**1. Introduction**

*Aurantiochytrium* sp. is a *Thraustochytrid* that has recently gained attention due to its high production of eicosapentaenoic acid and docosahexaenoic acid. They have emerged lately as an efficient economic alternative compared to other fish and microalgal oil sources by virtue of their simpler polyunsaturated fatty acids (PUFA) profiles and cost-effective culture conditions [1,2]. While the recovery of microbial oils avoids many of the problems

**Citation:** Reboleira, J.; Félix, R.; Félix, C.; de Melo, M.M.R.; Silva, C.M.; Saraiva, J.A.; Bandarra, N.M.; Teixeira, B.; Mendes, R.; Paulo, M.C.; et al. Evaluating the Potential of the Defatted By-Product of *Aurantiochytrium* sp. Industrial Cultivation as a Functional Food. *Foods* **2021**, *10*, 3058. https:// doi.org/10.3390/foods10123058

Academic Editors: Marco Poiana, Francesco Caponio and Antonio Piga

Received: 12 November 2021 Accepted: 4 December 2021 Published: 9 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

associated with the traditional sources of PUFAs, the leftovers generated by such largescale bioprocesses can still pose an environmental threat. A vast array of strategies and applications have been tested thus far in an attempt to add value to otherwise discarded microbial waste, with a large focus on the recycling of nutrients as a substrate for other economically feasible fermentations as well as the recovery of bioactive products [3–5]. Authors such as Medina (2015), Aida (2017), and Deshmukh (2021) have published distinct valorisation strategies applied to defatted microalgal or *Thraustochytrid* biomass, including the use as functional ingredients in biodegradable films, extraction of protein-rich antioxidant fractions, and direct use as nitrogen and phosphorous-rich additives to biofuel substrates [4,6,7]. The use of spent microalgal biomass has also been extended to livestock feed as either a soy or corn replacement as well as a nutritional supplement to traditional mixes [8].

Some of these applications fit under the designation of functional foods, which is a term that has acquired significant popularity in both social and scientific spheres. A recent definition given by Granato et al. (2020) states that functional foods, when regularly and efficaciously consumed as part of a diverse diet, can convey a positive effect on health beyond basic nutrition [9]. Said claims are regulated in most Western countries, limiting the classification to foods whose effects are verified via randomised, double-blind, and placebocontrolled clinical trials [10]. The potential held by functional foods in the prevention of many diseases deemed important in the 21st century, including obesity, type-2 diabetes, and several forms of cancer, has led to an enduring research interest over the course of the last 20 years [11–13]. The discovery of new functional foods is a large part of this effort, both as a way to find more bioactive ingredients and as a means to exploit new food resources in the form of new, high added-value products [13].

Due to the ever-growing demand for food of an expanding human population and their presence in an underexploited environment, marine food resources have found themselves under increased demand over recent years [14]. Among these, microalgae have confidently found their way into the niche market of food supplements. This was mostly due to exceptional amino-acid profiles in some cases comparable to terrestrial animal-sourced protein, as with the cyanobacteria *Arthrospira platensis* (commercial name "Spirulina") and the green algae *Chlorella* [6,15]. High dietary fibre content is also a highly desired feature in certain types of functional foods. These long-chain polysaccharides are incapable of being digested by the human digestive process and have been linked to numerous gastrointestinal health benefits [16,17]. Certain types of dietary fibre can be fermented by the gu<sup>t</sup> microbiota, selectively promoting the growth of beneficial *Bifidobacterium, Lactobacillus, Bacillus, Streptococcus, Saccharomyces* and *Lactococcus* strains [18]. In turn, the proliferation of these strains has been associated with improvements to gu<sup>t</sup> health via the suppression of pathogenic bacteria, improved gastro-intestinal flow, and short-chain fatty acid-driven immunomodulation, which are factors that are currently deemed essential in preventing intestinal and colonic cancers [18,19]. Carbohydrates that can reach the caeco-colon undigested and demonstrate the microbiota-enhancing effects described fall under the designation of prebiotics [20].

Given the recognition of how important gu<sup>t</sup> health is in the prevention of serious human disease, the study of prebiotics is currently at the height of its development [18]. The complexities of digestion and the changes it incurs on the chemistry and function of dietary compounds has led researchers to develop increasingly elaborate in vitro models when assessing the prebiotic potential of foods and supplements [19]. While these advances have greatly improved the authenticity of the attributed label of prebiotics and led to grea<sup>t</sup> new insights on the bioavailability of certain nutrients, they are often difficult to reproduce without highly specialised, often custom-made equipment or access to clinical samples of human faeces [18,21]. Thus, quick assessments of the prebiotic potential of new foods are difficult to execute with these methodologies. Simpler, in vitro batch fermentations are still considered valuable screening tools for this very reason, despite their inadequate simulation of the digestion process [17].

With the removal of its lipid content, the spent biomass of *Aurantiochytrium* sp. still holds the potential of a nutritionally and functionally valuable food product. Considering that these organisms can accumulate approximately 50% of their weight in lipids, the defatted and dried remainder is a highly concentrated mixture of proteins and carbohydrates of exotic origin and whose nutritional and functional potential remains unexplored [22]. The present study seeks to confirm these claims via chemical analysis of the spent *Aurantiochytrium* biomass in addition to a screening of its antioxidant and prebiotic potentials.
