Fp 4025

Raw amaranth (Amaranthus hybridus chlorostachys) grains were supplied by Darvash Giah Khazar medicinal herbs complex company (Ltf) (Gilan, Rasht, Iran), after a preliminary analysis of grain chemical composition as reported in our previous studies [8 (link),41 ] (Hosseintabar-Ghasmabad et al., 2020, Janmohammadi et al., 2022). In order to complete the nutritional analyses of amaranth, in the present study, squalene (method IOC, 2011) and phytosterols [42 (link),43 ] (Takatsuto and Abe, 1992; Bhandari et al., 2012) were determined via gas chromatography (model 6100, Younglin, Republic of Korea), while tocopherols were evaluated by RP-HPLC (Younglin Acme 9000 model, Republic of Korea) and a dual-channel fluorescence detector (Jasco FP-4025 model, Japan), as reported in Table 3. The enzyme blend used in this trial was Natuzyme P50 (Bioproton Pty Ltf., Sunny bank, QLD, Australia). Enzyme constituents and their respective activity (U/g) per kilogram of diet were as follows: xylanase 10⁷, cellulase 5 × 10⁶, pectinase 5 × 10⁴, and β-glucanase 10⁶, which is of Trichoderma reesei of fungal origin, and it included Trichoderma longibachiatum and α-amylase from Bacillus subtilis, and protease 6 × 10⁶ and phytase 5 × 10⁵ from Aspergillus niger.

pH values were measured after calibration (-58.2 mV/pH, 25 °C) of the pH electrode (phenomenal MIC220, Mikro) with a pH meter (MU 6100 L) and pH buffer standards from VWR. Fluorescence measurements of DCF were conducted with a fluorescence detector (FP-4025, Jasco) which was equipped with a square cell holder for 10 x 10 mm square cells (Type 3/G/10, Starna Scientific). The wavelengths for excitation and emission were 480 and 525 nm and a detector gain of 1 was used. In a typical measurement 800 μL of a sample were diluted with 2500 μL of a degassed 0.16 mM NaOH solution. In order to get reliable results it was crucial to protect the samples from light during the whole measurement process to prevent further DCHF oxidation. The fluorescence intensities correlated with the DCF concentration c_DCF in a linear fashion (S1 Fig). The quantitative analysis was done by external calibration with standards in three concentration ranges and duplicate measurements. In S1 Table, calibration parameters for the linear regressions are displayed.

P3 membranes were solubilized as described in the previous section and 200 µL of the detergent-solubilized supernatant was injected in a Superose 6 10/300 GL gel-filtration column (GE healthcare) equilibrated with 20 mM Tris-HCl pH 7.8, 150 mM NaCl, 10% (v/v) Glycerol, 0.1 mg/mL DDM, 0.02 mg/mL CHS. Fluorescence-detection size-exclusion chromatography (FSEC) experiments were performed in an ÄKTA^TM purifier chromatography system (GE healthcare) with a fluorescence detector (FP-4025, JASCO) connected “in line” with the column. The excitation and emission wavelengths of the fluorescence detector were set, respectively, to 460 and 520 nm. FSEC chromatograms were plotted using Plot2 software and data was normalized.