## 3 Results and Discussion  
### Encapsulation Efficiency of Spray-Dried Anthraquinone-Loaded Casein Micelles  
Figure 1 shows the encapsulation efficiency of spray-dried casein micelles loaded with AQ powder (CMAQP), freeze-dried powder (CMFDP) and gel (CMWLAG). The encapsulation efficiencies for spray-dried CMAQP were 88%, 72% and 67% for aloin, alocemodin and rhein, respectively. However, our earlier study found that the encapsulation efficiencies for aloin, aloce-emodin and rhein in the solution prior to spray-drying were 99%, 98% and 100%, respectively. It indicated an 11%, 26% and 33% decrease [28] for aloin, aloe-emodin and rhein, respectively. The decrease in encapsulation efficiencies after spray-drying was due to the thermal and air-interface-related stresses. The shear rate imparted on casein micelles resulted in the unfolding and exposing of the hydrophobic core of casein micelles [29] carrying anthraquinones (aloin, aloe-emodin, rhein), causing a decrease in EE%. However, the difference in EE% amongst aloin, aloe-emodin and rhein was due to the difference in hydroxyl groups attached to the C ring of aromatic anthraquinones [28] that makes hydrogen bonds with the carbonyl group of casein micelles. As reported previously, aloin, aloe-emodin and rhein are attached within casein micelles mainly through hydrophobic and hydrogen bonding while in solution [14]. Upon spray-drying, the aloin and aloe-emodin are less affected due to being attached to the hydrophobic core. However, rhein encapsulation efficiency decreased significantly, as hydrogen bonds  
are weak bonds and easily breakdown by thermal and dehydration stresses during spray-drying and some of these got attached to the surface of casein micelles, as evidenced by Figure 1b. Anthraquinone-loaded-casein micelles from freeze-dried powder (CMFDP) exhibited encapsulation efficiencies of 73%, 65% and 12% for aloin, aloe-emodin and rhein, respectively. In the solution phase, 100% of aloe-emodin was encapsulated only before spray-drying, and no aloin nor rhein was encapsulated into casein micelles [14]. The reason is that while in solution form, the aloin started to degrade quickly into aloe-emodin and rhein and could not be detected in the supernatant after centrifugation as per protocol in the previous study [14]. However, aloin and rhein encapsulation efficiency rose to 73% and 12% with spray-drying, respectively. So, the encapsulation efficiency of aloin increased from zero to 73% due to the relocation of free anthraquinones to the inner cluster of casein micelles, as spray-drying caused exposure of embedded hydrophobic regions of casein micelles. After spray-drying, the encapsulation efficiency of aloe-emodin decreased by almost 35%. This decrease in EE% was due to the unavailability of binding sites of casein micelles, which had already been saturated by aloin, as bulky compounds (higher molecular weight) have more affinity to bind with proteins [30] due to more bond formations. While spray-drying, the hydrophobic core of casein micelles gets exposed and could strengthen the interaction between anthraquinones and casein micelles. However, spray-drying did not modify the type of significant interactions [28]. The surface-attached aloin, aloe-emodin and rhein could be calculated in Figure 1b. The least EE (%) was observed in spray-dried anthraquinone-loaded casein micelles from whole leaf aloe vera gel (CMWLAG) with only 12% aloe-emodin, 8% rhein encapsulated with no aloin being encapsulated. However, according to our previous study, 98% of aloin was encapsulated in the solution phase [14]. This decrease in powder is because of two reasons: Firstly, the decomposition of aloin after harvesting and during extraction in methanol into its degraded isomers, aloe-emodin and rhein, as reported in the previous article [23].