Nanoemulsions (NEs) with extremely small droplet size (radius <100 nm) were found to possess characteristics that have many advantages over conventional emulsion systems. These nano-sized droplets were found to contribute to higher stability of the NEs and also found to improve the bioavailability of poorly water-soluble bioactive components. The overall aim of this thesis is to develop oil-in-water (O/W) NEs stabilized by a mixture of sodium caseinate (a dairy protein) and pea protein isolates (a pulse protein). The mixed protein stabilized NEs were utilized to encapsulate a bioactive compound, curcumin, where the goal is to investigate its stability, delivery and bioavailability through in vitro digestion studies.
Various concentrations (2.5 – 10 wt%) of sodium caseinate (SC) were used as the sole emulsifier in the development of 5 wt% O/W NEs and their long-term storage stability for 6 months was investigated. The sodium caseinate stabilized NEs (SCEs) developed in this work displayed an average droplet diameter less than 200 nm, which remained unchanged for an experimental time fame of 6 months. However, all of them displayed rapid creaming, which increased with an increase in protein concentration, in accordance with previous studies. It was postulated that excess unabsorbed protein caused depletion flocculation leading to creaming of oil droplets, which was confirmed using confocal laser scanning microscopy. Calculation of depletion interaction energy showed an increase in attraction with protein concentration and decrease with a reduction in droplet size, making NEs more resistant to flocculation than conventional emulsions.
Next, pea protein isolate (PPI), was utilized to partially replace SC and thereby PPIs efficacy in the formation and long-term stabilization of mixed protein NEs (MPEs) was investigated. Total aqueous phase-protein concentration of 5, 7.5 and 10 wt%, with SC and PPI in a 1:1 ratio, was used. As a control individual PPI-stabilized NEs (PPIE) were also prepared. PPI failed to produce stable flowable NEs displaying excessive droplet and protein aggregation. At higher concentrations of PPI (7.5 and 10 wt%), the emulsion transformed into viscoelastic gels. Interestingly, the mixed SC and PPI-stabilized NE did not display any creaming or aggregation and remained stable throughout the experimental timeframe of 6 months with average droplet diameter <200 nm. Results from interfacial protein composition (surface load) and SDS-PAGE indicated the presence of PPI at the interface along with SC confirming PPI’s ability to take part in droplet formation and stabilization. It was hypothesized that the mutual presence of SC and PPI during high-pressure homogenization led to interactions between the proteins, which was confirmed by FTIR spectroscopy, intrinsic fluorescence and surface hydrophobicity measurements. Interactions between the proteins not only prevented depletion flocculation effect of SC, but also interfered with PPI aggregation thereby preventing both the destabilizing mechanisms seen in individual protein-stabilized NEs. The mixed-protein stabilization could be a novel way to utilize plant proteins in the development of NEs.
In the final part, the efficacy of the MPE for stability, delivery and bioavailability of an encapsulated bioactive compound, curcumin was investigated and compared to the SCE . It was seen that over 54% of encapsulated curcumin was degraded in the MPE, while only ~42% was degraded in the SCE over a period of 8 weeks. In vitro digestion studies indicated that the amount of bioavailable curcumin from SCE was slightly higher (although not statistically significant) compared to that from MPE, which was attributed to a thicker droplet interface in the mixed protein NE (due to presence of globular protein PPI), thereby making the droplet less susceptible towards protein hydrolysis by pepsin in the stomach.
Overall, it was concluded that it is possible to develop mixed protein NEs utilizing PPI and SC, which displayed better stability when compared to the individual protein-stabilized NEs. The presented approach not only utilized pulse protein, PPI, in the development of NEs, but also showed good applicability in terms of encapsulating bioactive ingredients for prospective applications in food and pharmaceutical industries