*3.3. Release of Tamarillo Polypehnols from Cubosomes during Digestion*

In order to evaluate the stability of individual polyphenol compounds during digestion, a total of fourteen compounds were evaluated by LC-MS (Table 1). The CUB was also analysed as a control. The results showed that 11 phenolic compounds and three anthocyanins were released from the microcapsules after the digestion process, demonstrating that these phytochemical compounds were well encapsulated by the cubosomes.

The phenolics presented different behaviours during the simulated digestion (Table 1). Analysis of phenolics released from EXT during digestion showed a significant instability for the major phenolic acids (gallic acid, chlorogenic acid and *p*-coumaric acid), other phenolics (epicatechin, rutin and kaempferol rutinoside) as well as all anthocyanins after oral and gastric phases. For gallic acid, the concentrations in oral phase remained stable and only a significant (*p* < 0.05) increase was observed in the gastric phase (46.49%) when compared to the initial undigested EXT. Then, the concentration of this acid dropped down to 20.4%. Tagliazucchi, Verzelloni, Bertolini and Conte [37] reported the degradation (43%) of pure gallic acid after gastrointestinal digestion, while the total degradation for gallic acid from grape extract and carob pulp extract had been explored by Jara-Palacios et al. [38] and Ydjedd et al. [32], respectively. Meanwhile, caffeic acid showed insignificant changes during the digestion (24.13% at the oral phase, 31.42% after the gastric phase and 21.94% at the end of the intestinal phase). According to Wojtunik-Kulesza et al. [39], the remaining percentage of caffeic acid decreased to 75% and 78% after oral and gastric phases, respectively. Some studies have reported that the gastric phase has increased the bioaccessibility of some phenolic acids, while, during the intestinal phase, their levels could be decreased. This behaviour has been closely related to the stability and structural changes that each type

of polyphenolic acid undergoes [37]. Due to its low molecular weight, gallic acid has been better absorbed in humans compared to other phenolic acids, which makes it highly bioaccessible [36]. For chlorogenic acid, the highest concentration was detected in the gastric phase; then, the concentration of this compound reduced by 63% in the intestinal phase (Table 1). According to Tagliazucchi, Helal, Verzelloni and Conte [40], the degradation of chlorogenic acid during gastro-pancreatic digestion might be due to the oxidation and polymerization to form quinone in an alkaline environment. Significant reductions of free phenolic acids (gallic, chlorogenic, caffeic, *p*-coumaric acids) during in vitro digestion have been reported in previous studies [41–43]. These decreases in phenolic acids could be related to changes in pH and the presence of bile salts in the intestinal phase, which may lead to the formation of precipitates [42], which may explain the reductions observed at the end the intestinal phase of this study.

The concentrations of kaempferol rutinoside in EXT remained stable after the oral and gastric phases but decreased significantly (*p* < 0.05) at the end of the intestinal phase (Table 1). A similar trend for kaempferol rutinoside during in vitro digestion of the Cactus Cladodes plant had been observed [44]. Hydrolysis of glycoside flavonoids starts in the mouth by means of *β*-glycosidase action, but the degree of hydrolysis depends on the types of sugars present in the flavonoid compounds. For example, polyphenol compounds with more hydrophobic properties often interact more strongly with proteins [39]. Degradation of polyphenols with high molecular weights (such as kaempferol rutinoside) may be related to their strong affinities with human salivary proline- and histidine-rich proteins to form non-covalent and covalent associations [39].

All of the anthocyanins, especially delphinidin rutinoside and pelargonidin rutinoside, showed the same releasing behavior during in vitro digestion (Table 1). For these main anthocyanins in tamarillo extract, a significantly (*p* < 0.05) higher proportion of anthocyanins (43 to 76%) was released after the intestinal phase when compared to the undigested samples. The instability of anthocyanins at neutral or slightly basic pH has been observed for polyphenols from grape and chokeberry [37,45]. The instability can be explained by the formation of a colourless chalcone pseudo-base, resulting in the destruction of the anthocyanin chromophore [46]. The current results support these previous findings, suggesting that anthocyanins are stable in the acidic conditions of the gastric phase but are degraded in the alkaline/neutral conditions of the intestinal phase. The reduction of anthocyanins may also be related to the fact that, in aqueous solution in response to changes in pH, anthocyanins undergo structural rearrangements, change colour, may form complexes with proteins in food and digestate and be degraded to phenolic acids [42].

