Hp 5890 gas chromatograph

The cellular fatty acids were extracted from cells grown on (1) a nutrient broth, (2) mineral salt medium supplemented with diesel oil, (3) mineral salt medium supplemented with Triton X-100 and (4) mineral salt medium supplemented with Triton X-100 and diesel oil. Diesel oil was added to 1 % concentration and surfactant at 120 mg L⁻¹. Bacterial cells were harvested by a centrifugation (8000 g) at 4 °C for 20 min and then washed twice with 0.85 % NaCl to remove residues of the culture medium. Further fatty acid isolation and identification were conducted following the MIDI-MIS method according to Sasser (1990 ). The fatty acid methyl esters (FAMEs) analysis was performed using an HP 5890 gas chromatograph (Hewlett Packard, Rolling Meadows, IL, US) equipped with an HP 25 m × 0.2 mm cross-linked methyl-silicone capillary column. The initial oven temperature was 170 °C, increased every 5 °C min⁻¹ to 260 °C and then every 40 °C min⁻¹ and eventually held constant at 320 °C for 1.5 min. Helium was used as the carrier gas. FAMEs were identified using Sherlock software (TSBA library, version 3.9, Microbial ID, Newark, NJ, USA) based on the actual calibration retention times run prior to sample analysis.

The fatty acid composition of the oil obtained by extraction with sc-CO₂ was determined by gas chromatography according to the official method Ce 2-66 [26 ] using an HP-5890 gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) with a 50 m long bpx-70 fused silica column, 0.25 µm film thickness, and 0.25 mm internal diameter; along with an Fid detector and a split injection system calibrated at 90:10. The fatty acid methyl esters (FAMEs) obtained from Sigma-Aldrich (St. Louis, MO, USA) were prepared as follows: 100 mg of oil was mixed with 5 mL of 0.5 N sodium hydroxide solution in methanol and held in a thermoregulated bath for 5 min at 100 °C. Then, 5 mL of 12.5% boron trifluoride in methanol was added and heated for 3 min. Finally, 1.5 mL of petroleum ether and saturated sodium chloride solution were added. After gently shaking, the mixture was allowed to stand in order to promote phase separation to extract the FAME dissolved in petroleum ether.

For the analysis of skin phospholipids, a gas chromatograph with a flame ionization detector (GC-FID) was used as described elsewhere [24 (link),28 (link),29 (link)]. In summary, the dermal and epidermal compartments of the skin substitutes (previously frozen separately after cell culture) were incubated in a mixture of chloroform and methanol (2:1 vol/vol) in order to extract the lipids (a technique modified from the Folch method). Gas chromatography was performed using a HP5890 gas chromatograph (Hewlett-Packard, Toronto, ON, Canada) with an HP-88 capillary column (Agilent Technologies, Santa Clara, CA, USA) coupled with a flame ionization detector.

¹H- and ¹³C-NMR (400 and 100 MHz, respectively) spectra were acquired on a Bruker DRX400 spectrometer (Bruker, Karlsruhe, Germany). Data processing was performed using MestReNova 6.2 (Mestrelab Research SL, San Diego, CA, USA). NMR spectra were recorded in DMSO-d₆ and referenced to the residual non-deuterated solvent signal at δ_H 2.50 ppm. GC-FID and GC-O analyses were performed by using an HP 5890 gas chromatograph (Hewlett-Packard, San Diego, CA, USA). GC-MS (EIMS, 70 eV) analyses were carried out on a GC Agilent 7890B gas chromatograph equipped with a 5977A MSD mass selective detector (Agilent Technologies Inc., Wilmington, DE, USA). MS data were recorded between 40–400 u, and processed by Mass Hunter software. Wiley library 10ª edition with MS NIST 2011 (Ringoes, NJ, USA) was used to help in the compound identification. The analytical UHPLC-PDA-ELSD was performed on a Thermo Scientific Dionex UltiMate 3000 (Donierstr, Germany) system equipped with an autosampler, quaternary pump, PDA (Photodiode Array Detector) detector and ELSD detector (SEDEX, Alfortville Cedex, France). HPLC-ESI/MS was performed in a Shimadzu LCMS-2010 system (Shimadzu, Tokyo, Japan) equipped with a UV-VIS detector (SPD-10A) and two pumps (LC-10AD) coupled in-line with a MS-2010 mass spectrometer. The equipment also included an on-line DGU-14A degasser and a Rheodyne injection valve with a 5 μL loop.

