Prostar hplc

Analysis of metabolites was performed with the Varian ProStar HPLC (high-performance liquid chromatography) system equipped with a refractive index detector operated at 30°C and an Aminex HPX-87H column (1,300 by 7.8 mm; particle size, 9 µm; Bio-Rad Laboratories) kept at 30°C. Slightly acidified water was used (0.005 M H₂SO₄) as the mobile phase with a flow rate of 0.5 ml/min. To remove proteins and other cell residues, samples were centrifuged at 20,238 × g for 5 min and the supernatant was filtered with Spartan 13/0.2 RC filters. Ten microliters of the supernatant was then injected into the HPLC system for analysis. Measurements of headspace gas composition were carried out on a Varian CP-4900 micro gas chromatograph with two installed channels. Channel 1 was a 10-m Mol-sieve column running at 70°C with 200 kPa argon and a backflush time of 4.2 s, while channel 2 was a 10-m PPQ column running at 90°C with 150 kPa helium and no backflush. The injector temperature for both channels was 70°C. The run time was set to 120 s, but all of the peaks of interest eluted before 100 s.

UV absorption spectra were obtained using a Varian Cary 50 Bio UV−visible spectrophotometer (Agilent, Santa Clara, CA, USA). IR spectra were measured using a Perkin-Elmer FT-IR Spectrometer, SPECTRUM 2000 (Waltham, MA, USA). NMR spectra were acquired on a Bruker Avance III spectrometer (Billerica, MA, USA) operating at 600 MHz for ¹H and 150 MHz for ¹³C and equipped with a 3 mm cryogenically cooled probe. ¹H and ¹³C spectra were referenced to the residual deuterated solvent peaks. HRESIMS data were acquired on an Agilent 6530 Accurate Mass Q-TOF instrument (Santa Clara, CA, USA) and on a TripleTOF 5600 mass spectrometer (AB SCIEX, Framingham, MA, USA). Diol SPE fractionation of the extract was performed on DIO Spe-ed SPE cartridges (Applied Separations, Allentown, PA, USA), and subsequent fractions were separated on Sephadex LH-20 (GE Healthcare, Chicago, IL, USA). These fractions were then separated on silica gel. Sephadex LH-20 and silica gel columns were attached to a UA-6 UV detector and Foxy 200 fraction collector (Teledyne Isco, Lincoln, NE, USA). Final purifications were performed using a Varian ProStar HPLC with the indicated gradient and column. All solvents and chemicals used were of analytical grade.

Muscle concentration of vitamins and carotenoids was determined according to Serra et al. (37 (link)). Twenty microliters of each sample were analyzed using a Prostar HPLC (Varian) equipped with a UV-DAD coupled with a fluorescence detector spectra system (model FL3000, ThermoFinnigan, Whaltam, USA) and a C18 reverse phase column (ChromSep HPLC Columns SS 250 × 4.6 mm including Holder with ChromSep guard column Omnisphere 5 C18).
Two solutions were used as the mobile phase: one (A solution) composed of methanol:acetonitrile:water (10:70:20), the other (B solution) composed of methanol:ethyl acetate (70:30). The samples were injected into the column with 90% of solution A and 10% of solution B, maintaining a flow of 1 mL/min for 15 min. Then, a 50:50 ratio between solutions A and B at 1 mL/min flow was kept for 5 min. Finally, the flow rate was increased to 1.5 mL/min with 100% of solution B and maintained for 10 min before returning to the starting conditions. Carotenoids were detected by a UV-DAD detector at 450 nm, vitamin A was detected at 325 nm, while vitamin E was detected by the fluorimeter (excitation λ = 298 nm; emission λ = 340 nm). Quantification was obtained by an external calibration curve, obtained from the retinol, carotenoid and tocopherol standards at concentrations ranging from 0.045 μg/mL to 7 mg/mL. Carotenoids and vitamins were expressed as μg/kg of meat.

To characterize fermentation of glucose among the lab generated N6 first filial (F1) generation and the parental strains (FGSC 2223 and FGSC4825), fermentation was carried out in a 96-well format in deep-well plates. Biological replicates for each strain were collected from mycelial mats grown from spore suspension in high glucose liquid media (HGLM) with a 6 gauge punch and inoculated into 750μl of HGLM (2% glucose), sealed with aluminum Thermowell^TM seals, and allowed to ferment for 7 days in 12:12 Light/Dark conditions at 25°C. All samples were performed in biological quadruplicate. After fermentation, 600 μL of media was recovered and cell debris was removed by sequential centrifugation at 13.2k rpm for 5 min. The recovered supernatant was aliquoted into HPLC vials for ethanol analysis by HPLC. HPLC quantitation was performed using a Varian ProStar HPLC with a Varian ProStar Autosampler and a Varian 356-LC Refractive Index Detector. An isocratic elution was used with an Agilent Hi-Plex H⁺ (300 mm x 7.7 mm ion-exchange column) with 5mM H₂SO₄ at a flow rate of 0.7 ml/min at 60°C. The concentration of ethanol present was determined from a standard curve based on ethanol standards with a known concentration.