**4. Conclusions**

This study attempts an early exploration of functional food-related uses of spent *Aurantiochytrium* sp. biomass, which is a significant part of the industrial waste associated with the production of high added-value PUFAs from this source. Doing so could attribute this waste with value comparable to its lipid fraction and thus greatly increase the profitability and sustainability of all industrial exploitation of *Aurantiochytrium*. The chemical characterisations performed on the defatted biomass show a highly protein and dietary fibre-rich product that is simultaneously rid of most of its most caloric fraction. The amino acid profile revealed a fairly balanced distribution of essential variants, though it was not enough to warrant the use of this product as a protein supplement individually. High levels of glutamic and aspartic acid sugges<sup>t</sup> it could be used as a source of flavourenhancing umami compounds. The spent *Aurantiochytrium* was depleted of most of its lipophilic antioxidant compounds, but some residual activity was still registered by the FRAP assay, which was likely caused by antioxidant amino acids. The prebiotic potential assays revealed that after an in vitro enzymatic digestion, the sample held the capacity to enhance the growth of important probiotic strains. Together with its high dietary fibre content, these results point to a promising prebiotic supplement as one of the potential uses for the depleted biomass.

The results presented warrant a grea<sup>t</sup> deal of follow-up research, as many of its conclusions could be built upon a better understanding of the elemental composition of *Aurantiochytrium* sp., particularly of its non-lipid fractions. Additionally, the prebiotic potential demonstrated here is the result of a screening that is limited in scope and can be followed by either the testing of more strains and/or the use of a more in-depth digestion simulation and gu<sup>t</sup> microbiota consortia from clinical samples. With these next steps fulfilled, a valuable application of the main by-products of *Aurantiochytrium* cultivation could be quickly implemented in both existing industries and upcoming biorefineries looking to maximise their profit and sustainability.

**Author Contributions:** Conceptualization, C.M.S., J.A.S., N.M.B., M.C.P., J.C., M.M.R.d.M. and M.F.L.L.; methodology, J.R., B.T. and R.M.; validation, R.F., C.F., N.M.B. and M.F.L.L.; investigation, J.R. and R.F.; resources, M.F.L.L.; writing—original draft preparation, J.R.; writing—review and editing, R.F., C.F., N.M.B. and M.F.L.L.; supervision, C.F. and M.F.L.L.; project administration, C.M.S., J.A.S., N.M.B., M.C.P., J.C. and M.F.L.L.; funding acquisition, C.M.S., J.A.S., N.M.B., M.C.P., J.C. and M.F.L.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study had the support of Fundação para a Ciência e a Tecnologia (FCT) through the Strategic Project UID/MAR/04292/2020 to MARE, UIDB/50011/2020 & UIDP/50011/2020 to CICECO, and the University of Aveiro and FCT/MCT for the financial support for LAQV-REQUIMTE research Unit (FCT UIDB/50006/2020) through national founds, and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement. This work was also funded by the project Algavalue—Valorizaç<sup>ã</sup>o dos subprodutos do processo biotecnológico de produção de esqualeno e DHA pela microalga *Aurantiochytrium* sp. (POCI-01-0247-FEDER-017680) supported by COMPETE 2020, and Integrated Programme of SR&TD "SmartBioR" (reference Centro-01-0145-FEDER-000018) cofunded by Centro 2020 program, Portugal 2020, European Union, through the European Regional Development Fund.

**Data Availability Statement:** Data is available upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.
