Up200s ultrasonic processor

Three aqueous phospholipid premixes were chosen for the emulsion development. In total, 12 emulsions were prepared that consisted of a phospholipid premix and an oil phase (white soft paraffin (PO), medium-chain triglycerides (MCT) and refined castor oil (CO)), as well as a cosurfactant. The cosurfactants used were Plantacare^® 1200 UP (main component: Lauryl glucoside (LG)) and TEGO^® Betain F 50 (main component: Lauramidopropyl betaine (LAPB)).
During the first emulsification step, the respective oil was added to the aqueous phospholipid premix and homogenized for 4 min at 9000 rpm and 2 min at 13,400 rpm using an Ultra Turrax T 25 disperser (IKA-Werke GmbH & Co. KG, Staufen, Germany) equipped with a S 25 N-18 G dispersing tool. After adding the cosurfactant, the preparation of the emulsions was completed using an ultrasonic homogenizer (UP200S ultrasonic processor, Hielscher Ultrasonics GmbH, Teltow, Germany) equipped with a S7 titanium sonotrode with an amplitude setting of 50% (0.5 cycle) for 5 min. All emulsions were analyzed 24 h after preparation.

All applied chemicals were purchased from Sigma Aldrich (Germany), if specified in purities ≥95%. The cell disruption by sonification was performed on a UP 200s ultrasonic processor (Hielscher Ultrasonics GmbH, Teltow, Germany) with an S1 sonotrode (Hielscher Ultrasonics GmbH, Teltow, Germany). The reactions were incubated in an Eppendorf thermomixer comfort (Eppendorf, Hamburg, Germany) and samples were centrifuged in an Eppendorf centrifuge 5424 (Eppendorf, Hamburg, Germany). Sample analysis was performed on an Agilent technologies 1260 infinity high performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA). Metabolic profile analysis was performed on an Agilent Technologies 6890N gas chromatography system (Agilent Technologies, Santa Clara, CA, USA) coupled to a GCT Premier mass spectrometer (Waters Corporation, Milford, MA, USA). Pipetting assistance was provided by Tecan Freedom Evo 1 (Tecan, Männedorf, Swiss).

The anthranilate phosphoribosyltransferase overproducing strain Tp679 (pCES208-trpD) was inoculated from an overnight culture and was cultivated for 24 h in LB medium at 30°C with 120 rpm before cells were centrifuged for 10 min at 4°C and 4,000 rpm and stored at −20°C. After resuspension in 100 mM Tricine buffer (pH 7.0), the cells were sonicated for 9 min at 55% amplitude and 0.5 cycles on ice in the UP200S Ultrasonic Processor from Hielscher Ultrasound Technology. The supernatant obtained after centrifugation (60 min, 4°C, 16,400 rpm) was used as crude extract for the enzyme assay. The activity was assayed fluorometrically by monitoring the decrease of anthranilate (Ant) at room temperature. The reaction mixture with a final volume of 1 mL contained 100 mM Tricine buffer (pH 7.0), 15 μM Ant, 0.3 mM PRPP, 10 mM MgCl₂, and the crude extract and was filled in a quartz glass cuvette (Hellma Analytics, High Precision cell, Light Path 10 × 4 mm). Ant was detected by fluorescence at 325 nm excitation and 400 nm emission wavelength with the Shimadzu Spectrofluorophotometer RF-5301PC. Protein concentrations were determined by the Bradford method (Bradford, 1976 (link)) with bovine serum albumin as reference. Means and errors from triplicates were calculated.