Casein micelles as a delivery system for aloe-vera extracted anthraquinones
Creators: name: Sadiq, Uzma
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300602
subject: Food chemistry and food sensory science
classificationCode: 400405
subject: Food engineering

description: Food bioactive components benefit human health, but are susceptible to light, pH, temperature, and oxygen degradation. Thus, technologies capable of retaining, protecting and enabling their targeted delivery need to be researched. Microencapsulation is one of these technologies that has gained considerable attention within the food industry in recent years. Having a proper delivery vehicle with less toxicity and high biocompatibility is essential during microencapsulation. Milk proteins are widely used as delivery vehicles, particularly casein micelles due to their porous, self-assembling, water-binding, and surface-active properties.

Aloe vera (Aloe barbadensis miller) plant is characterized by its polysaccharides, vitamins, enzymes, amino acids, phenolic compounds, phytosterols and high concentrations of anthraquinones. Aloin (A and B), barbaloin, isobarbaloin, emodin, aloe-emodin, rhein, chrysophanol, physcion, and chrysophanol are some of the anthraquinones present in Aloe vera. In addition to being laxatives, anthraquinones possess antibacterial, antiviral, antiinflammatory, antifungal, and anticancer properties, which makes them highly versatile to be used in functional foods and nutraceuticals. Anthraquinones in Aloe vera are highly sensitive to pH and temperature. Therefore, their stability is essential for preserving their efficacy and quality while developing functional foods and nutraceuticals. 

The aim of this project is to fundamentally design a stable delivery system for anthraquinones extracted from Aloe vera. However, understanding the stability of these bioactive compounds under various processing and storage conditions needs to be analyzed thoroughly. Thus, in the first experimental chapter of the thesis, the stability of three anthraquinones (aloin, aloeemodin, rhein) extracted from whole-leaf aloe vera gel was compared with their spray-dried and freeze-dried powders as a function of pH and storage temperature conditions. Different processing conditions affected the behavior of anthraquinones. 

Whole leaf aloe vera gel (WLAG) had the most significant amount of aloin, followed by freezedried powder (FDP) and spray-dried powder (SDP) due to poor preservation of phenolic glycosides and exposure to high temperatures, respectively. A high level of aloe-emodin was detected in WLAG, followed by spray-dried powder, while FDP did not contain aloe-emodin due to insufficient aglycon extraction. Regarding storage stability, FDP retained more aloin than SDP, while WLAG retained the least aloin over seven days of storage at room temperature and natural pH of 4.5. All samples exhibited the highest stability of aloin at pH 3.5, while pH 6.7 exhibited the lowest stability. In contrast, aloin content decreased significantly at 50°C and 70°C irrespective of pH because of oxidative degradation of aloin into aloe-emodin. Initially, rhein was not detected, but over time, the presence of rhein was shown due to oxidative degradation of aloin into aloe-emodin and rhein. 

The second experimental chapter of the study investigated the use of ultrasonication in encapsulating Aloe vera-extracted anthraquinones into casein micelles. WLAG, Anthraquinone powder (AQ powder), SDP, FDP, pure aloin, pure aloe-emodin and pure rhein were encapsulated within casein micelles (CM) by applying an ultrasonic energy density of 39.74 W. The prepared nanocapsules in their liquid state were characterized for their encapsulation efficiency (EE), yield, and CM-anthraquinone binding interactions. CM loaded with pure Aloin (CMA), aloe-emodin (CMAE), and rhein (CMR) displayed the highest EE for aloin and the lowest EE for rhein. The AQ powder showed the highest EE for aloin (99%), aloe-emodin (98%) and rhein (100%) due to increased hydrophobic interactions, while WLAG had the least EE. However, SDP and FDP had the highest EE for aloe-emodin but zero for aloin affixed by hydrogen bonding to the hydrophilic surface of CM during encapsulation. The FTIR spectra of CM nano capsules confirmed that amide bands (I, II) remain unchanged while encapsulating anthraquinones from AQ, SDP and FDP powders, indicating no change in CM structure. The results showed that different anthraquinone types have different binding affinities and ratios to CMs, affecting the size, zeta potential and EE% of the nanoparticles. The study confirmed that anthraquinone type, casein micelle ratio, structure of anthraquinone and pH of the solution plays significant roles. 

In order to develop functional foods, these liquid nanocapsules are unsuitable due to their perishability and their effects on the consistency and texture of the final products. Thus, the dry state is preferred by the food industry, which has logistical advantages such as easy handling, fewer transport costs, and longer shelf life. Therefore, the effect of spray drying (SD) on the encapsulated nanocapsules was investigated. The findings demonstrated that after the spray drying process, casein micelles loaded with AQ powder (CMAQP) had the highest EE% based on the solution pH prior to SD, although a slight reduction in EE% was observed as compared to liquid state. The EE% of casein micelles loaded with FDP increased significantly compared to the liquid state. A higher EE% of CMFDP could be attributed to the inclusion of a hydrophobic binding site embedded in the CM structure after SD, which was attached to the core of the casein micelles. With no aloin encapsulated, spray-dried anthraquinone-loaded casein micelles (CMWLAG) yielded the lowest EE (%) compared to the liquid state, where 98% of aloin was encapsulated. The reason is that spray-drying does not encapsulate aloin residues after decomposition but instead binds them to the powder surface due to their hydrophilic properties. According to FTIR and SEM results, the amide I and amide II bands of CMAQP have moved to higher and lower energy levels due to rapid CM aggregation after SD. CMFDP showed casein micelle structural changes due to the folding and unfolding of CMs. Both amide I and amide II vanished for CMWLAG after SD, indicative of the denaturation of CM due to heat, dehydration, and interface-induced stresses. It was found that pH, spray-drying conditions, and degree of Maillard reaction affected EE% of CMAQP, CMFDP and CMWLAG, respectively.

The last experimental chapter was performed to study how changes in the structure of casein micelles, and their interactions with anthraquinones, could affect the release of anthraquinones from casein micelles during oral, gastric, and intestinal digestion. The CMAQP has the slowest release percentages of anthraquinones in the oral and gastric phases, followed by CMFDP. The strong hydrophobic interactions between anthraquinones and CM delayed diffusion resulting a slower release. A controlled release of anthraquinones in the small intestine was achieved by non-covalent bonding between casein micelles and anthraquinones. The CMFDP microcapsules were easily rehydrated during gastric digestion, leading to the release of more anthraquinones. Trypsin, hydrolysed casein micelles in the small intestine, releases anthraquinones rapidly. In contrast, CMWLAG microcapsules released anthraquinones in the mouth phase and within 60 minutes of gastric digestion. As a result, they are not protected by casein micelles and are unstable, resulting in significant losses during in vitro digestion. The FTIR spectra suggested that the secondary structure of the CMAQP and CMFDP is retained until it reaches the stomach, where breakdown starts. The study found that the degree of dynamic release of anthraquinones depends on the structural characteristics of wall materials, the porosity of the wall material, its surface roughness, and the size of microcapsules. By understanding the release behaviors of anthraquinones in spray-dried microparticles during in vitro digestion, researchers can design an effective casein micelle delivery system to deliver aloe vera extracted anthraquinones to their target sites in the body.