Accq tag eluent a concentrate

Fresh oil palm nuts were obtained from PT Wilmar Nabati, Indonesia. The palm nuts were a hybrid from Dura and Pisifera variety of Elaeis guineensis species harvest in 2016. After the hulls were removed, the PK, with a moisture content of 100 g/kg, was released.
The freeze-dried yeast culture, food grade Y. lipolytica NCYC 2904 was purchased from National Collection of Yeast Cultures (NCYC) (Norwich, UK). It was propagated in sterile yeast malt (YM) broth (2% glucose, 0.25% yeast extract, 0.25% bacteriological peptone and 0.2% malt extract, all w/v, pH 5.0) at 30 °C for 48 h under aerobic conditions to obtain a pure culture with a cell population of about 7 log CFU/mL. Glycerol was added to the pure culture at 15% v/v and it was stored at −80 °C before use.
Standards of sugars including arabinose, xylose, glucose, fructose, mannose, galactose, maltose and sucrose were purchased from Sigma Aldrich (St Louis, MO, USA). Standards of amino acids were obtained from Thermo Scientific (Rockford, IL, USA). Acetonitrile of HPLC grade was acquired from Tedia (Fairfield, OH, USA) and petroleum ether of ACS grade (boiling point of 35–60 °C) and ethanol were purchased from Merck (Darmstadt, Germany). AccQ-Fluor reagent kits and AccQ-Tag eluent A concentrate for amino acids analysis were provided by Waters (Dublin, Ireland).

Sulfuric acid, hydrochloric acid, formic acid, perchloric acid, sodium nitrate, sodium hydroxide, potassium hydroxide, potassium chloride, sodium sulfate anhydrous, potassium dihydrogen phosphate, chloramine T trihydrate, trichloroacetic acid, 2-thiobarbituric acid, ethylenediaminetetraacetic acid (EDTA), propylgallate, 4-(dimethylamino)benzaldehyde, petroleum ether (b.p. 40–60 °C), methanol, ethanol, hexane, acetonitrile, 2-propanol, chloroform, sodium methoxide solution, amino acid standard, L-tryptophan, hydroxyproline, L-2-aminobutyric acid, and 1,1,3,3-tetraehoxypropane were purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA).
Supelco 37 comp. FAME mix, linoleic acid methyl ester isomer mix were purchased from Supelco (Supelco Analytical, Bellefonte, PA, USA), trans FAME mix K110 from Grace (Grace, Deerfield, IL, USA).
AccQ Fluor reagent kit and AccQ Tag eluent A concentrate was purchased from Waters (Waters Corp., Miliford, MA, USA). Boric acid was purchased from AFT (Bratislava, Slovakia), Kjeltabs from Velf Scientifica (Velf Scientifica srl, Usmate, Italy).

For the variation of amino acid concentrations in DM media, samples were subjected to an AccQ-Fluor Reagent (AQC, 6-aminoquinolyl-N-hydroxysuccinimide carbamate) derivatization (Waters Corp., Milford, MA, United States) according to the manufacturer’s protocol. AQC was reconstituted at a final concentration of 10 mM in acetonitrile, included in the AccQ-Fluor Reagent Kit (Waters Corp.). Briefly, 10 μL of samples were derivatizated with 70 μL of AccQ-Fluor Borate Buffer (Waters Corp.) and 20 μL of reconstituted reagent. The samples were heated to 55°C for 10 min. The amino acids content was analyzed using an HPLC (PU-1580 Intelligent HPLC pump, Intelligent Fluorescence Detector FP-1520 and Intelligent Sampler AS-2055 Plus, with 10 μl loop; Jasco Corp.). Separation of amino acids was obtained using AccQ-Tag^TM column (3.9 mm × 150 mm) for amino acid analysis (Waters Corp.). A gradient elution was performed maintaining a column temperature of 30°C and using two mobile phases: A (100 ml of AccQ-Tag Eluent A concentrate (Waters Corp.), diluted 1:10 with H₂O for chromatography (Sigma-Aldrich, St. Louis, MO, United States) and B (60% acetonitrile and 40% H₂O for chromatography) (Sigma-Aldrich, St. Louis, MO, United States) with a flow rate of 1 ml/min. The fluorescence detector was set at excitation wavelength of 250 nm and emission wavelength of 395 nm.