*3.2. Amino Acid Profile*

The amino acid profile of the undigested spent *Aurantiochytrium* is shown in Table 2, listing the concentrations of 16 amino acids. Among these amino acids, eight essential amino acids were found. The major amino acids were glutamic acid (18 g/100 g) and aspartic acid (7.0 g/100 g), followed by serine, lysine, leucine, and proline. Cysteine, valine, and histidine were the among the least prevalent amino acids. The total amount of essential amino acids was higher than that of non-essential amino acids, with the high levels of leucine hinting at a potential use of this biomass in mid-workout energy snacks that aids muscle recovery and build-up [4]. While histidine levels were relatively low, they remain above the relative values of common plant or algae-based protein-rich supplements such as spirulina, soybean, and flaxseed. Histidine is a nutritionally essential amino acid that is also a precursor for several hormones (e.g., thyrotropin-releasing hormone), and critical metabolites affecting renal function, neurotransmission, gastric secretion, and the immune system [40]. The lysine/arginine/(Lys/Arg) ratio has been shown to positively affect the metabolic pathways of hypertension and have a positive effect on hypercholesterolemia, imparting lipidemic and atherogenic effects in rats even though the effects on humans were modest [26,41]. Although the exact mechanisms that lead to its positive effects are unknown, Yang et al. (2011) proposed that this amino acid ratio could limit the absorption rate of cholesterol [42]. The authors proposed either the slowdown of lipid absorption or promotion of 7 α-hydroxylase activity, which is a hepatic enzyme that limits the rate of cholesterol to bile acid conversion, as the mechanisms for this effect. In this study, the Lys/Arg ratio was found to be 3.73, which is quite similar to that of flaxseed (3.90) and a favourable ratio to be a useful protein ingredient in formulations intended to improve human health [40]. It should also be noted that the comparatively high amounts of glutamic and aspartic acid will likely contribute to an intensely umami flavour, which could gran<sup>t</sup> any spent *Aurantiochytrium*-based supplement with desirable flavour-enhancing characteristics [43]. Such potential would need to be further investigated via sensory analysis.


**Table 2.** Amino acid profile of the defatted *Aurantiochytrium* sp. biomass in g/100 g of extracted protein and as a percentage of total detected amino acids. A collection of comparable values reported in the literature was included for both *Aurantiochytrium* sp. and other *Thraustochytrids*.

### *3.3. Antioxidant and Lipid Protective Activities*

Table 3 lists the results of the three antioxidant assays performed on the digested defatted *Aurantiochytrium* sp. sample. There was no lipid oxidation prevention demonstrable using the LPIP method, which is unsurprising given the nature of the spent *Aurantiochytrium* sample. The majority of bioactive compounds, including those with antioxidant potential, whose presence in *Aurantiochytrium* sp. was previously reported, are lipophilic carotenoids and sterols [45]. These are expected to be mostly absent from the defatted biomass. Any residual activity, such as that which was detected in the FRAP assay, was likely caused by amino acids containing sulphur side chains, such as cysteine and methionine, or aromatic side chains, such as tyrosine, phenylalanine, and tryptophan [46,47]. The contribution of these effects to the overall antioxidant activity is heightened by the lipid-removal process. DPPH radical reduction activity was, similarly to the LPIP assay, indicative of a lack of lipophilic antioxidant compounds. In this instance, the ethanolic nature of the reaction medium results in the precipitation of most protein, and thus, any amino acid-driven activity is unrepresented [47].

**Table 3.** Antioxidant potential of the post-digestion defatted *Aurantiochytrium* sp. biomass, according to the DPPH radical reduction potential, ferric-reducing antioxidant potential (FRAP), and lipid peroxidation inhibitory potential (LPIP) assays. Each result is the average of at least three measures ± standard error.


The presence of compounds with antioxidant activity in foods, regardless of their status as either functional or nutritious is, in most cases, highly desired. While the effects of dietary antioxidants in human health is still a contentious topic, their contribution as a positive factor in food preservation is generally well understood [48,49]. The presence of antioxidants in fatty foods, either as an ingredient or additive, is particularly desired, as these can greatly delay the loss in quality related to the oxidation of lipids [50]. While there are other compounds vulnerable to degradation under oxygen exposure, the defatted nature of the samples studied here means that lipid peroxidation phenomena are not as significant a concern for their long-term stability, and thus, a loss of antioxidant compounds may not be as sorely missed in a product based in defatted *Aurantiochytrium* [34]. Regardless, the presence of antioxidant proteins suggested by the FRAP activity assay could still provide a health benefit and warrants further research to determine their true chemical nature and concentrations.
