Rezex roa organic acid column

The frozen GL and WL material was rapidly homogenised in liquid nitrogen and extracted in 75% methanol and centrifuged at 10,000× g for 10 min at 4 °C. The obtained pellet was re-extracted, and the extracts and re-extracts were pooled together. The extractions were carried out in duplicate.
Organic acids were analysed by HPLC coupled to a photodiode array detector (LC-20AB Prominence liquid chromatograph, Shimadzu, Kyoto, Japan) on a Rezex ROA organic acid column (300 × 7.8 mm, 8 μm; Phenomenex, Torrance, CA, USA) maintained at 25 °C. Isocratic elution system (95% of 5 mM H₂SO₄ and 5% of acetonitrile) with stepwise flow rate gradient: 0.2 mL min⁻¹ in the first 45 min and 0.5 mL min⁻¹ from 45 to 60 min [82 (link)]. Organic acids were detected at 210 nm and identified by comparing the absorption spectra with authentic standards and by spiking. Quantification was based on peak area using Shimadzu LC Solution software (Shimadzu, Kyoto, Japan).

DDE were centrifuged at 13 000 g for 5 min to remove all particulate matter and supernatant fractions were filtered through 0·2 µm acrodisc filters before injection (20 µl) onto the HPLC system (MERCK) equipped with refractive index (RI) detection. Separation of compounds was achieved using an ion-exclusion REZEX-ROA Organic acid column (Phenomenex) maintained at 85°C. Sulfuric acid in HPLC-grade water (0·0025 mmol/l) was used as an eluent and the flow rate was maintained at 0·5 ml/min. Quantification of the samples was obtained through calibration curves of lactic, acetic, propionic, butyric and valeric acids in concentrations between 12·5 and 100 mm.

For extraction of sugars and organic acids, 0.05 g of powder was weighed and poured over with 2 ml of bidistilled water. Only edible parts (pericarp and placenta) were used for total sugars and organic acids analysis. Samples were then shaken on an orbital shaker for 30 min. After shaking, they were placed in a cooled centrifuge in which the samples were rotated at 10,000 rpm for 8 min. Samples were filtered through 25 µl cellulose filters (Chromafil A-25/25; Macherey–Nagel, Düren, Germany) and saved at – 20 °C until analysis on the Thermo Finnigan Surveyor HPLC system (Thermo Scientific, San Jose, USA). Two columns were used for the analysis: for sugars (Rezex RCM-monosaccharide (30 × 0.78 cm; Thermo Scientific, San Jose, USA)) and for organic acids (Rezex ROA organic acid column (30 × 0.78 cm; Phenomenex, Torrance, USA). Extraction method and HPLC settings were based on Zamljen et al.^{12 (link)} The results are reported in g/kg dry weight (DW) for sugars, citric, malic and quinic acid and in mg/100 g DW for succinic, fumaric and oxalic acid. All sugars and organic acids (including ascorbic acid) were determined only in edible parts (pericarp and placenta).

HPLC analyses were performed to quantify the amount of glucose, sucrose, arabinose, fructose, and acetic acid. In brief, 1-mL samples from each of the three different streams of production (enzymatically hydrolyzed Camelina meal, SHF or SSF) were collected and centrifuged twice (7000 rpm, 7 min, and 4 °C), and then analyzed by HPLC using a Rezex ROA-Organic Acid column (Phenomenex, Torrance, CA, USA). The eluent was 0.01 M H₂SO₄ pumped at 0.5 mL/min and column temperature was 35 °C. Separated components were detected by a refractive index detector and peaks were identified by comparing with known standards (Sigma-Aldrich). Optical density (OD) of R. toruloides was measured spectrophotometrically at 600 nm. The pH was measured with indicator strips at the beginning and at the end of enzymatic hydrolysis to assess suitability of the initial conditions and to foresee possible toxic effects of the final medium.
The titer of carotenoids extracted in acetone from R. toruloides was determined spectrophotometrically (UV-1800; Shimadzu, Kyoto, Japan) based on the maximum absorption peak for β-carotene (455 nm). A calibration curve with standard concentration of β-carotene was obtained.

High-performance liquid chromatography (HPLC) was performed to determine fecal SCFA concentration. Aliquots (1 ml) of 10% (w/v) fecal suspension were centrifuged at 13000 g for 10 min and the supernatant was stored at −20°C for up to 3 months. Supernatants were then filtered using 0.2 mm polycarbonate syringe filters (Whatman, Maidstone, Kent, UK) and injected (20 μl) into an HPLC system (Merck, Whitehouse Station, NJ, USA) equipped with refractive index detection. For the preparation of the external standard containing the SCFA; acetic, propionic and butyric acid were added to give a final concentration of 25 mM to HCl (6 M) and HPLC gradient water. Dilutions of the external standards were prepared and added to the internal standard (ratio 4:1) to give a final concentration for the internal standard of 20 mM ethyl butyric acid, and a final concentration of external standards as 80, 40, 20, 10, 5.0, 1.0, and 0.5 mM. The column used was an ion-exclusion REZEX-ROA organic acid column (Phenomenex, Inc., Torrance, CA, USA) maintained at 85°C. H₂SO₄ in HPLC-grade H₂O (0.0025 mmol/l) was used as the eluent, and the flow rate was maintained at 0.5 ml/min. Quantification of the samples was obtained through calibration curves of acetic, propionic and butyric acids.

Glucose and xylose monomers were quantified by ion chromatography (Dionex 5000 + , Thermo Fisher Scientific, USA) using a 4 × 250 mm analytical CarboPac PA1 column. Analytes were separated isocratically at 30 °C using 50 mM NaOH as eluent at a flow rate of 1.0 mL/min.
Lactic acid and acetic acid were quantified by HPLC (Agilent 1200 system) using a 300 × 7.8 mm REZEX ROA organic acid column (Phenomenex, USA). Analytes were separated isocratically at 65 °C in 5 mM H₂SO₄ at a flow rate of 0.6 mL/min (Wright et al. 2018 (link)).