**2. Materials and Methods**

### *2.1. Recovery of Spent Aurantiochytrium sp. Biomass*

*Aurantiochytrium* sp. biomass under the commercial label Algamac 3050 was purchased from Pacific Trading–Aquaculture Ltd. (Dublin, Ireland). The biomass was supplied in vacuum-sealed plastic bags and as coarse flakes approximately 1.5 mm in length and 0.5 mm in thickness. Removal of the lipid fraction was carried out to simulate its industrial processing for the recovery of PUFAs using a lab-scale Soxhlet extraction apparatus. Samples of 5 g of biomass were loaded in a Soxhlet cartridge and extracted with n-hexane for 6 h. At the end of the extraction, the spent biomass was recovered from the cartridge solvent and dried overnight in a 50 ◦C oven.

### *2.2. Chemical Analysis of the Spent Aurantiochytrium sp. Biomass*

Protein content was estimated using a LECO FP-528 DSP nitrogen analyser (LECO, St. Joseph, MI, USA). Fat content of both whole and spent *Aurantiochytrium* was determined according using the Bligh and Dyer technique with the modifications employed by Burja et al. (2007) applied to the standard method [23]. Ash and fibre content were determined according to the Association of Official Analytical Chemists (AOAC) standard methods 942.05 and 985.29, respectively [24,25]. A protein-rich fraction obtained from the defatted *Aurantiochytrium* sp. was prepared according to the procedure detailed by Vallabha et al. (2016), with some modifications [26]. These included a more prolonged extraction time (overnight) and the combination of all precipitated protein fractions. These were analysed using reverse-phase high-performance liquid chromatography following the method of Bidlingmeyer et al. (1984) after being subjected to a 24 h acid hydrolysis with 6N HCl with 0.1% phenol under vacuum and derivatisation with phenyl thiocarbamoyl [27].

### *2.3. Enzymatic Digestion of the Spent Aurantiochytrium sp. Biomass*

A two-step simulated digestion of the defatted *Aurantiochytrium* sp. biomass was performed with a procedure adapted from Gawlik-Dziki et al. (2009) [28]. A solution of simulated saliva was prepared by dissolving 2.38 g Na2HPO4, 0.19 g KH2PO4, and 8 g NaCl in 1 L of distilled water and adjusting its pH to 6.75. Then, the solution was supplemented with 200 U of α-amylase (EC 3.2.1.1.). The simulated gastric digestion solution was an acidic (pH 1.2) 0.32% pepsin (porcine stomach mucosa, pepsin A, EC 3.4.23.1) dilution in 0.03 M NaCl. In 50 mL plastic centrifuge tubes, an approximate weight of 10 g of defatted *Aurantiochytrium* sp. flakes were mixed with 50 mL of simulated saliva and incubated in a 37 ◦C water bath for 10 min with occasional stirring using a steel spatula. Then, the slurry was brought to a pH of 1.2 using 5 M HCl, after which 50 mL of the simulated gastric solution were added. Then, a 120 min, 37 ◦C water bath incubation took place with occasional manual stirring. Afterwards, the digestion was halted via a short exposure to a 70 ◦C water bath (approximately 60 s), and the pH was brought up to 6.0 with a 1 M solution of NaHCO3. Then, the entire digested slurry was treated as the digested defatted *Aurantiochytrium* sp. sample, from which 2 mL aliquots were gathered and stored at −20 ◦C prior to analysis.

### *2.4. In Vitro Prebiotic Potential Assay*

The digested samples' potential to promote the growth of probiotic lactic acid bacteria was evaluated using a simplified and miniaturised method based on publications of Wichienchot (2010) and Liu et al. (2016) and represented in Figure 1 [29,30]. The three bacterial strains selected for this assay include *Lactobacilus delbrueckii* subspecies *bulgaricus* DSMZ 20081, *Bifidobacterium bifidum* DSMZ 20456, and *Weissella cibaria* DSMZ 14295. Precultures of *W. cibaria*, 48 h, 30 ◦C and pre-cultures of *B. bifidum* and *L. delbrueckii,* 72 h, 37 ◦C were prepared in MRS agar plates, with the latter two cultures having been maintained under anaerobic and oxygen-depleted atmospheres respectively using Mini Anaerocult A and C kits (Merck, Darmstadt, Germany). All pre-culture plates were prepared in triplicate as independent replicas. From these, 2.5 × 10<sup>6</sup> CFU/mL (*W. cibaria*) and 5.0 × 10<sup>6</sup> CFU/mL (*L. delbruecki* and *B. bifidum*) cellular suspensions were prepared in saline solution (0.85% NaCl; VWR). A set of master mixes were prepared encompassing all the necessary sample, blank, and control conditions in a 3:1:1 ratio of MRS medium, inoculum suspension, and sample, respectively. Digested *Aurantiochytrium* sp. samples were used without any further dilution, and digestion blanks provided a measure of growth induced by the enzymatic mixture (vehicle). Negative controls used the appropriate volume of saline solution instead of the digested sample. Then, 200 μL of each mixture were transferred to sterile roundbottom 96-well microplates and incubated for 72 h, with optical density (OD) readings occurring every 24 h at 600 nm using a microplate reader (EPOCH 2, BioTek Instruments, Winooski, VT, USA). Incubation of *W. cibaria* was conducted in aerobic conditions at 30 ◦C, while *B. bifidum* and *L. delbrueckii* microplates were enclosed in sealed bags under anaerobic and oxygen-depleted atmospheres at 37 ◦C as stated above.

