*4.2. Construction of Carriers Based on Bio-Macromolecules*

## 4.2.1. Chemical Interaction

Several technologies have been widely used to construct biomacromolecule-based carriers for ascorbic acid (Table 1), in order to shield the unfavorable environmental factors and improve the taste of the product. These processes involve physical and chemical interactions between carriers and ascorbic acid; chemical interactions mainly refer to covalent and non-covalent bonds.

Proteins are generally recognized as safe (GRAS) and have high nutritional value. The delivery systems based on proteins have received widespread attention in food field due to their biocompatibility, biodegradability and tunability. Ascorbic acid binds to βlactoglobulin (β-LG) through ion contact to form a more stable conjugate than human serum albumin (HSA) and bovine serum albumin (BSA). β-LG, HSA and BSA can, respectively, bind about 50–60%, 40–55% and 35–50% of ascorbic acid, and the proteins can be used to deliver vitamin C in vitro [86]. Through a cationization reaction, the quaternary ammonium salt cationic group was attached to the soybean protein isolate (SPI) chain, which increases the solubility of the protein and favors the encapsulation of ascorbic acid [87]. However, the low loading capacity and carrier instability in the stomach and intestines are the main challenges that restrict proteins from being ideal delivery vehicles for ascorbic acid. Since the excellent hydrophilicity of ascorbic acid and its same charge as most proteins (isoelectric point, pI < 7) at physiological pH, their interactions such as hydrophobic interaction, electrostatic interaction, hydrogen bonds and van der Waals forces are usually weak or absent. This results in a low encapsulation efficiency and rapid release of ascorbic acid from protein nanoparticles in the aqueous solutions.

Chitosan is a cationic polysaccharide with excellent chelating and cross-linking properties and is widely used as a delivery vehicle in the food field. The formation of chitosan nanoparticles requires cross-linking with polyanions, such as tripolyphosphate (TPP). The amino groups of chitosan in the polymer backbone can interact with ascorbic acid to form a strong hydrogen bond, which captures and retains ascorbic acid on the polysaccharide [10,88]. The formed chitosan-ascorbic acid complexes have high singlet oxygen scavenging ability and then maintain the high antioxidant capacity of ascorbic acid. The nanoscale size and positive charge of the particles are very important for their adsorption on the mucosa, which is conducive to achieving a high uptake rate of the loaded ascorbic acid by the intestinal cells. Chitosan-ascorbic acid complex nanoparticles increase the residence time of ascorbic acid in the digestive tract of trout. Compared with protein nanoparticles, chitosan nanoparticles strengthen the interaction with ascorbic acid through electrostatic interaction, but the encapsulation efficiency is still relatively low [11]. This is related to the molecular weight and concentration of chitosan, the addition of ascorbic acid and the measurement method of the encapsulation. There are two views about the influence of chitosan molecular weight on the encapsulation efficiency of ascorbic acid. One is that high-molecular-weight chitosan has more surface charges to bind with more ascorbic acid molecules, thus the long backbone can capture more ascorbic acid. As the molecular weight of chitosan increased from 65 kDa to 110 kDa, the content of ascorbic acid loaded increased from 30% to 70%, respectively. With the further increase in chitosan molecular weight, the particle size increases but the overall surface area decreases, resulting in a decrease in the encapsulation efficiency of ascorbic acid [16]. Short fragments of low-molecular-weight chitosan are easier to protonate free amino groups, thereby complexing with ascorbic acid through electrostatic interactions. The average diameter of 55-kDa chitosan complex particles is 70.6 nm, and the loading efficiency of ascorbic acidic is about 66% [89].

#### 4.2.2. Physical Barrier

In order to maintain the stability of ascorbic acid in food applications, ascorbic acid can be loaded into biomacromolecule-based delivery vehicles through physical encapsulation and adsorption. Compared with ascorbic acid nanoparticles chelated with protein and chitosan, the construction of physical barriers such as microcapsules based on protein and polysaccharide, solid lipids and liquid state multiple emulsions have a better loading capacity of ascorbic acid in the core and hence, this improves stability.

