## 3 Antioxidant Properties of C\({}_{30}\) Carotenoids  
The antioxidant activity of a compound is the constant reaction rate between the compound and reactive species that include both radicals and nonradicals [13]. The physiological health benefits of carotenoids are attributed to the length of their conjugated backbones, which makes them highly reactive molecules capable of scavenging free radicals and quenching singlet oxygen. Carotenoids respond to ROS by donating electrons and, through oxidation, creating carotenoid-radical cations, thus lowering the oxidation state of the oxidizer [47]. Carotenoids also scavenge reactive oxygen species through the hydrogen atom transfer (HAT) mechanism in which a chemical transformation occurs, whereby there is movement of a proton and an electron between two substrates [48]. Currently, there are no universal measures for the determination of antioxidant activity. The antioxidant activity of C\({}_{30}\) carotenoids is more commonly determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPPH) free-radical scavenging activity assay and the singlet oxygen quenching assay, which is performed by measuring the methylene blue-sensitized photooxidation of linoleic acid. While the DPPH assay is commonly used for antioxidant capacity determination with absorbance typically read at 515 nm, some carotenoids such as \(\beta\)-carotene and lycopene produce dark brown colors when mixed with DPPH. The color has been observed to interfere with the absorbance readings at 515 nm, but a change to 540 nm allowed for readings as there was still significant absorption at that wavelength [49; 50]. The carotenoid antioxidant activity and reactivity in quenching ROS stem from the high electron density of the compounds, especially C=C bonds, and occur via physical or chemical ROS quenching. Singlet oxygen (\({}^{1}\)O\({}_{2}\)) originates from triplet molecular oxygen (\({}^{3}\)O\({}_{2}\)) in the presence of light, or superoxide and hydrogen peroxide in the absence of light. During physical quenching, carotenoids convert singlet oxygen to its triplet ground state, which is less reactive. The energy taken on by the carotenoid leaves it in an excited state, which dissipates as it releases the energy as heat into the environment and returns to its ground state. During chemical quenching (or scavenging), there is a chemical reaction between the carotenoid and the ROS, and barring enzymatic recycling of the carotenoid, it is oxidized and consumed completely. Carotenoid antiradical capabilities have also been studied by analyzing the carotenoid-hydrogen bond dissociation energy via the HAT antiradical mechanism [51]. [PERSON] et al. [52] developed a method to distinguish between hydrogen and electron transfer by using a galvinoxyl radical (G\({}^{\bullet}\)) and magnesium ions. If electron transfer is the rate-determining step, the mechanism is accelerated by the magnesium via stabilization of the reduced radical anion. A subsequent proton transfer occurs from the radical cation, resulting in a neutral antioxidant radical and GH. However, information on this as applied to C\({}_{30}\) carotenoids is lacking. The structure of a carotenoid has significant bearing on its antioxidative efficiency. Depending on their structure, carotenoids can be classified as carotenes, which only have hydrocarbon backbones; and xanthophylls, which possess functional groups such as hydroxy, keto, and methoxy groups [53]. In certain microorganisms, the arrangement and yield of carotenoids are affected by their growth conditions, with different parameters eliciting different effects [54]. Stereoisomerism also plays a role in carotenoid biofunctionality and affects both solubility and absorbance. The rigidity of the _trans_ forms increases their propensity for aggregation and crystallization. Additionally, certain _trans_ forms have greater antioxidant activity than their _cis_ counterparts, although exceptions such as _cis_-lycopene have greater bioavailability than _trans_-lycopene [55].