Agilent 5975 mass spectrometer

IR spectra were recorded with a NICOLET iS50 FT-IR spectrometer (Thermo Scientific, Waltham, MA, USA). NMR data were collected on Bruker Avance-400FT and Avance-600 NMR spectrometers (Bruker Corporation, Billerica, MA, USA). GC–MS analyses were performed on an Agilent 7890A GC system connected to an Agilent 5975 mass spectrometer with a HP-5MS column (30 m × 250 µm × 0.25 µm, Agilent Technologies, Santa Clara, CA, USA). HREIMS spectrum was collected on a Hybrid Quadrupole-Orbitrap GC-MS/MS System (Thermo Scientific, Waltham, MA, USA) equipped with a TraceGOLD TG-5HT column (30 m × 250 µm × 0.1 µm, Thermo Scientific, Waltham, MA, USA). Size-exclusion chromatography was carried out with Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) columns. HPLC analysis was performed on an Agilent 1260 series (Agilent Technologies, Santa Clara, CA, USA) with a C18 RP-column (Extend-C18, 250 × 4.6 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA). Semi-preparative HPLC was performed on an SSI 23201 system (Scientific Systems Inc., State College, PA, USA) with a YMC-Pack ODS-A column (250 × 10 mm, 5 µm, YMC CO., LTD. Shimogyo-ku, Kyoto, Japan). All fermentations were carried out in MQD-B1R shakers (Minquan Instrument Co., Ltd., Shanghai, China).

Sample processing protocols for cecal contents were according to the method described with some minor modifications.^{38 (link)} Briefly, ~30 mg of fresh cecal content was mixed with 300 μL of 1 M NaOH solution, homogenized with 1 mm zirconium beads and centrifuged at 16,000 × g at 4°C for 20 min. A volume of 200 μL of supernatant was collected into an autosampler vial. Then, the residue was re-extracted with 200 μL of cold methanol with the same steps of homogenization and centrifugation. The first supernatant in the sample vial was combined with 200 μL of supernatant from the second extraction. Subsequently, 300 μL of chloroform containing 30 µM indole-d6 was added to the mixed supernatant and samples were shaken for 10 s. Another 400 μL of 50 mM sodium bicarbonate solution was added again, and samples were shaken for another 10 s. A volume of 100 μL chloroform phase was transferred to GC vials for further analysis after centrifugation at 2000 × g for 10 min at 4°C. Indole and 3-methylindole were quantified with an Agilent 7890A gas chromatograph coupled with an Agilent 5975 mass spectrometer (Agilent Technologies Santa Clara, CA, USA).

To quantify the total lipid in cells, 50 mL of fermentation broth was centrifuged to collect the cells, which were then washed with 0.9% (w/v) NaCl and lyophilized to determine cell dry weight. 3 mL of chloroform and methanol mixture (chloroform: methanol = 2:1, v/v) was added to cells in a sealed testing tube and incubated at 30 °C, 180 rpm for 3 h, followed by adding 0.5 mL of deionized water. After phase separation, the bottom phase containing lipids was extracted again with 2 mL of chloroform and methanol mixture (chloroform: methanol: water = 3:48:47, v/v/v). The lower phase was collected and evaporated under N₂ atmosphere and the weight of lipids was determined [15 (link)]. The weight of lipids was divided by cell dry weight to obtain the total lipid yield (g lipid/g CDW). Then, the incremental lipid yield (g lipid/g CDW) of cells was calculated by deducting the initial lipid yield (at 0 h fermentation) from the total lipid yield. The fatty acid methyl esters (FAMEs) were obtained by the sulfuric acid–methanol method [1 (link)]. The FAMEs distribution was determined using an Agilent 7890 GC coupled with Agilent 5975 mass spectrometer as previously reported [6 (link)] (Additional file 5).

The GC-MS system used was an Agilent 6890 GC equipped with an Agilent 5975 mass spectrometer (Agilent Technologies Santa Clara, CA, USA). The column used was a 60 × 0.25 mm HP-INNOWAX capillary with 0.25 μm film thickness (J & W Scientific, Folsom, CA, USA). The carrier gas was helium at a flow rate of 1 mL/min. Samples were injected by placing the SPME fiber at the GC inlet for 25 min with the splitless mode. The oven’s starting temperature was 50 °C, which was held for 1 min, then raised to 220 °C at a rate of 3 °C/min and held at 220 °C for 5 min, transfer-line temperature was 105 °C. The mass spectrometry in the electron impact mode (MS/EI) at 70 eV was recorded in the range m/z 20 to 450 u.m.a. The mass spectrophotometer was operated in the full scan and the selective ion mode (SIM) under autotune conditions at the same time. The area of each peak was determined by Chem. Station software (Agilent Technologies). Analyses were carried out in triplicate. Retention indices were calculated after analyzing C8-C24 n-alkane series under the same chromatographic conditions. Identifications were based on MS matching in the standard NIST05 library, retention indices of reference standard in authors’ laboratories and a comparison of retention indices reported in the literature. Retention indices were listed in Table 2.

Conjugated diene value (CDV) was determined according to White [22 (link)]. The oleogel sample (0.01 g) was weighed carefully into a 25 mL volumetric flask. The sample was dissolved in iso-octane (2, 2, 4-Trimethylpentane) and gently mixed. The absorbance of the sample was measured at 233 nm using an Agilent 5975 mass spectrometer (GC–MS; Agilent Technology, CA, USA). The blank sample with iso-octane was measured using a quartz cuvette. The absorbance of the dissolved samples was measured using a quartz cuvette.