*3.5. Total Phenol Content, Antioxidant Activity and Release of Polyphenol Compounds in Yoghurt Fortified with CUBTAM during Digestion*

In a yoghurt matrix, catalase and super oxidase enzymes, casein as well as lactic acid bacteria which have antioxidant properties are present [50]. Without digestion, as expected, the addition of CUBTAM led to a dose-dependent increase in TPC and total antioxidant capacity; i.e., as expected, a higher level of fortification led to a higher TPC as well as antioxidant activity (*p* < 0.05). Furthermore, fortification in PRE resulted in higher TPC and antioxidant activity than in POS at the same concentration (Figure 4A).

**Figure 4.** Total phenolic content (TPC) and antioxidant activity (CUPRAC and FRAP assay) of yoghurts fortified with cubosome containing tamarillo extract before digestion (**A**), after oral (**B**), gastric (**C**) and intestinal (**D**) phases of in vitro digestion. The units of TPC (-), CUPRAC (-) and FRAP (-) were mg GAE/100 g yoghurt, μmol TEAC/100 g yoghurt and μmol TEAC/100 g yoghurt, respectively. Data are presented as mean and error bar (standard deviation) (*n* = 3). Different alphabets indicate statistical difference (*p* < 0.05) for each assay.

After the oral phase, TPC and total antioxidant capacity determined using CUPRAC and FRAP assays in both POS and PRE samples at all fortified concentrations reduced by 7.6–8.9%, 12.9–15.5% and 7.8–9.4%, respectively, compared to undigested samples (Figure 4B). After the gastric phase, significant increases in TPC and total antioxidant capacity were obtained compared to those of the oral phase (up to 3-, 2- and 3-fold, respectively), (*p* < 0.05) (Figure 4C). After simulated intestinal digestion, further significant increases in TPC (64–71%) were observed (*p* < 0.05). Furthermore, total antioxidant activity resulted in an additional 39–50% and 63–70% increase for CUPRAC and FRAP, respectively (Figure 4D). These results were in line with the measures of TPC and antioxidant activity of encapsulated tamarillo extracts (CUBTAM) (Figure 3) as well as polyphenol concentration determined by LC-MS (Table 1), in which the TPC and antioxidant activity increased greatly during the gastric and intestinal phases of in vitro digestion. Previous research showed that the antioxidant activity of the yoghurt samples containing encapsulated phenolics was increased due to the controlled release of the phenolic components from the encapsulation network [50]. Some researchers have considered the Folin–Ciocalteau assay as an antioxidant capacity test since this assay not only measures phenolic compounds but also the total reducing capacity of a sample and hence [42]. This may explain the difference in polyphenols content measured by LC-MS and by the colorimetric tests. Considering the pH conditions of the total antioxidant capacity assays performed in this study, the CUPRAC assay (pH 7.0) could be more appropriate for evaluating total antioxidant activity after the oral and intestinal phases, while the FRAP assay (pH 3.6) could be more suitable to assess total antioxidant activity after the gastric phase.

The data in Table 3 are described as a percentage of each digestion phase in the supernatant compared to the content of the undigested sample. The TPC and antioxidant activities measured by CUPRAC and FRAP between CUBTAM and yoghurt fortified with CUBTAM were relatively similar at each phase of the digestion simulation (Table 3), demonstrating that yoghurt was a suitable carrier of cubosome without significant interference. For the oral phase, the TPC released and antioxidant activities in CUBTAM fortified yoghurt were higher than that for the CUBTAM itself by 1.5–1.7, 1.2–1.5 and 1.1–1.4 times, respectively. For the gastric phase, the amount of TPC released for CUBTAM was slightly higher than the fortified yoghurts, whereas the opposite trend was observed for the antioxidant activities. For the intestinal phase, the TPC and FRAP of CUBTAM were lower than that for CUBTAM fortified yoghurts, whereas, for the CUPRAC, the CUBTAM showed a similar value to PRE5, which were both much higher compared to the rest of the samples. Overall, cubosomes containing tamarillo extract showed effective protection for polyphenols in the oral and gastric phases.

The differences in the LC-MS profiles between PRE and POS were significant (*p* < 0.05). Major polyphenols in tamarillo were also detected in CUBTAM fortified yoghurts (both from PRE and POS) before digestion (Table S2). Thus, the yoghurt matrix as well as encapsulation had helped to retain these individual polyphenols during processing. Encapsulation of bioactive compounds promoted lower loss of polyphenols under refrigerated conditions [51]. At the same concentration of CUBTAM yoghurt, addition of tamarillo polyphenol loaded-cubosomes prior to fermentation was associated with a higher concentration of major polyphenols than addition post fermentation. The concentrations of chlorogenic acid, kaempferol rutinoside and delphinidin rutinoside (accounted for over 65% of the total polyphenol content in yoghurts) were higher for pre-fermentation versus the post fermentation approach (Table S2). Delphinidin rutinoside was the dominant anthocyanin in fortified yoghurts that was in agreement with the main anthocyanin in Laird's Large tamarillo pulp reported in our previous study [13]. The results showed that the fermentation process appeared to have little impact on anthocyanins present in both yoghurts, which might be due to the encapsulation of anthocyanins.