The isolated strain was phenotypically and biochemically characterized using standard techniques (Gram staining, colony shape, size, and color on nutrient agar plate, etc.), according to Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994 ). Additional biochemical and physiological characteristics were determined using the API Coryne system (BioMerieux, Lyon, France). Isolation of fatty acids was performed according to Sasser (1990 ). Analysis of FAMEs was performed using an HP 5890 gas chromatograph (Hewlett Packard, Rolling Meadows, IL, USA) equipped with an HP 25 m × 0.2 mm cross-linked methyl-silicone capillary column. The initial oven temperature was 170 °C, increased 5 °C min ⁻¹ to 260 °C, the increased 40 °C min⁻¹ and held constant at 320 °C for 1.5 min. Helium was used as the carrier gas. Fatty acid methyl esters (FAMEs) were identified with Sherlock software (TSBA library, version 3.9. Microbial ID, Newark, NJ, USA) based on the actual calibration retention times run prior to sample analysis.

Prior to the structural NMR analysis of the P-1 fraction, we prepared an asialo P-1 fraction in addition to the intact P-1 preparation. Sialic acid was released from the P-1 fraction by adding 5 mM HCl at 80 °C for 50 min under N₂ gas. The reaction was terminated by adjusting the pH to 7.5 at 0 °C followed by desalting. ¹H- and ¹³C-NMR and 2D NMR experiments (¹H-¹³C HSQC, COSY, H2BC, HMBC, TOCSY and ROESY) were performed using JNM-α500 or JNM-ECA920 spectrometers (JEOL Ltd., Tokyo, Japan). The oligosaccharide preparation was permethylated following the method of Ciucanu and Kerek [14 (link)] prior to GC-MS analysis. Hydrolysis of the permethylated oligosaccharide was conducted with 2 M trifluoroacetic acid at 121 °C for 1 h. Reduction was performed by adding DMSO-NaBD₄ at 40 °C for 1.5 h, and peracetylation was performed by adding acetic acid, 1-methyl imidazole and acetic anhydride successively. The GC-MS system consisted of a HP5890 gas chromatograph (Hewlett-Packard Co., Palo Alto, PA, USA), a JMS DX-303 mass spectrograph and a JMA DA5000 data module (JEOL).

The laccase-pretreated samples were hydrolyzed with a cocktail containing commercial enzymes (from Novozymes, Bagsvaerd) with cellulase (Celluclast 1.5 L; 2 FPU · g^-1) and β-glucosidase (Novozym 188; 100 nkat · g^-1) activities, at 1% consistency in 3 mL of 100 mM sodium citrate (pH 5) for 72 h at 45°C, in a thermostatic shaker at 170 rev · min^-1 (in triplicate experiments).
The different monosaccharides released were determined as alditol acetates [53 (link)] by GC. An HP 5890 gas chromatograph (Hewlett-Packard, Hoofddorp, The Netherlands) equipped with a split-splitless injector and a flame ionization detector was used. The injector and detector temperatures were set at 225°C and 250°C, respectively. Samples were injected in the split mode (split ratio 10:1). Helium was used as the carrier gas. The capillary column used was a DB-225 (30 m × 0.25 mm internal diameter, 0.15 μm film thickness) from Agilent J&W (Folsom, CA). The oven was temperature-programmed from 220°C (held for 5 min) to 230°C (held for 5 min) at 2°C min^-1. Peaks were quantified by area; glucose, xylose and arabinose were used as standards to elaborate calibration curves. The data from the three replicates were averaged.