In this study, the effect of three self-assembly sequences on the structural properties, three-dimensional (3D) printing characteristics, storage stability and freeze-thaw stability of high internal phase Pickering emulsions (HIPPEs) stabilized by ternary aggregates of lactoferrin, epigallocatechin-3-gallate (EGCG) and low methoxylated pectin was evaluated. The results showed that self-assembly sequence directly affected the ability of ternary aggregates to stabilize HIPPEs. The particle sizes of the ternary aggregates were between 1.7 and 2.9 μm, and they showed different network structures and surface wettabilities (between 55.62° and 68.15°). Optical microrheology and texture analysis showed that the decreasing order of the viscoelasticity, hardness, adhesiveness and chewiness of HIPPEs was lactoferrin-EGCG-low methoxylated pectin > lactoferrin-low methoxylated pectin-EGCG > low methoxylated pectin-EGCG-lactoferrin. The HIPPE stabilized by lactoferrin-EGCG-low methoxylated pectin was solid-like and more suitable for 3D printing. The microstructure showed that these HIPPEs were oil-in-water Pickering emulsions. It is particularly noteworthy that the HIPPE stabilized by lactoferrin-EGCG-low methoxylated pectin showed excellent long-term storage stability and freeze-thaw stability, which is of great significance for its potential application in cold chain logistics. In conclusion, when using ternary complexes to stabilize HIPPEs, self-assembly sequence is an important factor that cannot be ignored.

In this study, self-assembled quaternary complexes of lactoferrin (LF), epigallocatechin-3-gallate (EGCG), high methoxylated pectin (HMP) and β-cyclodextrin (β-CD) were constructed and characterized, and successfully utilized for stabilizing high internal phase Pickering emulsions (HIPPEs). In comparison with ternary complexes, the quaternary complexes had lower interfacial tension (reduced by about 3 mN/m) and more appropriate wettability (contact angle close to 90°), and were more effective in stabilizing HIPPEs (smaller droplet size and enhanced homogeneity). The dense cross-linking network of the quaternary complexes endowed HIPPEs with higher viscoelasticity (increasing elasticity and viscosity by up to 1.9 and 5.3 times, respectively), textural properties, 3D printing characteristics, and refrigeration stability. β-CD significantly improved the antioxidant activity of HIPPEs (up to 2.1 times) by interfering with the combination of EGCG with LF or HMP. In addition, the self-assembly sequence could flexibly regulate the structures and emulsifying properties of the quaternary complexes, with the LF-β-CD-EGCG-HMP complex having the most pronounced effect. In conclusion, this study provides a theoretical basis for the construction of protein-polyphenol-polysaccharide-polysaccharide complexes and their applications in HIPPEs.