The yields of polyphenols were associated with the extractability of polyphenols from the original tamarillo extract. According to Sun-Waterhouse, Zhou and Wadhwa [52], during fermentation, the yoghurt starter cultures could transform the polyphenols into other forms/types of compounds, e.g., via flavonoid glycosides hydrolysis or C-ring cleavage. Such a conversion may result in the deactivation of bioactive compounds or activation of previously inactive compounds, e.g., polyphenol glycosides are hydrolyzed into their aglycones of higher free radical scavenging ability, and procyanidins break down to flavan-3-ols or to smaller molecular phenolic acids. Acidity of yoghurt may have induced acid hydrolysis of polyphenols, hence this could explain an increased amount of hydroxycinnamates such as caffeic acid, chlorogenic acid, ferulic acid and p-coumaric acid in the fortified yoghurts. Sun-Waterhouse, Zhou and Wadhwa [53] stated that the yields of hydroxycinnamic acids and flavonols detected might be dependent on the extractability of these polyphenols from different product matrices.


**Table 3.** Measures of total phenolic content and antioxidant activity of supernatant of cubosome encapsulated tamarillo and yoghurt fortified with encapsulated tamarillo during in vitro digestion.

Data are presented as % of the sample before digestion. 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.

Despite the importance of the recovery in each digestion phase, bioactive compounds will need to be released from their food matrix and reach the intestine where they can be absorbed and be metabolised [54]. The release rate during in vitro digestion has been considered as an indicator to assess the effectiveness of compound carriers [55]. In general, non-encapsulated phenolic compounds in drinking yoghurt were highly degraded after digestion [56] while microencapsulated formulation showed the ability to preserve the antioxidant activity of extract in yoghurt when compared with the free form [51,57]. The amount of individual polyphenol in yoghurt samples was significantly different (*p* < 0.05) after each phase of in vitro digestion. The quantity of polyphenols released at each stage is dependent on the time at each phase, pH and the concentration of CUBTAM (Table S3). Most polyphenol components were detected in both PRE and POS in each digestion phase. The oral digestion lasts for a few minutes; the encapsulated polyphenol in yoghurts release from oral digestion was significantly lower than the gastric (post to 2 h) and intestinal (post to 3 h) simulated digests. These data are in line with the findings from Section 3.3 that encapsulating polyphenol in cubosome particles could effectively protect the bioactive compounds from the gastric enzymes and facilitate the utilization of polyphenols in the human body. Studies on the metabolism of bioactive compounds in the humans have shown that bioactives are mainly metabolised by a large number of small and large intestinal bacteria, and the metabolites are absorbed into the human blood [58]. Therefore, for the polyphenols to be absorbed by the human body or to be active in the microbiome of the small and large intestines, the polyphenols should be protected in encapsulated form until completely released in the intestinal tract. When the polyphenol capsules were present in the simulated oral phase, the concentration of all bioactive compounds was low, indicating good retention in the cubosomes. Furthermore, compared to undigested samples, a significantly (*p* < 0.05) lower amount of most polyphenols (percentage loss < 10%) were released during the oral phase (except for epicatechin, rutin and kaempferol rutinoside). The findings provide evidence that cubosomes protect bioactive compounds from interaction with, for example, milk proteins and digestive enzymes, reducing the risk of polyphenol degradation.

To our knowledge, most of reports of the loads of cubosomes are for proteins or small molecules such as drugs while research about encapsulation of hydrophilic polyphenols, mainly presented in tamarillo, using the cubosome is limited. Effects of the digestion process on properties and stability of cubosome as well as application of cubosome in food are still scarce. Hence, a strength of this study is it is the first attempt to encapsulate polyphenols from tamarillo and has demonstrated the proof of principle that this technique can be used to fortify yoghurt with a fruit extract. Entrapment efficiency was greater than 50% and, together with enhancement of antioxidant effects, stability and bioavailability of polyphenols in vivo and in vitro [2], the results showed the potential of cubosome to minimize degradation of polyphenols and contribute to controlled release of these and other bioactives during digestion. Application of encapsulated polyphenols into yoghurt did not significantly change texture and rheology of yoghurts when compared to the control, except for 15% fortification, where higher texture and rheology values were observed. However, there are still challenges about applications of cubosomes, including a deeper understanding of the stabilizer and possible cytotoxicity. In the future interaction between yoghurt components (mainly protein), starter culture and encapsulated bioactives should be evaluated with longer intestinal digestion and possible effects on the microbiota explored. According to Wei et al. [21], monoolein has been easily hydrolysed to free oleic acid due to the presence of pancreatic lipase and bile salt; therefore, it can be assumed that cubosome particles (with 60% of monoolein in components) can only be degraded in intestinal phase. However, degradation or stability of cubosome after each phase of digestion should be evaluated to ensure the safety, effectiveness and acceptability to the consumer of this approach. It is a limitation that we did not validate the correct encapsulation of the tamarillo polyphenols in the cubosome. In future work, we could confirm the encapsulation with confocal Raman/FTIR microscopy.
