*3.2. Stability of Lutein*

The percentage of encapsulated lutein in the microfluidic noodle with four different treatments are presented in Table 1. The highest level was with combination-flow + SO, being 92.1 ± 1.6%, followed by co-flow + SO and co-flow + OL, being 88.7 ± 3.6% and 83.3 ± 1.5% respectively. Among which, combination-flow + OL showed the lowest encapsulated lutein level, being 80.0 ± 0.6%. Compared to OL, SO resulted in a higher lutein encapsulated content in the microfluidic noodle. It is worth noting that here the encapsulated lutein percentages were the same data present as the stability of lutein on day 1. Given that the microfluidic noodle was subjected to thermal processing (80–85 ◦C) for six minutes on day 1, the differences in the encapsulated content were assumed mainly to be attributed to the different degradation rate of lutein under high temperatures. Therefore, our findings sugges<sup>t</sup> that SO may be beneficial in protecting lutein from degradation during heating. Theoretically, the encapsulated lutein content is approximately 17.4 mg in the 100 g microfluidic noodle, and 15.0 ± 0.5 mg of lutein was detected in the extruded noodle (86.0 ± 2.7%), calculated by the average level with four different treatments. After up to seven days of storage, although the stability of lutein experienced a decreasing trend, the retention of lutein in all four types of microfluidic noodle still maintained above 60% (Figure 5). This result indicates that microfluidic encapsulation may be a good strategy to ensure the stability of bioactive compounds.

**Table 1.** Encapsulated lutein in noodle-like product via microfluidic techniques.


Notes: Tukey tests were carried out and significant differences (*p* < 0.05) exist among those with different letters (a, b). OL: olive oil; SO: safflower oil.

**Figure 5.** Storage stability of lutein in the microfluidic noodle. Notes: Tukey tests were carried out and bars with different letters (a, b, c, d) indicate significant differences among each storage day (*p* < 0.05). OL: olive oil; SO: safflower oil.

In this study, SPI was used to create oil-in-water emulsion and served as a vehicle for encapsulation of lutein. However, the interfacial layer formed by SPI is electrically charged and is particularly sensitive to pH and ionic strength [37], leading to an unstable emulsion system. For example, oil–water phase separation was observed during the storage [38], and significant droplet flocculation occurred following the simulated gastric digestion [39]. Additionally, the stability of the encapsulated compounds is hardly ensured with these protein-stabilized emulsions. This is because only a thin layer of protein molecules coat the lipid droplets. Therefore, any encapsulated compounds located close to the droplet surfaces are prone to chemical degradation, which could be promoted by compounds such as acids, transition metals, or enzyme components present in the aqueous phase [40].

To overcome this shortage, this study fabricated a core-shell structured food material, comprising a gelled alginate shell to protect the SPI core from any adverse environmental conditions and improve its stability during the long-term storage. A previous study reported that trapping β-carotene-loaded oil droplets in protein-alginate microparticles was observed with less oil-water phase separation and had a relatively constant particle size, as compared to the emulsion without the alginate coating [38]. Similarly, another study described that the microbeads maintained good physical integrity when they were coated with alginate [31]. These suggested a potential advantage of alginate in stability improvement. During seven days of storage, no free oil fortified with lutein was observed in the water medium where the noodle samples were stored. Consistent with our observation, Liu et al. reported that nearly all of the β-carotene-loaded oil droplets were fully trapped inside the alginate microparticles [38]. This indicated that alginate coating ensured a good encapsulation efficiency. Ivana et al. also demonstrated that the free oil enriched with carotenoids was only about 10%, and this value was measured as the free oil left on the surface [41]. However, it is worth noting that in our study, the amount of free lutein left on the surface was minimized by using the microfluidic technique, given that lutein was directly extruded into the core of the noodle-like sample. Meanwhile, studies showed that applying an interfacial layer of alginate outside the protein-stabilized emulsions was more effective at inhibiting carotenoids degradation [37,38]. In our study, the retention of lutein was still above 60% in all four types of microfluidic noodle after seven days of storage. Consistent with our result, a study reported that the content of β-carotene was able to maintain around 55% with an alginate coating after storage for twelve days. However, it fell down to only 0.2% with the emulsion alone [40]. Moreover, emerging evidence demonstrated the improvement in lutein stability with multilayered emulsions by covalently attaching polysaccharides to proteins [42]. A previous study fabricated the whey protein isolate-flaxseed gum-chitosan stabilized lutein emulsions by using layer-by-layer electrostatic deposition, and they observed that the retention of lutein was as high as 69% after seven days of storage at a higher temperature 70 ◦C [43]. This is possibly attributed to the multilayer biopolymer, which provides a physical barrier to the diffusion of oxygen, pro-oxidant, and free radicals [36], and thus inhibits the oxidation of carotenoids.

