Amino acid standard h

The total nitrogen of the T. versicolor biomass was determined by the Kjeldahl method [45 ]. The total protein content was calculated by multiplying the total nitrogen by 4.38. The result was expressed as a percentage.
The amino acid composition was determined by the method described by Tumbarski et al. [48 (link)]. The lyophilized biomass was subjected to acid hydrolysis using 6N HCl for 24 h at 105 °C. An aliquot of the hydrolysate was derivatised using AccQ-Fluor reagent Kit (Waters). The derivative was separated on RP AccQ-Tag™ silica-bonded amino acid column C18, 3.9 mm × 150 mm (Waters, Etten-Leur, The Netherlands), and conditioned at 37 °C using an ELITE LaChrom HPLC system (VWR™ Hitachi, Tokyo, Japan). A sample of 20 μL was injected and the elution of the amino acids was performed by the following gradient system: eluent A, buffer WAT052890 (Waters, Etten-Leur, The Netherlands) and eluent B, 60% acetonitrile (Merck KGaA, Darmstadt, Germany) with a constant flow rate of 1.0 mL/min. The amino acids were detected using a diode array detector (DAD) at 254 nm. The amino acid peaks were then analyzed using EZChrom Elite™ software [49 ] and the amino acid content was calculated based on the amino acid standard calibration curve (amino acid standard H, Thermo Fisher Scientific, Waltham, MA, USA). The results were expressed as mg AA/g sample and as a percentage [50 (link)].

The amino acid content of olive pomace and the LMW peptides (<3 kDa) extracted from olive pomace was determined after hydrolysis in 6 N HCl plus 1% phenol in sealed Pyrex tubes (Corning, NY, USA) at 110 °C for 24 h by high-performance liquid chromatograph (HPLC) according to the Waters Pico Tag method [47 ] as described in [48 (link)], using a Waters 2695 separation module (Waters Corporation, Mildford, MA, USA). L-alpha-amino adipic acid and DL-norleucine were used as internal standards. Pre-column derivatization was carried out with phenyl isothiocyanate. The cysteine and methionine contents were determined as cysteic acid and methionine sulphone, respectively, after oxidation of samples with performic acid prior to the protein hydrolysis step, as described in [49 (link)]. For quantification, seventeen amino acid standards (Amino Acid Standard H, Thermo Fisher Scientific, Waltham, MA, USA), L-Cysteic acid, and L-Methionine sulfone (Sigma-Aldrich, St. Louis, MO, USA) were used at concentrations of 0.1, 0.2, and 0.3 mM. Tryptophan was not determined. A Millennium 32 chromatography manager system was used for gradient control and data processing. Each sample was analysed three times with two technical replicates.