Ultra turrax homogeniser

The extraction of metabolites from both milk creams and ricotta samples was carried out as previously reported [19 (link),20 (link)], with some modifications. Briefly, 2 g of each sample was extracted using a 10-millilitre mixture containing 80:20 (v/v) methanol:water, added with 0.1% of formic acid, with an Ultra-Turrax homogeniser (IKA-Werke, Staufen im Breisgau, Germany) for 4 min at room temperature. Next, the samples were centrifuged at 12,000× g for 10 min at 4 °C and the supernatants were incubated overnight in a freezer (−18 °C) following the addition of a 5% TCA solution, to remove large biomolecules (such as proteins). The supernatants were then filtered through 0.2-micrometre cellulose membranes and transferred to amber vials for the further metabolomic analysis. Each sample was analysed considering three biological replications.

Frozen colonic tissues were homogenised on ice with an ultra-turrax homogeniser (IKA-Werke GmbH & Co., Germany) in a cold buffer (composition: sucrose 250 mM, Tris 5 mM, EGTA 1 mM, Triton X-100 0.02%, pH 7.4). Then, homogenates were centrifuged at 12,000× g for 15 min at 4 °C (EuroClone, Speed Master 14 R centrifuge, Italy). The supernatant was used for determination of the citrate synthase activity, and the protein concentration in the supernatant was determined spectrophotometrically by Bradford assay (Bio-Rad, USA), using a microplate reader (EnSpire, PerkinElmer, USA). Then, proteins were diluted in Tris-buffer 100 mM (pH 8.2) containing 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB, 100 µM) and acetyl-coenzyme A (100 µM). The assay was performed in 96-well plates (1 µg of proteins per well) and the reaction was initiated by addition of oxalacetate 500 µM. The absorption of the reaction product was measured spectrophotometrically at 30 °C and 412 nm every 30 s for 15 min. Citrate synthase activity was determined by comparing the activity in the samples to that of known concentrations of the isolated enzyme (Sigma-Aldrich, St. Louis, MI, USA). Citrate synthase activity was expressed in mU/µg protein. Data were analysed by a computer fitting procedure (software: GraphPad Prism 5.0).