Gas chromatograph

OAs and FAAs were analyzed using HPLC (Agilent, 1260 Infinity, GER) with a DAD detector. OAs detection: Briefly, 1 g of broad bean paste-meju was taken into a 50-ml plastic tube and vortex-mixed with 10 ml of ddH₂O; ultrasonic extraction was performed for 30 min (100%, 30°C). Then the mixture was centrifuged at 8,000 r/min for 10 min, and the supernatant was filtered for detection. The detection was performed on a column sepaxme-h (NP) 7.8 × 300 mm (US) at 55°C, separated by a mobile phase consisting of 0.02 mol/L H₂SO₄ at a flow rate of 0.6 ml/min, and the detection wavelength was 210 nm.
Volatile flavor components and FAAs were detected using the method described in our previous research (Li et al., 2023 (link)). FAAs were detected with HPLC (Agilent 1260 Infinity II, GER), performed on a column (4.6 × 250 mm, 5 μm) (Ultimate Amino Acid, Welch, USA) at 40°C. Volatile flavor components were detected by a Shimadzu gas chromatograph (Shimadzu, Kyoto, Japan) with a DB-WAX column (Agilent, Santa Clara, USA).

A Shimadzu gas chromatograph coupled to a quadrupole mass spectrometer QP-2010 (Shimadzu Corporation, Kyoto, Japan) was used. The column was a 30 m × 0.25 mm i.d., 0.25 μm film thickness, fused silica SPB 5 (Supelco, Sigma-Aldrich, Milan, Italy). The transfer line and ion source temperatures were 300 and 200 °C respectively. The mass spectrometer operated in electron impact ionization mode at 70 eV.
For the analysis of FAME, the column temperature was programmed from 80 °C to 250 °C at 5 °C/min, from 250 °C to 300 °C at 10 °C/min and held at 300 °C for 20 min. The injector temperature was 280 °C. The carrier gas helium at a flow rate of 1.0 mL/min. Split ratio was 1:30 and injection volume 1 μL.
For the analysis of hydrocarbons, the column temperature was programmed from 60 °C to 300 °C at 5 °C/min and held at 300 °C for 15 min. The injector temperature was 300 °C. The carrier gas was helium at a flow rate of 1.0 mL/min. Split ratio was 1:30 and injection volume 1 μL.
For the analysis of sterols, the column temperature was programmed from 260 °C to 280 °C at 2 °C/min and held at 280 °C for 30 min. The injector temperature was 300 °C. The carrier gas was helium at a flow rate of 1.0 mL/min. Split ratio was 1:30 and injection volume 1 μL.
The compounds were identified by comparison of chromatographic behaviour and mass spectra with authentic standards, library data and literature.

The SCFAs acetate, butyrate, and propionate in cecal suspensions were assayed on a gas chromatograph (Shimadzu) using a capillary free fatty acid packed column (Achrom, Zulte, Belgium; 30 m × 0.33 mm × 0.25 μm), a split injector, and a flame ionization detector. The results are reported in mmol per gram of wet caecal material.

An Agilent gas chromatograph coupled to a mass QP 2010 SE spectrometer (Shimadzu, Maryland City, MD, USA) was used. The mass spectrometer was used in EI mode (ionization energy of 70 eV) and set to scan a mass range from 45 to 700 amu. The samples were introduced through the gas chromatograph, equipped with a 30-m × 0.25-mm I.D., an Optima-5 silica capillary column (Restek), and a 0.25-μm thick film. The oven temperature was increased from 60 to 300 °C at 4 °C/min. The injector temperature was 300 °C and the carrier gas was helium. The injector was set to 280 °C, and the injections were done using the splitless mode.

Gas Chromatographic analysis of volatile compounds was performed using a GC-MS Shimadzu QP 2010 PLUS Mass Spectrometer coupled with Gas Chromatograph (Shimadzu equipped with an AOC-20i+s injector and a highly polar polyethylene glycol stationary phase MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness, ZB-Wax Phenomenex) [31 (link)]. The temperature of the injector and the MS transfer line was set to 220 °C. The starting oven temperature was 40 °C, for 5 min, then programmed to rise from 40 °C to 220 °C, at 4 °C/min, and held at this temperature for 15 min. The carrier gas was helium at a constant flow rate of 0.8 mL/min. The injection volume was 1 µL, made in split mode (20:1) at 220 °C. The mass spectral detector was a quadrupole mass spectrometer operated in the full spectral acquisition mode. The mass spectrometer was equipped with an electron ionization source, which was operated with an electron energy of 70 eV.

Totals solids (TS), volatile solids (VS), chemical oxygen demand (COD), electrical conductivity, and total organic carbon (TOC) were determined according to German standard measurement methods [47 ]. Total nitrogen was determined as previously described (VDLUFA-Methodenbuch II, 3.5.2.7). The VFA spectrum was determined with a gas chromatograph (Shimadzu, Japan). The flame ionization detector was equipped with a DB.1701 column (Machery-Nagel/Germany).

Lipids were derivatized by a direct acid/basic double transesterification of freeze-dried RL (Alves et al., 2013 (link)). Identification of fatty acid methyl esters (FAMEs) and DMAs was performed by thin-layer chromatography purification of the esterified fraction (Alves et al., 2013 (link)). The identification of DMAs was obtained by gas chromatography–mass spectrometry (GC–MS), according to Alves et al. (2013) (link).
The composition of FAs was characterized using a GC2010 Shimadzu gas chromatograph (Shimadzu, Columbia, MD, United States) as previously reported (Alves et al., 2013 (link); Cappucci et al., 2018 (link)). Every single FAME was identified through comparison to a standard FAME mixture containing 52 standards (Nu-Chek-Prep Inc., Elysian, MN, United States). Nonanoic acid and nonadecanoic acid were used as internal standards. The identification of the 18:1 isomers was based on a mixture of commercial standards (Supelco, Bellefonte PA, United States) and on the basis of the isomeric profiles (Kramer et al., 2004 (link)). Individual FA and DMA profiles were expressed in g/100 g of total FAs and DMAs, respectively.