The process of encapsulating ascorbic acid in a core walled by polymers coating to isolate it from the external adverse factors is microencapsulation. The current preparation methods of microcapsules mainly include spray chilling, spray drying and complex coacervation. Among them, spray drying is one of the most common techniques due to its low cost, continuity and easy industrial scale production [90]. The selection of wall materials includes various proteins and polysaccharides, such as gum Arabic, maltodextrin, pectin, xyloglucan, sodium alginate. Gum Arabic and sodium alginate are low-cost and GRAS category polysaccharides, which are often used as food additives. The sodium alginate/gum Arabic microcapsules prepared by spray drying have an excellent loading capacity of ascorbic acid, which can reach more than 90%. Meanwhile, the thermal stability temperature of ascorbic acid is increased to 188 ◦C, which is higher than the temperature required for product preparation [91]. The xyloglucan extracted from *Hymenaea courbaril var. courbaril* seeds is a water-soluble polysaccharide containing gum Arabic, which is used as a thickener, stabilizer and crystallization inhibitor in the food industry. The spray-dried microcapsules can encapsulate around 96% of ascorbic acid. The system shows strong antioxidant activity and inhibits the formation of furan, an ascorbic acid degradation product, during the preheated process of products. After 60 days of storage at room temperature, the retention of ascorbic acid in the system is still around 90% [92]. However, the high viscosity of high-concentration polymers limits the granulation by spray-drying. To a certain extent, the loading capacity is related to the wall-to-core ratio and increases with the increase in the coating of wall materials [15]. Complex coacervation is the phase separation of at least two hydrocolloids from the initial solution, and then the coacervate phase is deposited around the suspended or emulsified bioactive compounds. One of the hydrocolloids is in the colloidal state. On the contrary to hydrophobic bioactive compounds, hydrophilic ascorbic acid needs to be emulsified before it is prepared [93]. Compared with spray drying, this method does not involve a heat treatment process and is more suitable for encapsulating thermally unstable ascorbic acid [94]. The microcapsules prepared with gelatin and pectin as wall materials improve the thermal stability of ascorbic acid, although the solubility of the microcapsules is relatively low [15]. The encapsulation efficiency of ascorbic acid using gelatin and acacia as wall materials is about 97% [93].

The systems based on lipids, such as solid lipid microcapsules and emulsions, can be obtained by high-pressure homogenization, microfluidics, and solvent evaporation. The solid lipid microcapsules prepared by polyglyceryl monostearate (PGMS) have the encapsulation capacity of ascorbic acid up to about 94%. The system can be added in to fortify milk, significantly inhibiting the Maillard reaction between milk proteins and ascorbic acid. Sensory analysis showed that there was no significant difference in most aspects between the control sample and the fortified sample encapsulated with ascorbic acid after 5 days of storage [95]. As reported, palm fat was used as wall material to fabricate the solid lipid microcapsules to encapsulate and protect ascorbic acid using a microfluidic technique. The internal phase was added with salt or chitosan to further improve the encapsulation efficiency of ascorbic acid. The two different mechanisms involve pore blockage and ascorbic acid chelation [6]. This system has better physical isolation performance than protein and/or polysaccharide solid microcapsules. However, the operation process includes thermal melting and ice bath cooling of liposomes. This method is limited to the laboratory scale and is difficult to industrialize. On the other hand, the storage stability of ascorbic acid in oil-containing systems may be affected by lipid oxidation and thermodynamic instability of emulsions, which is lower than that of carrier-stable protein and polysaccharide microcapsule systems [96,97].

The microcapsule system based on the physical barrier has a better loading capacity of ascorbic acid than complex nanoparticles (Table 1). In addition to the properties of the delivery carriers, it may also be related to the different measurement method of encapsulation efficiency. For delivery systems in micro-scale, the measurement conditions for encapsulation efficiency of ascorbic acid are gentler than those of protein and/or polysaccharide nanoparticles. The determination method includes separation by standing, ultrasonic and filter paper filtration [82–86]. Compared with the ultra-isolation method [79,80] used in the nanoparticle system, these methods reduce the release and diffusion of ascorbic acid during the measurement process.


**Table 1.** Different types of bio-macromolecule delivery vehicles of ascorbic acid.