#### *3.3. Bioaccessibility, Release and Micellarization of Lutein*

The co-flow and combination-flow devices did not result in a difference in lutein bioaccessibility (co-flow: 3.1 ± 0.5%; combination-flow: 3.6 ± 0.6%). SO and OL also showed no differences in lutein bioaccessibility (SO: 3.4 ± 0.8%; OL: 3.3 ± 0.4%). These results sugges<sup>t</sup> that both types of oil and device do not influence on the bioaccessibility of lutein. However, during the gastrointestinal digestion, the co-flow device showed higher lutein release (co-flow: 64.3 ± 4.5%; combination-flow: 44.3 ± 1.6%), while lower micellarization (co-flow: 4.8 ± 0.2%; combination-flow: 8.1 ± 0.7%) as compared with the combination-flow device. Moreover, compared to OL, SO resulted in less lutein released from the noodle matrix (SO: 48.7 ± 3.0%; OL: 59.9 ± 6.3%) but greater lutein formed into micelles (SO: 7.2 ± 1.0%; OL: 5.7 ± 0.5%). Specific data of the bioaccessibility, release and micellarization of the encapsulated lutein are presented in Table 2.


**Table 2.** Bioaccessibility, release and micellarization of lutein in microfluidic noodle following the in vitro digestion.

Notes: Theoretically, 870 μg lutein was initially added in every 5 g of microfluidic noodle. Lutein content in micelles and digesta were calculated based on every 5 g of the noodle sample. The bioaccessibility, release and micellarization of lutein were all determined on day 1. Lutein bioaccessibility was determined as the fraction of lutein solubilized in the mixed micelles after passing through the simulated in vitro digestion. Lutein release was determined as the lutein content in the digesta from the initial food matrix. Lutein micellarization was determined as transfer of lutein from the digesta to the mixed micelles. Tukey tests were carried out in each column and significant differences (*p* < 0.05) exist among those with different letters (a, b, c). OL: olive oil; SO: safflower oil.

> Compared to the co-flow, the combination-flow device had an approximately 31% lower lutein release rate. This is possibly because the droplet of lutein-fortified oil is tightly trapped within the SPI layer and further surrounded by an alginate layer when the noodle is created with the combination-flow device. As described above, the luteinfortified oil droplet was shown to be encapsulated in a core-shell structure with SPI being the core and gelled alginate being the shell. In particular, the droplet of the lutein-fortified oil (0.2 mm in diameter) was tightly trapped at the center of the SPI core (0.5 mm in diameter), and a relatively thick alginate shell (0.9 mm in diameter) fully covered the SPI core. Thus, it may make lutein difficult to be released from this complex multilayer structure. However, lutein is relatively easier to be released when the noodle is created with the co-flow device. Since lutein-fortified oil was pre-mixed with SPI solution and formed an emulsion, to be released from the noodle matrix, lutein only has to penetrate the outer alginate layer. On the contrary, the co-flow device resulted in about 41% lower lutein micellarization as compared with the combination-flow device. This is mainly explained by the SPI-stabilized emulsion as an inner layer of the noodle made using the co-flow device. When subjected to in vitro digestion, especially during the gastric phase, due to the weakening of electrostatic repulsion and protein hydrolysis, protein stabilized emulsions are prone to flocculate [44,45]. This flocculation phenomenon may further restrict the lipase access to lipid droplet surfaces [38], resulting in less generation of lipid-digestion products, which are the components of micelles. Consequently, the formed mixed micelles could be insufficient to solubilize the lutein molecules, and thereby result in lower lutein micellarization with the co-flow device.