The quantity of individual bioactive compounds from the CUBTAM at the end of each digestive phase varied by compound (Table 1). Catechin, epicatechin, isorhamnetin rutinoside and all anthocyanins (delphinidin rutinoside, cyanidin rutinoside and pelargonidin rutinoside) were released after the gastric phase in acidic medium; gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, rutin, kaempferol and kaempferol rutinoside were released into the neutral medium after oral and intestinal phases. It is worth noting that the percentage of free polyphenols was lower in CUBTAM (encapsulated) than in EXT (non-encapsulated) ones, and remained fairly constant along different in vitro digestion phases. These results were also expected because the initial amount of polyphenols in the encapsulated sample was lower owing to the encapsulated efficiency (over 50%). According to Ydjedd et al. [32], the properties of encapsulating material play a significant role in enhancing the entrapment efficiency and controlled release of the core compounds. They reported a slow release of some phenolics (gallic acid, p-coumaric acid, and kaempferol) from the microcapsules and a period of more than 3 h in the intestinal phase (neutral medium) has been necessary for complete release of these compounds, when the encapsulating material was completely degraded [32].

The present study is the first to report the proportion of cubosome encapsulated polyphenols released after each phase of in vitro digestion, demonstrating the potential of cubosomes to protect bioactive compounds in their matrix. Similarly, reduction of the degradation in cubosome encapsulated bioactive antimicrobial peptide has been reported, showing resistance towards the enzymatic degradation [18]. Cubosomes have a high viscosity which hinders the diffusion of polyphenols into the release medium and slows the entry of water, which sustains the slow release profile [47]. The rate of release controlled by the structure also depends both on the partition coefficient and on the diffusion of the drug through the hydrocarbon tail region [48].

#### *3.4. Physicochemical Properties of Yoghurt Fortified with CUBTAM*

The addition of 5%, 10% and 15% CUBTAM to yoghurt was associated with a small but statistically significant fall in pH and reduced syneresis (Table 2). This can be explained by the use of the freeze-drying treatment to prepare the powder for the cubosome, which would result in an increase in total dry solids, which in turn would increase the water holding capacity, reduce porosity and reduce the syneresis.

**Table 2.** Physicochemical properties of yoghurt fortified with CUBTAM (5, 10 and 15%) in PRE and POS. Control yoghurt contained no CUBTAM.


N/A: not applicable. Data are expressed as Mean ± SD (*n* = 3). Different alphabetic superscripts indicate statistical difference (*p* < 0.05) across each row. CUBTAM: tamarillo polyphenols loaded-cubosomes. POS5, POS10, POS15: addition of 5%, 10%, 15% of CUBTAM post to fermentation process, respectively. PRE5, PRE10 and PRE15: addition of 5%, 10%, 15% of CUBTAM prior to fermentation process, respectively.

Viscosity of yoghurt increased with the increase of the concentration of CUBTAM, showing significant (*p* < 0.05) differences across the yoghurt samples (Table 2). Within the same % CUBTAM fortification, there was no significant (*p* > 0.05) difference in viscosity between PRE and POS. Based on the Oswald–de Waele power law model, yoghurts fortified with CUBTAM made from both fermentation processes can be considered as non-Newtonian fluids with shear-thinning behaviour due to the flow behaviour index (n) below 1. The breakage of bonds between the protein aggregates as a consequence of shear stress led to the pseudoplastic behaviour of the yoghurt samples [49]. The consistency index (K) and flow behaviour index (n) of yoghurts were not significantly influenced by the fermentation process, whereas the increase of encapsulated powder concentration led to the increase of K and decrease of *n* values. The increase of K value might be attributed to the water holding capacity, caused by the addition of powder.

The elastic modulus of all yoghurts was very low, indicating the same relatively weak structure with or without CUBTAM (Table 2).
