidic pH of 3, 4 and 5, indicating the aggregation of EWP. At alkaline conditions of pH 7, 8, 9 and 10 the EWL solutions remained to be transparent. The effect of heat treatment and holding time on the denaturation of EWP in EWL was also studied at different temperatures (57-62oC) and heating times (0-19 minutes). Higher turbidity due to protein aggregation was observed as temperature increased from 57 to 62oC and the heating time increased from 5 to 19 minutes. It is therefore concluded that EWL can be safely pasteurized with little or no denaturation or aggregation at around 57-58oC for less than 5 minutes. At 60oC, it was observed that EWL began to thicken and after 5 minutes coagulation and gelation occurred rapidly. Studies were also carried out to determine the cause of visible large aggregates formed in emulsions prepared with EWL using various factors, such as heat treatment, oil concentration and protein concentration. It was found that heat treatment (60oC for 30 minutes) of 1% (w/w) EWP solution prior to homogenisation had no effect on reduction of aggregates in emulsions containing 5, 10, 15 and 20% (w/w). However, the formation of aggregates was reduced significantly as oil concentration was reduced to 5%. Therefore, the effect of lower oil concentrations (1, 3, 5, 6, 7 and 10% w/w) on the formation of aggregates in emulsions prepared with 1% or 3% EWP concentrations was also investigated. Little or no visible aggregates were formed when emulsions were prepared with 1% EWP and ≤ 5% oil or 3% EWP and 1% oil. Therefore, the results indicated that both protein and oil concentrations played a significant role in the formation of visible aggregates in emulsions prepared with EWP as an emulsifier. The effect of EWP concentrations (0.1, 0.3, 0.5, 0.8, 1 and 2% w/w) on the formation and properties of 5% oil emulsions at ~pH 8 was then investigated. It was discovered that little or no aggregates were produced in emulsions when prepared at 0.1-1% EWP while large aggregates were formed at 2% EWP concentration. The size of emulsion droplets was observed to increase significantly from 242.1 to 703.7 nm as protein concentration increased from 0.1 to 2%. ζ-potential was however not significantly affected by protein concentration and ranged from -35.3 to -39.2 mV. The emulsions prepared were also heat treated at 60-90oC for 30 minutes. No sign of instability with a significant change in the size of emulsions due to heat treatment was observed from all emulsion samples prepared at different EWP concentrations (0.1 - 2%). However, phase separation of the emulsions was observed upon freezing at -20oC and thawing at 4 and 20oC, respectively, at all protein concentrations used. Also, the stability of emulsions was affected by the addition of salts, such as CaCl2 (5-100 mM) and NaCl (50-600 mM), with an increase in droplet size and phase separation. However, the emulsions were relatively more stable to salt-induced flocculation, especially against NaCl, at higher protein concentration (1-2%) than lower protein concentrations (0.1-0.8%). Lastly, the effect of pH 2-10 was also determined from the emulsions prepared at 1% EWP and 5% oil. Extensive droplet aggregation was observed at pH 4 and 5 as expected which is around the pI of most egg white proteins. On the other hand, it was not observed at extremely acidic pH 2.0 and alkaline pH 9-10 and in the control emulsion prepared at pH 8.3. In another part of the study, the effects of enzyme type (bromelain, ficin and papain), enzyme concentration (0.3, 0.5, 1, 2 and 4% w/w; enzyme/substrate (E/S) ratio) and hydrolysis time (0, 30, 60 and 120 minutes) on the degree of hydrolysis (DH) of EWP were investigated by diluting EWL containing 10% EWP to different EWP concentrations followed by adding enzymes into the EWL solutions. DH was observed to increase significantly (p < 0.05) with increasing enzyme concentration and hydrolysis time. A significant difference (p < 0.05) among the different types of enzymes was only observed from the samples with 4% E/S ratio at 120 minutes of hydrolysis time. Papain yielded the highest DH of 7.69% while bromelain and ficin yielded similar DH levels of 5.03% and 4.99%, respectively. The results of SDS-PAGE revealed that the protein bands corresponding to ovalbumin and ovotransferrin disappeared due to their enzymatic hydrolysis into smaller peptides but it was not significantly different between the samples treated with different E/S ratios and hydrolysis reaction times. The effects of enzyme concentration, DH and hydrolysis time on the emulsifying properties of hydrolysed EWP prepared with bromelain and ficin were investigated. Surprisingly, enzymatic hydrolysis significantly improved the appearance of emulsions prepared with EWL containing hydrolysed EWP by producing an emulsion free of aggregates compared to the control emulsions prepared from original EWP which had lots of large aggregates in it. For example, emulsions containing 10% oil and various EWP concentrations (1, 5 and 10%) prepared with hydrolysed EWP (4% E/S, DH 5.16%) yielded smaller droplet size (0.66-0.98 μm) than those of original EWP emulsions (1.22-39.35 μm). However, phase separation occurred immediately after preparation at all protein concentrations (1, 5 and 10%) used while phase separation occurred in only emulsions stabilised with 5 and 10% original EWP. When the emulsions were heat treated at 60-90oC for 0-30 minutes, gelation occurred in the emulsions prepared with 5 and 10% EWP concentrations while the emulsions prepared with 1% EWP had no gelation but had aggregation and phase separation after heat treatment. Emulsions prepared with 1% E/S ficin (DH 4.03% and 4.96%, respectively, after 2 and 4 hours of hydrolysis time) yielded smaller droplets size (0.75-0.87 μm) than droplet size (6.40-7.37 μm) of emulsions prepared with 1% E/S bromelain (DH 4.10% and 4.87% after 2 and 4 hours of hydrolysis time). Droplet size decreased as hydrolysis time increased from 2 to 4 hours for both ficin and bromelain hydrolysates with phase separation occurring the following day after the preparation of emulsions. Thus, DH and enzyme type had some influence on the emulsifying properties of EWP hydrolysates. In conclusion, this study demonstrated that egg white emulsions can be prepared with little or no aggregates using low oil (≤5%) and low protein (1%) concentrations and by enzymatic hydrolysis of EWP. Emulsions containing 5% oil prepared with a relatively higher protein concentration (1-2%) were more stable to destabilization to ionic strength (salt concentration), especially against NaCl. These could lead to production of egg white protein based-emulsions with distinct appearance and characteristics