> Compared to OL, SO showed an approximately 19% lower lutein release rate. Linoleic acid is a polyunsaturated omega-6 fatty acid and accounts for 55–77% fatty acids in the SO [46]. Dietary oils rich in polyunsaturated fatty acids (PUFAs) were reported to make carotenoids more susceptible to oxidation. PUFAs are prone to oxidation and the generated radicals are likely to attack carotenoids, thereby leading to a variety of carotenoid decomposition products [47,48]. Since SO is rich in PUFAs, greater lutein degradation may happen when lutein was co-digested with SO than OL. This may also explain the lower lutein content determined in the digesta which was considered as the released fraction. On the contrary, SO resulted in about 26% higher lutein micellarization as compared with OL. A previous study reported that PUFAs-rich oils promoted the formation of smaller droplet size in emulsions because of the presence of more unsaturated linkages increasing the interface flexibility [49]. Emulsions with a smaller droplet size tend to have a higher extent of lipid digestion, and consequently, a greater mixed micelles formation [50,51]. Although the detail underlying the mechanism explaining the effect of oil types on lutein micellarization cannot be elucidated from this present study, further research may be required to investigate the differences (if any) of lipid digestion extent and the formed micelles size between OL and SO.

> Lutein generally shows a low bioaccessibility, which ranges from 9 to 59% determined from raw fruits and vegetables [52]; in this study, the bioaccessibility of lutein from the lutein-fortified noodle was about 3 to 4%. A previous study formulated a carotenoids-rich

milk beverage and the bioaccessibility of lutein was also reported to be lower or equal to 7% [27]. Several reasons possibly attribute to this relatively low lutein bioaccessibility. Firstly, lutein degradation may happen during the noodle thermal processing, considering carotenoids are sensitive to high temperatures [53,54]. Therefore, lutein loss is more inclined to happen with the processed food rather than in raw fruits and vegetables. Secondly, lutein can remain in the lipid droplets and be trapped inside the alginate layer. Thirdly, trapped and undigested lipids can result in insufficient fatty acids and monoacylglycerols to form mixed micelles [55], and in turn hinder the micellarization of lutein. Lastly, the increased viscosity due to the presence of alginate may induce lutein aggregation and precipitation, thereby making lutein hard to solubilize in the mixed micelles [56].

As mentioned earlier, an advantage of this fabricated multilayer structure lies in the release rate of its encapsulated compounds could be engineered to fit different purposes. A previous study evaluated the stability of alginate-coated microbeads with different pH to mimic the gastrointestinal tract, and reported that the alginate-coated microbeads maintained physical integrity in acidic conditions but started to disintegrate when pH reaches 7 [31]. This indicates that the multilayer structure of the noodle may be able to delay the release of lutein in the gastric phase at a pH of about 3. This may in turn help protect lutein from degradation. When following the intestinal phase near pH 7, the structure may disintegrate and lutein will be released for absorption. Furthermore, abundant evidence has been reported that consuming 10–20 mg lutein supplementation resulted in a beneficial effect on visual health [57–59]. A meta-analysis reported that every 0.3 mg increment of lutein/zeaxanthin intake reduced the risk of nuclear cataracts, cortical cataracts and posterior subcapsular cataracts by 3%, 1% and 3%, respectively [60], and lutein intake was reported to be safe up to 20 mg per day [61]. According to USDA, a serving size of dry pasta is about two ounces (57 g); after considering the morphology and compositions of this microfluidic noodle, we recommend one cup as a portion, which is about 128 g for consumption. This present study managed to encapsulate about 15 mg lutein per 100 g of the new noodle-like food products, which indicates that this lutein-fortified noodle may be a possible alternative food item to compensate for the insufficient lutein intake in humans. However, it is also worth noting that only around 0.5 mg lutein may be successfully incorporated into the mixed micelles upon consuming 100 g of this noodle. Therefore, further study on the application of microfluidic technology in encapsulation is necessary, especially to improve the bioaccessibility of encapsulated compounds.
