Sorvall lynx 6000 superspeed centrifuge

At baseline and conclusion of each study phase, participants visited the Brain, Performance and Nutrition Research Centre (BPNRC/NUTRAN, Northumbria University, Newcastle, UK) having fasted overnight (Fig. 1). At each visit, a first spot urine sample and stool sample (collected ≤ 10 h prior to study visit) were collected from participants. Urine samples were immediately put on ice, then aliquoted into sterile 1.5 ml Eppendorf tubes and stored at − 80 °C until analysis. Stool samples were put on ice at collection and then aliquoted into sterile 2 ml tubes and stored at − 80 °C for gut microbial analysis. The remainder of the sample was weighed, diluted 1:1 with PBS (Sigma) and homogenised in a stomacher (Seward Stomacher 400 Circulator) at 200 bts/min for 2 min. The homogenates were transferred into polypropylene tubes and centrifuged at 65,000 ×g, for 2 h, at 4 °C using an ultra-speed centrifuge system (Thermo Scientific Sorvall LYNX 6000 Superspeed Centrifuge). The supernatants were then filtered through a 0.44 µm polyethersulfone (PES) syringe filter prior to a second filtration through a 0.22 µm PES syringe filter (Fisher Scientific). The supernatants, representing the faecal water fraction, were aliquoted into sterile 1.5 ml Eppendorf tubes and stored at − 80 °C for analysis of faecal water genotoxicity and metabolites.

Around 20 l of spent brewer’s yeast (S. cerevisiae) were obtained from a local brewing company at the end of beer brewing process and centrifuged in 1-liter aliquots to separate yeast biomass and liquid brewing residues using a Sorvall Lynx6000 superspeed centrifuge (Thermo Fisher Scientific, United States) at 17,568 x g for 20 min. Cell pellets obtained after centrifugation were washed by resuspension in 1 liter deionized water followed by centrifugation. This washing step was repeated three times to remove remaining alcohol and sugars from the brewing process. After centrifugation, the yeast pellets were frozen at −20°C. Frozen yeast biomass was lyophilized using a CHRIST^® Alpha 1-4 LDplus freeze dryer (CHRIST, Germany), ground to a fine powder using a kitchen blender and stored in a benchtop desiccator to protect it from moisture. Sieve analysis was used to obtain the particle size distribution using a stacked sieve tower with mesh sizes ranging from 2 mm to 0.1 mm.

Graphene oxide was prepared via a modified Tour et al. [4 (link)] synthesis in a custom-built rig designed to employ up to 10 L of concentrated acid in two jacketed reactors with overhead stirrers. In a typical synthesis, a 10:1 mixture of concentrated acids (3 L H₂SO₄:0.3 L H₃PO₄) was added to 24 g of natural graphite flakes (150–500 µm; Sigma-Aldrich, UK) under vigorous stirring, followed by the addition of 144 g of KMnO₄ (6 weight equivalent) in small portions. The reaction mixture was then kept at 50 °C under vigorous stirring for 18 h. The mixture was cooled to room temperature, and the oxidation reactions were stopped by a drop-wise addition of 1.72 L of 2 wt% aqueous H₂O₂. The GO suspension was washed by repeated centrifugation and re-dispersion in distilled water, using a Sorvall LYNX 6000 Superspeed Centrifuge (Thermo Scientific, UK). This washing procedure was repeated until the pH of the supernatant matched that of the used distilled water, which typically occurred after 16 washing cycles. Typically two low-speed (<1000 rpm) centrifugation cycles were then performed to remove un-exfoliated graphite particles.