*2.10. Total Phenolic Content (TPC) and Antioxidant Activity*

The TPC of extracts and digests at each stage of digestion was determined using a Folin–Ciocalteu assay as described in our previous study [13]. Two different methods were used to determine the antioxidant activity, namely cupric ion reducing antioxidant capacity (CUPRAC) and ferric-reducing antioxidant power (FRAP) assays, which were mentioned in our previous study [13]. Results of TPC and antioxidant activity are presented as mg gallic acid equivalent per 100 g of tamarillo or yoghurt (mg GAE/100 g) and μmol Trolox equivalent antioxidant capacity per 100 g of tamarillo or yoghurt (μmol TEAC/100 g), respectively.

#### *2.11. Statistical Analysis*

Measurements of all the analytes were undertaken in triplicate, and the results are presented as mean ± standard deviation (SD). For comparison among different samples, one-way analysis of variance (ANOVA) was applied using SPSS 25.0 (IBM Corp., Armonk, NY, USA). Fisher's (LSD) multiple comparison tests were used to determine the magnitude of differences between means. A *p*-value of <0.05 was considered statistically significant.

#### **3. Results and Discussion**

#### *3.1. Characterization of Cubosomal Suspensions Containing Tamarillo Extract (CUBTAM)*

Adapting the temperature–composition phase diagram of a monoolein/water system [27], pure monoolein cubosomes (CUB) and tamarillo polyphenols loaded-cubosomes (CUBTAM) were prepared in a top–down approach. This method allows the formation of reproducible, stable cubosomes without adding solvents. Therefore, it is unnecessary to reinvestigate phase behaviour, and there are no solvent concerns for cellular toxicity [2]. The concentration of the surfactant pluronic F127 was chosen to be 2 wt%, which yields stable cubosome dispersions [28]. The CUB dispersion appeared homogenously milky white and CUBTAM appeared semi-opaque pink (picture not shown).

Figure 1 shows the PLM and SEM photos of CUB and CUBTAM while their particle size distribution (PSD) is summarized in Figure S1 and Table S1. The addition of tamarillo extract did not significantly affect the morphology of cubosome particles. The initial cubic periodicity can be clearly visualized for both samples using PLM and SEM. Because tamarillo extracts are mainly water-soluble compounds (phenolics and anthocyanins), they should be dispersed in the water channel of the cubosome and should minimally affect the structure of the nanoparticles [19].

**Figure 1.** (**A**) PLM and (**B**) SEM micrographs of CUB (**left**) and CUBTAM (**right**).

For particle size distribution, CUBTAM had a unimodal curve and its polydispersity index (PDI) was quite small (below 0.3), as shown in Figure S1. The mean hydrodynamic diameter of liquid crystal particles increased significantly (from 270 to 327 nm) with the addition of the tamarillo extract. In general, this parameter depends on several factors such as the concentration of amphiphile (lipid and polymer), the presence of charged lipids, the ionic strength and the interactions between groups [23]. For CUBTAM, the addition of hydrophilic groups contributed to increasing electrostatic interaction as well as the coalescence between colloidal particles resulting in a bigger average particle size. However, most of the cubic particles in CUBTAM were still limited to a sub-micron range (100–1000 nm). According to Danaei et al. [29] the small particle size and the narrow size distribution (small PDI) create a large surface area that benefits cellular uptake.

Entrapment efficiency (EE%) of bioactive compounds from CUBTAM ranged from 19.8 (catechin) to 87.7% kaempferol rutinoside (Figure 2). Twelve of the fourteen tamarillo bioactive compounds had an EE of more than 50%. In addition, it is noteworthy that we show high EE% (>69%) for the major polyphenols in tamarillo (chlorogenic acid, kaempferol rutinoside, delphinidin rutinoside, cyanidin rutinoside and pelargonidin rutinoside). The high EE in the CUBTAM could be attributed to the fact that polyphenols in tamarillo extract are water-soluble compounds which embed in the water channels. The EE difference between polyphenols encapsulated by cubosome might also depend on the number of -OH groups in molecular structure. For example, the hydroxycinnamic acids chlorogenic acid, caffeic acid and ferulic acid (with >2 -OH groups) showed higher EE than p-coumaric acid (which has only 1 -OH group). More -OH groups will more easily attach in the aqueous channel of cubosome particles. Furthermore, different polyphenol classes showed different EE. For instance, the hydroxycinnamic acids (chlorogenic acid, caffeic acid and ferulic acid), hydroxybenzoic acid (gallic acid), flavonol glycosides (rutin, kaempferol rutinoside, isorhamnetin rutinoside) and anthocyanins (delphinidin rutinoside, cyanidin rutinosid, pelargonidin rutinoside) showed higher EE than flavanols (catechin, epicatechin). According to Patil et al. [23], the EE is dependent on particle size rather than the amount of poloxamer (pluronic F127) used to stabilise the cubosome. The larger the nanoparticles, the higher entrapment efficiency for the polyphenols. This is because surface area to volume ratio of large particles is less than that of smaller particles and exposure to water of active compounds also decreased. Thus, the active compound loss due to diffusion also decreased in larger particles.

**Figure 2.** Entrapment efficiency of polyphenols from tamarillo extract using lyotropic liquid crystalline nanoparticles. Data are presented as mean and error bar (standard deviation) (*n* = 3).
