Th two temperature-controlled 25 mL stress vessels (Sitec, Maur, Switzerland), which are
Th two temperature-controlled 25 mL stress vessels (Sitec, Maur, Switzerland), that are made for pressures up to a limit of 700 MPa. Prior to the pressure application, the samples were filled into polypropylene test tubes (two mL) and wrapped with sealing film. The stress was enhanced in the rate of 200 MPa/min, whilst it was released quickly at the end on the holding time by valve opening. The decompression time was much less than 10 s. Kale puree samples had been treated for time periods involving five and 40 min, at pressures up to 600 MPa at RT. All treatment options have been performed in duplicate and analysed thrice. 2.five. Determination of Carotenoids, Vitamin E, and Chlorophyll 2.five.1. Extraction Procedure Kale samples (0.five g) were weighed into conical test tubes (50 mL). Then, 200 mg of magnesium carbonate, 200 mg of sodium sulfate, and 25 of an internal regular (Lucantin-Yellow, -tocopheryl acetate) were added. Afterwards, 20 mL of a mixture of MeOH/MtBE (50:50 = v/v), which includes 0.1 wt BHT, served as extraction solvent, following 5 s of vortexing. All samples have been then sonicated four occasions in an ice bath beneath decreased daylight situations. Centrifugation at 7000 rpm was Inositol nicotinate supplier applied for phase separation in between repetitions of extractions. Consequently, combined upper phases had been evaporated below decreased stress utilizing a rotary evaporator at 30 C. Afterwards, the residue was dissolvedAntioxidants 2021, 10,four ofin MeOH/MtBE (70:30 = v/v), following centrifugation (14,000 rpm, five min) for further HPLC evaluation. two.5.two. Identification and Quantification of Carotenoids and Chlorophyll HPLC-DAD Kale extracts had been analyzed making use of a VWR Hitachi Chromaster (5000 series) reversedphase HPLC method (Develosil C30, 250 4.6 mm, 5 , Phenomenex, JPH203 manufacturer Aschaffenburg, Germany) at a column temperature of 13 C and 20 injection volume. Both an eluent gradient as well as a flow gradient have been applied. At 0 min, the eluent gradient started at 9 of solvent A (MeOH) and 91 of solvent B (MtBE), at a flow rate of 0.43 mL min-1 . Solvent A was then improved to 50 over 23.5 min at constant flow prices. Afterwards, solvent A was elevated to 70 till 38 min, with an rising flow price of 0.6 mL min-1 , which was held till 40 min. Subsequently, solvent A was decreased to 9 , with an elevated flow price of 1.0 mL min-1 and following a holding time of 12 min for equilibration. A diode array detector served for identification, in regards for the characteristic spectral absorbance profiles and quantification of carotenoids (450 nm ), chlorophyll a (662 nm ), and chlorophyll b (644 nm ), in comparison to external standards applying 5-point calibration curves (r 0.999). Recovery in the internal regular (Lucantin-Yellow) was regarded as. Chromaster program manager (Version two.0, Hitachi High-Tech Science Corporation, Tokyo, Japan) was applied for data evaluation. HPLC S/MS Mass spectral evaluation was applied to support final results from identification via diode array detection. As a result, a Shimadzu HPLC program (LC-20 series) was hyphenated having a triple quadrupole mass spectrometer (API 2000, AB Sciex). Kale extracts (50 ) had been injected onto a reversed-phase column (YMC C30, 250 4.six mm, five , YMC Europe, Dinslaken, Germany), applying a gradient elution with MeOH/water (80:20 = v/v; A) and MtBE/MeOH/water (78:20:2 = v/v/v; B) at 30 C. Pumping flow mode was kept isocratic at 1.three mL min-1 . The gradient elution began with an increase of solvent B to 30 for five min, which was increased to 60 till 35 min. Lastly, s.