Hp 6890 series

UV spectra were recorded using a Shimadzu UV2450 spectrophotometer. The ¹H, ¹³C and 2D NMR spectra were obtained on a Bruker Avanc3–400 MHz NMR spectrometer or Varian Mercury-300 NMR spectrometer using TMS as an internal reference. TLC analysis was carried out on silica gel plates (ACROS organics, New Jersey, USA). Silica gel 60 (230–400 mesh or 70–230 mesh, 47 cm by 2.5 cm, ACROS organics, New Jersey, USA) was used for column chromatography. GCMS analysis was carried out using Hewlett Packard, HP6890 series, fitted with a fused silica HP5-MS 5% phenyl methyl siloxane cap column (30 m × 0.25 mm i.d., film thickness 0.25) and directly coupled to the MS. The ELIZA microplate reader was purchased from BioTek (Winooski, VT, USA).

The chromatographic analysis was carried out on a HP 6890 series gas chromatograph (Hewlett Packard, Palo Alto, CA, USA), equipped with a DB-5 (5% phenylmethylsiloxane) capillary column (30 m × 0.25 mm × 0.25 µm film thickness), an FID detector set at 250 °C and lied by a mixture of H₂/air gas. The injection mode is split; the carrier gas used is nitrogen with a flow rate of 1.7 mL/min. The column temperature was programmed with a rise of 4 °C/min from 50 °C to 200 °C for 5 min. The device was controlled by a HP Chemstation computer system managing the operation of the device and allowing to follow the evolution of chromatographic analyses. The GC-MS was coupled to a mass spectrometer (HP 5973 series). The fragmentation was carried out by electronic impact at 70 eV. The column used is a capillary type DB-5SM (30 m × 0.25 mm × 0.25 µm). The column temperature was programmed at a rate of 4 °C/min from 50 to 200 °C for 5 min. The carrier gas used is helium with a flow rate of 1.7 mL/min. The injection mode is split. The identification of the constituents of the essential oils studied was carried out both by the index identification method of Kovàts [57 ], and Adams [58 ], and from the mass spectral database.

Approximately 10 mg of lyophilized cells were used for the extraction of cytoplasmic metabolites. Lyophilized cells were mixed with extraction buffer (1:1 ice cold methanol/water), vortexed, placed in liquid nitrogen and kept at −20 °C for 30 min to let the thawing process occur slowly. The contents of the solution were then separated by the centrifugation method to eliminate cell debris. The supernatant enclosing the metabolites was transferred into new 50 mL falcon tubes and placed in a drier vacuum to eradicate the methanol/water residues. Cytoplasmic amino acids were then extracted using EZ:faast kit (Phenomenex^® EZ: faast™) and subsequently applied to Agilent Gas Chromatography–Flame Ionization (GC-FID) (Hewlett Packard HP 6890 series) as earlier described [18 (link)].

GS Caltex Corp. provided bio-based levo-2,3-BDO and meso-2,3-BDO samples with 99% purified (2R,3R)-2,3-BDO or (2R,3S)-2,3-BDO. Briefly, the samples were prepared via fermentation by B. licheniformis 4071, with separation and purification according to previous reports [18 (link), 25 (link)]. Three stereoisomers of 2,3-BDO, (2R,3R)-2,3-BDO, (2R,3S)-2,3-BDO, and (2S,3S)-2,3-BDO, were determined by gas chromatography with flame ionization detector (GC-FID; HP 6890 series, Hewlett Packard, USA) equipped with an HP-chiral 20ß column (30 m, 0.32 mm internal diameter, 0.25 μm film thickness; Agilent Technologies, Germany). The oven temperature was initially set at 40°C for 5 min and increased to 160°C at a gradient of 15°C/min, at which point it was maintained for 2 min. The temperature of the injector and detector was set at 230°C. Argon was used as the carrier gas and run through the column at 2 ml/min. The sample injection volume was 0.2 μl.

The free amino acid concentration in the M. longissimus thoracis was analyzed using ultra high-performance liquid chromatograph (Waters ACQUITY UPLC I-Class, Waters, United States) and high-resolution mass spectrometer (Q-Exactive, Thermo Fisher, United States) as previously described (18 (link)). About 300 mg muscle sample were dissolved in water with methanol (water: methanol = 2:8, v/v). After ultrasonic treatment for 5 min, the sample was placed at room temperature for 1 min, and the above operation was repeated for six times. The sample was then placed on ice for 2 h and centrifuged at 10,000 g, 4°C for 10 min to collect the supernatant.
Total amino acid (mainly protein-bound amino acids) profile of the M. longissimus thoracis was determined based on the standard methods in AOAC (14 ).
The composition of fatty acids of the M. longissimus thoracis was analyzed as described previously (19 (link)). About 150 mg lyophilized muscle sample was added with 4 ml chloracetyl methanol (1:10, v/v), 1 mL n-hexane and 1 mL internal standard FA solution (1 mg/mL C11:0). The mixture was then kept in a water bath at 75°C for 2 h. After cooling to room temperature, the mixture was added with 5 mL carbonate solution (70 g/L) and centrifuged at 800 g for 3 min. The supernatant was analyzed by the Gas Chromatography (HP 6890 series, Hewlett Packard, Avondale, PA, United States).