Avanti j 25

Cells were cultivated on 50 mM glycine betaine in 0.5 to 2 l carbonate buffered complex medium to late exponential growth phase (OD₆₀₀ of 0.3) and then harvested by centrifugation (Avanti J-25 and JA-10 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) at 8000 rpm and 4 °C for 10 min. The harvested cells were subsequently washed with 30 ml of buffer containing 50 mM imidazole (pH 7.0), 20 mM KCl, 20 mM MgSO₄, 4 mM DTE and 4 µM resazurin by centrifugation at 8500 rpm and 4 °C for 10 min (Avanti J-25 and JA-25.50 Fixed-Angle Rotor; Beckman Coulter, Brea, CA, United States) and resuspended in 5 ml of imidazole buffer and kept in 16-ml Hungate tubes. All the steps were performed under strict anoxic conditions in an anoxic chamber (Coy Laboratory Products, Grass Lake, MI, United States) filled with N₂/H₂ (96–98%/2–4%; v/v). The total protein concentration in the resting cells was determined according to a previously described method [38 (link)].

First, 0.5 g of ground whole grain was extracted with 10 mL of methanol:1 M HCl 85:15 (v/v) and maintained under stirring in the dark for 30 min. Samples were centrifuged at 8000 g for 20 min at 4 °C (Avanti J-25, Beckman Coulter, CA, USA), filtered with a 0.45 μm PTFE filter (VWR International, Fontenay-sous-Boys, Francia), and stored at −20 °C until used. Each extraction was performed in triplicate.

Soluble phenolic compounds were extracted as follows: samples of 0.5 g of ground whole grain were added with 10 mL of ethanol:water 60:40 (v/v) and maintained under stirring in the dark for 2 h. Samples were centrifuged at 2000 g for 15 min at 4 °C (Avanti J-25, Beckman Coulter, CA, USA). The extracts were filtered with 0.45 μm filters (VWR International, Fontenay-sous-Boys, Francia) and stored at −20 °C until used. Each extraction was performed in triplicate. These extracts were used to determine soluble phenolic content and in vitro antioxidant activity, as well as for the separation of different classes of molecules by High Performance Thin Layered Chromatography.

At the beginning and end of the study, ten ccs of venous blood samples were taken, and after the separation of serum, SIRT and Fetuin levels were measured in the plasma of the participants. Blood samples were collected into tubes with and without EDTA. Blood samples without EDTA Centrifuged (Beckman Avanti J-25, USA) at a rate of 3000 rpm for 10 min in order to the separation of serum. Blood samples were kept at − 70 °C (Snijdes, Germany) in Diabetes Research Center and then transferred to the laboratory of Velayat Hospital of Qazvin University of Medical Sciences, and the measurements were carried out. Fasting plasma glucose (FPG) concentration was measured by the enzymatic method using an Abbot ModelAclyon 300, USA auto analyzer with Pars-Azmone kit (Tehran, Iran). Plasma insulin was measured by using a chemiluminescent immunoassay method (LIAISON analyzer (310360) Diasorin S.P.A., Verecelli, Italy). Insulin resistance (HOMA-IR) was calculated according to the following formula: HOMA-IR = (fasting insulin (U/ml) × FPG (mg/dl)/405) [17 (link)]. Serum levels of SIRT1 and Fetuin-A in participants’ plasma were measured using a special kit and by ELISA method (Diameter, Italy and Bioassay Technology Laboratory, China).

Approximately 500 mg of PL were homogenized in TN buffer and clarified at 1.400×g for 15 min using a JA 25.50 Beckman rotor in a Beckman Avanti J25 centrifuge. Supernatant was collected and a second clarification was performed at 10,000×g for 15 min. For pelleting, the supernatant was then centrifuged at 150,000×g for 4 h in a Ti 70 rotor in a Beckman ultracentrifuge L8-70M. After re-suspension in TN buffer, the final pellet was layered onto 15–30 % (w/w) sucrose in a TN buffer gradient and run at 135,000×g for 3 h using an SW 40 Ti rotor. The 0.5 ml fractions were collected from the gradient manually. The presence of viruses in fractions was examined using a spectrophotometer (Beckman DU-600) at 260 nm and 280 nm. The desired fractions were diluted in TN buffer, pelleted at 150,000×g for 4 h in a Ti 70 rotor and finally suspended in water.

Two grams of commercial crab shell chitin (CC) was added to 200 mL of GE and allowed to interact on a magnetic stirrer cum heater, set at 70 °C for 1, 2, 3, 4, 5, 7 days. The retrieved samples were centrifuged (Beckman Coulter Avanti J-25, USA) at 3000 rpm for 3 min to pellet the bigger debris. The pellet was discarded and the supernatant was centrifuged at 19,000 rpm for 15 min and the pellet was suspended in distilled water. One set of the pellet was dried and used for FTIR analysis.