Universal 320r

Venous blood samples were drawn intraoperatively by venipuncture using 1.6 mL EDTA tubes (S-Monovette, Sarstedt, Sarstedt-Straße 1, 51588 Nümbrecht, Germany) 5–20 min after the end of infusion. Blood samples were then centrifuged at 4900 rpm for 10 min at 4 °C (Hettich Universal 320 R, Andreas Hettich GmbH & Co.KG, Tuttingen, Germany). The obtained plasma was separated from the cellular components and divided into four aliquots of 100 μL each. Aliquots were stored at − 80 °C until the day of analysis.
Blood sampling for PRF was performed together with blood plasma collection using at least four and up to six sterile vacuum PRF tubes (Dr. Choukron Glass A-PRF Tubes, 10 mL, Process for PRF, Nice, France) and centrifuged with a Duo Quattro centrifuge (PRF, Nice, France) at 2300 rpm for 12 min and RCFmax = 652 g. Samples were stored at − 80 °C for further processing in the Institute for Pharmacy and Food Chemistry of the University of Würzburg or directly processed for agar diffusion tests in the Institute for Hygiene and Microbiology of the University of Würzburg. Due to a limited amount of PRF membranes, it was not possible to proceed membranes of each patient for mass spectrometry (Bioanalysis of plasma and PRF) and agar diffusion tests (Agar diffusion test with CLI-loaded PRF membranes). Therefore, membranes acquired from different patients were used for the two methods.

Beer bitterness was measured according to EBC 9.8/2020 standard [61 ], by centrifuging with Hettich Universal 320 R (Hettich GmbH & Co., Tuttlingen, Germany) the beer at 4000 rpm for 20 min at 20 °C, 1 mL of 3 M HCl and 20 mL isooctane were added to 10 mL beer sample and it was mixed with IKA KS 260 basic shaker (IKA^®-Werke GmbH & Co., Staufen im Breisgau, Germany) at 450 rpm for 25 min. The samples were left in complete darkness for 30 min and were measured spectrophotometrically (Hach Lange DM 6000, Hach Lange GmbH’s, Düsseldorf, Germany) at λ = 275 nm wavelength. Triplicate measurements were performed for all samples.

Cells of R. erythropolis strain MI2 were cultivated in mineral salt medium (MSM) [22 (link), 23 (link)] containing 60 mM sodium succinate or 30 mM DTDB as carbon source in baffled flasks at 30°C and 120 rpm on a rotary shaker (New Brunswick Scientific Co., Inc., NJ, USA). Growth was monitored using a Klett-Summerson photometer (Manostat Corporation, NY, USA). 150 ml pre-cultures were cultivated for 20 h, and after normalizing the cell density mass to 0.1 at OD₆₀₀, the 500 ml volume main cultures were inoculated. Samples for proteomic analysis were withdrawn during the exponential growth phase (harvest of 300 ml culture) and 7 h after entering the stationary phase (harvest of 200 ml culture). For cell harvest, the samples were transferred to 50 ml falcon tubes and were centrifuged for 30 min at 4°C and 7000 x g (Hettich Universal 320 R, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany). The supernatant was discarded, and the remaining cell pellets were washed with fresh MSM without carbon source. Afterwards they were stored at −20°C until further use.

Intact biomass, controls, and freeze-dried hydrolysates (from both blades and PI) were dispersed at 20 mg/mL in deionized water or in the appropriate buffer for each bioassay and the samples were centrifuged (21,250× g, 5 min, at room temperature; Hettich Universal 320R, Andreas Hettich GmbH & Co. KG, Germany) to remove insoluble material. Supernatants were then diluted as needed. Hydrolysates and controls were characterized for their in vitro antioxidant activity using a range of bioassays. The oxygen radical absorbance capacity (ORAC) and ferric reducing activity power (FRAP) were assessed as described by Harnedy and FitzGerald [25 (link)]. Samples were also assessed for their ability to scavenge DPPH· [47 (link)], HOCl [1 (link)] as well as for their Trolox equivalent antioxidant capacity (TEAC) using potassium persulfate to generate the radical (ABTS·+), according to the procedure described by Kleekayai et al. [48 (link)]. Experiments were performed in triplicate (n = 3).

Healthy olive fruits from Coratina cv. olive trees were harvested manually during the second half of October 2019 and 2020 (three trees per treatment) in similar ripening stages (ripening index, RI = 1.5–2.0) determined by the method of Beltrán et al. [33 (link)]. The fruits collected from each tree were processed within 24 h of harvesting into oil, separately, to obtain three oil samples per treatment. Oil samples were obtained by processing olive fruits (3 kg per repetition) by centrifugal extraction at the Abencor laboratory oil mill (MC2, Ingenierias y Sistems, Seville, Spain). The olives were ground with a metal hammer mill, while mixing was carried out at a temperature of 25 ± 1 °C in thermostatted vertical mixers for 40 min. After mixing, the olive paste was centrifuged for 90 s at a speed of 3500 rpm, and the oil together with the vegetable water was discharged into separation cylinders. The decanted oil was clarified by additional centrifugation using a Hettich Universal 320R (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany) for 1 min at 4000 rpm after which it was separated from the vegetable water by decantation and used as a sample. All samples were subsequently stored in dark glass bottles during analyses, which started immediately after oil production.

All strains used in this study are listed in Table 1. Cells of E. coli were inoculated in Lysogeny Broth medium [31 ] at 37°C on a rotary shaker (New Brunswick Scientific Co., Inc., NJ, USA) at 130 rpm with addition of applicable antibiotics (ampicillin (Ap) 75 μg ml^-1; tetracycline (Tc) 12.5 μg ml^-1; chloramphenicol (Cm) 34 μg/ ml), if necessary.
Cells of A. mimigardefordensis strain DPN7^T were cultivated with nutrient broth or mineral salt medium (MSM) [35 (link)], which was supplemented with either 60 mM succinate, MS or DTDP, at 30°C. Liquid cultures were incubated on a rotary shaker in Erlenmeyer flasks without baffles. Growth was monitored via a Klett-Summerson photometer (Manostat Corporation, NY, USA) or an Ultrospec 2000 photometer (Pharmacia Biotech, Uppsala, Sweden). Precultures were incubated for 20 h, and main cultures of 400 ml for growth experiments and proteomic studies were inoculated to 30–40 KU at t = 0 h. For proteome analysis cells were harvested 6 h after the cultures had entered stationary phase by centrifugation for 30 min at 4°C and 7,690 x g (Hettich Universal 320 R, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany). Cell pellets were stored at -20°C if not used immediately, whereas the supernatants were discarded. Solid media contained 20 mM carbon source and 1.8% (wt/vol) Bacto-Agar^™ (Becton, Dickinson and Company, New Jersey, USA).

The oil binding capacity was achieved by subjecting the spreads to a centrifugal force according to the method described by Okuro et al. [42 (link)]. Approximately 5 g of each oleogels was introduced after melting into conical tubes with a screw cap, Falcon type (50 mL), crystallized at 4 °C and placed in the Hettich Universal 320R centrifuge (Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany). These were centrifuged for 30 min at a maximum speed of 9000 rpm corresponding to a relative centrifugal force of 13.1 × 10³ g. The temperature during centrifugation was set at 4 °C, but because no oil release was observed, the experiment was conducted at 21 °C in order to observe the stability of the product under ambient temperature. After centrifugation, the released oil was drained by inverting the tubes for 30–35 min. The comparison between the mass of the sample before and after a centrifugation cycle allowed the determination of the oil loss as a percentage of the initial oleogel mass. The analysis of the oil binding capacity was performed in duplicate.