A validation trial was performed using a 5.0 × 10<sup>6</sup> CFU/mL suspension of *L. delbrueckii* and inulin as a reference probiotic. Inulin concentrations ranging from 0.01 to 1% (*w/v*) were prepared in master mixes with identical MRS media and inoculum ratios as the main assay.

### *2.5. Antioxidant and Lipid Oxidation Protective Assays*

The digested and defatted *Aurantiochytrium* sp. sample was subjected to a set of three antioxidant potential assays. Ferric-reducing antioxidant potential (FRAP) activity assay was performed according to Dudonné et al. (2009) with slight modifications to sample dilution rates [31]. First, 195 μL of ferric 2, 4, 6-tri (2-pyridyl)-s-triazine (TPTZ) along with 5 μL of either sample or iron sulphate standard were incubated for 30 min at 30 ◦C. The concentrations of the latter ranged from 20 to 1000 μM. A minimum of three independent assays were performed for each extraction condition tested. The 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical reduction assay used a 96-well microplateadapted protocol [32,33]. The working reagen<sup>t</sup> was prepared by dissolving DPPH radical in absolute ethanol at a concentration of 0.1 mg/mL. The assay was conducted by pipetting 10 μL of each standard's or sample's concentration and of the digestion vehicle as control per well (8 wells each). In four wells, 190 μL of working reagen<sup>t</sup> was added, and in the other four, 190 μL of ethanol was added. The plate was incubated in the dark for 60 min at room temperature, after which its absorbance (Abs) was read at 515 nm (EPOCH 2 microplate reader, BioTek® Instruments, Winooski, VT, USA). The amount of DPPH radical reduced by the standard per samples was calculated using a standard curve previously obtained, following the formula:

$$[DPPH](mM) = \frac{Abs\_{Sample} - 0.0391}{5.1238}.\tag{1}$$

**Figure 1.** Flowchart representation of the miniaturised prebiotic potential assay employed in this study. Bacterial cultures were pre-grown in their respective optimal conditions for up to 72 h prior to the assay. The first step of the serial dilutions was performed identically for all cultures, with the following step adjusting for the required concentration. Inulin concentrations listed under "Tested mixtures" are higher than the tested concentrations as to account for the dilution occurring in the microplate well. Oxygen-depleted and anaerobic conditions were achieved using Merck's Mini Anaerocult A and C kits following manufacturer specifications. The incubations were prolonged for up to 72 h.

For each sample, Abs (515 nm) was calculated by subtracting the mean absorbance of the wells containing the sample and ethanol to the mean absorbance of the wells containing the sample and working reagent. The assay was performed in triplicate. The lipid peroxidation inhibitory potential (LPIP) was evaluated by a method adapted from Félix et al. (2020) and Yen and Hsieh (1998) [32–34]. The method was designed based on the auto-oxidation of a pure suspension of PUFAs in contact with air, in which case the only peroxides present are the lipid peroxides, which are quantifiable by the thiocyanate method. Briefly, a linoleic acid (LA) suspension was prepared (20 mM LA in Tween 20 at 5.6 mg/mL prepared in phosphate-buffered saline (PBS) at 20 mM, pH 7.1) and used as substrate. Then, 25 μL of extract at 1 mg/mL were pipetted onto a 2.0 mL microtube, in triplicate, and 125 μL of LA suspension and 100 μL of PBS (same as above) were added. As blanks, tubes with extract but with LA suspension's solvent (Tween 20 in PBS) instead of LA suspension were used (to determine peroxides native to the extract and subtract them from final result). As positive control (maximum peroxidation), 25 μL of extract vehicle (extract's solvent) and 125 μL of LA suspension were used (along with 100 μL of PBS), and as negative control, 25 μL of vehicle as 125 μL of LA suspension's solvent with 100 μL of PBS was used. All microtubes were incubated at 37 ◦C for 48 h in the dark and well capped. The experiment was performed in triplicate. Then, each tube was used to quantify peroxides by the thiocyanate method. From each tube, 20 μL (in triplicate) were sampled and added to a tube containing 940 μL of ethanol at 75% (v/v) and 20 μL of ammonium thiocyanate at 30% (w/v). Then, 20 μL of iron (II) chloride at 20 mM prepared in HCl at 3.5% (w/v) were added to each tube, and the mixture was properly homogenised using a vortex. Afterwards, each tube was used to read the absorbance in a microplate reader by pipetting 4 wells of 200 μL with the mixture. The absorbance was read at 500 nm, and the inhibitory potential was calculated:

$$LPIP\left(\%\right) = 100 \frac{1 - Abs\_{SampleBlank}}{Abs\_{Pos.ClrI} - Abs\_{Nç.Clrl}}\tag{2}$$
