Mini star

The ACE-inhibitory activity was performed as described by Hayakari [95 (link)] and used by Hell et al. [43 (link)]. Briefly, 20 μL of ACE (250 mU per mL of borate buffer, 1 M NaCl, pH 8.3) and 80 μL of phosphate buffer (pH 8.3) were added to 20 μL of water-soluble macroalgae extract at concentrations of 8 and 10 mg prot/mL (A_sample), or to enalapril which was used as the positive control (A_control), then incubated at 37 °C for 10 min. Simultaneously, a replicate of each sample was incubated at 95 °C for 10 min to inactivate ACE and used as negative control (A_blank). Then, 40 μL of Hippuryl-L-histidyl-L-leucine hydrate 6.25 mM (ACE substrate) was added to all samples and incubated for 60 min at 37 °C. The enzymatic reaction was stopped by heating to 95 °C for 10 min. Volumes of 360 μL of 2,4,6-trichloro-s-triazine (TT, 3% v/v in 1,4-dioxane) and 480 μL of phosphate buffer (pH 8.3) were added to each sample, vortexed, and centrifuged at 2000 ×g for 30 s (MiniStar, VWR, St. Catharines, ON, Canada). A transparent 96-well microplate (Clear F-Bottom, Greiner Bio-One GmbH, Frickenhausen, Germany) was filled with 200 μL of each sample per well and the absorbance was read at 382 nm (xMark, Biorad, Hercules, CA, USA). The ACE-inhibition rate (%) was calculated using the following formula: $$\mathrm{ACE}\mathrm{inhibition}(\%)=\frac{A_{control}-A_{sample}}{A_{control}-A_{blank}}\times 100$$

The cultures sampled (50 mL) were centrifuged for 10 min at 3500 rpm in VWR Mini Star microcentrifuge (VWR, Radnor, PA, USA). The supernatant was collected to quantify glucose, phosphate, and ammonium concentrations.
When necessary, the supernatant was diluted in a saline solution (10% sodium chloride, 90% distilled water). Freestyle precision Neo kit (Abbott, Witney, Oxon, UK) was used to determine glucose concentration in g/L.
Ammonia and phosphate Sera Tests (Sera, Heinsberg, Germany) were used to determine ammonium and phosphate concentrations, respectively. The supernatant was diluted with distilled water when necessary. The absorbance was measured at the wavelength of 697 nm for ammonium and 716 nm for phosphate. The absorbances were measured using Genesis 10S UV–Vis (Thermo Scientific, Waltham, MA, USA).