*3.2. TPC and Antioxidant Activities of Encapsulated and Non-Encapsulated Extracts during In Vitro Digestion*

The impact of digestion on TPC and antioxidant activities of EXT and CUBTAM is shown in Figure 3. The TPC and antioxidant activities decreased significantly after digestion for both non-encapsulated and encapsulated in comparison to the undigested samples.

**Figure 3.** Changes in the total phenolic content and antioxidant activities of tamarillo extract (**A**) and tamarillo polyphenol loaded-cubosomes (**B**) before and after in vitro digestion. The units of TPC (-), CUPRAC (-) and FRAP (-) were mg GAE/100 g tamarillo, μmol TEAC/100 g tamarillo and μmol TEAC/100 g tamarillo, respectively. Data are presented as mean and error bar (standard deviation) (*n* = 3). Different alphabets indicate statistical difference (*p* < 0.05) for each assay.

For both EXT and CUBTAM, there were significant differences (*p* < 0.05) between the amounts of TPC in the supernatant after each stage of digestion. Gastric digests recorded the highest TPC for EXT, while, in the oral and intestinal phases, no significant differences were observed (Figure 3A). Low values of TPC in the supernatant of the oral digest (after 2 min of digestion) are related to the short time for diffusion and low solubility of polyphenols. The loss of polyphenols during digestion could be explained by physicochemical transformations, such as oxidation or the presence of yoghurt molecules (fats and proteins) in the digestion mixture. Furthermore, the decrease of bioactive content could arise from precipitation of several compounds with proteins or enzymes in the digest [30].

However, for the CUBTAM, the release of polyphenols increased during the digestion and was greater in the intestinal phase than the gastric phase (Figure 3B), which was not seen for the non-encapsulated extract, demonstrating a protective effect of the cubosome encapsulation technique against digestive enzymes and pH changes during gastric digestion. Results obtained from LC-MS (Table 1) further support the protective effect of the cubosomes on polyphenols. Similar findings have been reported on release properties of encapsulated blueberry extract [31] and carob pulp extract [32] during simulated gastrointestinal digestion. Both studies showed that TPC in the supernatant increased throughout gastric to intestinal digestion. The materials used for lipid bilayer and stabilisation of cubosomes determine susceptibility of polyphenols to digestive enzymes as well as pH at each stage [33]. The reduction in TPC of tamarillo extract during in vitro digestion might be associated to sensitivity of phenolic compounds to higher pH (>6), since, at that pH, monomers obtained by hydrolysis from larger molecules might be less stable [34]. The increase in the TPC of CUBTAM could be related to the release of complexed bioactive compounds as a result of the digestive process [35].

**Table 1.** Phenolics and anthocyanins in tamarillo extract and tamarillo polyphenol loaded-cubosome released during three stages of in vitro digestion.


Results are expressed as % with respect to the initial concentration. Data are expressed as Mean ± SD (*n* = 3). Different letters of the alphabet superscripts indicate statistical difference (*p* < 0.05) across each row.

The antioxidant activity of tamarillo fruit phenolic extracts is mainly linked to their anthocyanins, chlorogenic acid and kaempferol rutinoside compounds. However, due to the chemical transformations from different mechanisms, the antioxidant properties of these compounds might change during digestion. Thus, the influence of digestion on the antioxidant capacity of tamarillo pulp extracts in non-encapsulation and encapsulation form was assessed by using CUPRAC and FRAP assays (Figure 3).

All activities tested significantly (*p* < 0.05) decreased after digestion in comparison to the raw material, which coincide with the decrease in bioactive compounds, mainly polyphenols, after digestion (Table 1). There were substantial and significant differences in CUPRAC values between the non-encapsulated and encapsulated extracts (*p* < 0.05) throughout the process of in vitro digestion. Tamarillo extract had the highest CUPRAC value in the gastric phase, whereas, for CUBTAM, the highest supernatant activity was noted in the intestinal medium. EXT and CUBTAM presented significant differences (*p* < 0.05) in FRAP values during the digestion process. The highest FRAP value was observed in the oral phase for non-encapsulated extract, whereas, for encapsulated samples, this activity increased with the progress of digestion with the highest FRAP activity of the supernatant was recorded at the end of the intestinal phase. In other studies, an increase in the FRAP with digestion was most pronounced at the intestinal phase for both encapsulated blueberry extract [31] and carob pulp extract [32].

The difference in FRAP and CUPRAC activities, in the oral and intestinal phases, respectively, may not be due to the content of phenolics and anthocyanins, but rather to the diversity and characteristics of the polyphenols present. However, the highest activities (FRAP and CUPRAC) after the gastric phase might be due to the higher release of phenolics and anthocyanins content and the quenching and reducing properties of the acidic medium of the sample. The effect of the pH could also be different among various polyphenols. At neutral pH, some polyphenols have exhibited pro-oxidant activities, whereas, at lower pH, others have demonstrated antioxidant activities [32]. Furthermore, the difference might be related to in vitro digestion conditions used and/or change of polyphenol availability related to the release of matrix associated compounds [36]. In fact, free polyphenols have shown higher antioxidant activity than iron-phenol chelates. Together with the enzymatic action, the pH influence within the gastrointestinal digestion and the presence of compounds that were not analysed in this study (e.g., peptides or complex polyphenols) enhance antioxidant activity [35]. The increase in antioxidant power of the supernatant between the acidic gastric phase and alkaline intestinal phase environments, as seen in this study, can be partially explained by the deprotonation of the hydroxyl groups on the aromatic rings of the polyphenols [36].

Under the intestinal conditions, the decrease in antioxidant activity (CUPRAC and FRAP) for the non-encapsulated extract would be related to the lower TPC alongside transformation of some polyphenols into conformations related to the neutral pH (Figure 3A). Meanwhile, the highest antioxidant activities for CUBTAM supernatant, in the intestinal phase (Figure 3B), could be explained by their release from the microcapsules as they are degraded in the neutral pH. The weak activities recorded in oral and gastric phases of digestion might be due to a small amount of polyphenol release from the microcapsule surface and/or via the penetration of salivary and gastric fluids into the microcapsules through their surface pores.
