Gc fid 7890a

The total lipid content was measured by Soxhlet extraction (Velp Scientifica 148, Italy) and calculated on a gravimetric basis. It is expressed in g of lipids/100 g of dry sample [19 ].
The fatty acid profile was obtained by extracting the lipids and quantified by GC-FID (GC-FID 7890 Agilent Technologies, Santa Clara, CA, USA) [20 (link)]. A total of 1 g of sample (accurately weighed) was put into contact with 50 mL of isooctane and 2.5 mL of methanolic solution of KOH, and the resulting solution was homogenized in an ultrasound bath at 80 °C for 20 min. The filtered solution was neutralized with 1 g of NaHSO₄ × H₂O. A volume of 1 μL of the neutralized solution was introduced into a FAME gas chromatograph with the flame ionization detector GC-FID 7890A Agilent equipped with db-wax silica capillary column (30 mm × 0.25 mm i.d. × 0.25 μm film thickness). The working conditions were as follows: the injector temperature was set at 250 °C, He gas was used a carrier with a flow rate of 1 mL · min⁻¹, and the applied column temperature ranged from 100 °C to 180 °C with a speed of 7 °C/min and a 5 min isothermal regime, followed by an increase from 180 °C to 240 °C at a speed of 10 °C/min and another 10 min isothermal regime. The results are expressed as the fatty acid percentage of the total fatty acids.

Mature brown rice grains were obtained by manual de-hulling and ground with a CapMixTM capsule mixing device (3 M ESPE, Seefeld, Germany). Total lipids from ~300 mg above prepared rice flour samples were extracted with a mixture of chloroform/methanol/0.1 M KCl (at a ratio of 2/1/1, by volume). Fatty acid methyl esters (FAME) were prepared by incubating lipid samples in 1 N Methanolic-HCl (Supelco, Bellefonte, PA) at 80 °C for 2 h. TAG and polar membrane lipid pools were fractionated from total lipids in thin layer chromatography (TLC) (Silica gel 60, Merck, Darmstadt, Germany) using a solvent mixture of hexane/diethylether/acetic acid (at a ratio of 70/30/1, by volume) and individual membrane lipid classes were separated by TLC using a solvent mixture of chloroform/methanol/acetic acid/water (90/15/10/3, by volume). Authentic lipid standards were loaded and were run in separate lanes on the same plates for identification of lipid classes. Silica bands, containing individual class of lipid were used to prepare FAME as mentioned above and were analysed by gas chromatography GC-FID 7890A (Agilent Technologies, Palo Alto, CA) that was fitted with a 30 m BPX70 column (SGE, Austin, TX) for quantifying individual fatty acids on the basis of peak area of the known amount of heptadecanoin that was added in as an internal standard [50 (link)].

At 6, 12, 24, 36, and 48 h time points after inoculation, we measured total gas production from fermentation as overpressure using a graduated syringe and passing a needle through the rubber stopper prior to unsealing the tubes. For pH measurements, the supernatant was transferred to a separate 15 ml Falcon tube and measured using a pH meter. We collected two aliquots from each tube, one for SCFA (0.4 ml) and other for DNA extraction (1 ml) and stored the aliquots at −80°C. An internal standard (157.5 μl of 4-methyl valeric acid, 1.47 ml of 85% phosphoric acid, 39 mg of copper sulfate pentahydrate in a total volume of 25 ml) was immediately added to the SCFA aliquots samples before vortexing.
We measured SCFAs as previously described (Tuncil et al., 2018b (link)). Briefly, 4 μl of the supernatants were analyzed on a fused-silica capillary column (Nukon^TM, SUPELCO No: 40369-03A, Bellefonte, PA, United States) using a gas chromatograph (GC-FID 7890A, Agilent Technologies, Inc.) (Tuncil et al., 2017 (link)). We used 4- methylvaleric acid (Fisher Scientific) as an internal standard and acetate, propionate, and butyrate (Fisher Scientific, Hampton, NH, United States) to generate standard curves.

SCFA analyses were performed as previously described^{35 (link)}. Briefly, the samples were thawed at room temperature and centrifuged at 13,000 rpm for 10 mins. Supernatants (4 µl) were analyzed using a gas chromatography (GC-FID 7890 A; Agilent Technologies Inc.) on a fused silica capillary column (Nukon^TM SUPELCO No: 40369-03A, Bellefonte, PA) under the following conditions: Injector temperature at 230 °C; initial oven temperature at 100 °C; temperature increase of 8 °C/min to 200 °C with a hold for 3 min at final temperature. Helium was used as a carrier gas at 0.75 ml/min. Acetate (catalog number: A38S), propionate (catalog number: A258), and butyrate (catalog number: AC108111000) purchased from Fisher Scientific (Hampton, NH) were used as external standards. 4-methylvaleric acid (catalog number: AAA1540506, Fisher Scientific) was used as an internal standard for quantification.

Samples for SCFA analyses were prepared as previously described (10 (link)) and analyzed using a gas chromatograph (GC-FID 7890 A; Agilent Technologies Inc.) on a fused silica capillary column (Nukon Supelco no. 40369-03A; Bellefonte, PA) under the following conditions: injector temperature at 230°C, initial oven temperature at 100°C, and temperature increase of 8°C/min to 200°C with a hold for 3 min at final temperature. Helium was used as a carrier gas at 0.75 ml/min.
Quantification was performed based on relative peak area using external standards of acetate (A38S), propionate (A258), and butyrate (AC108111000) and an internal standard of 4-methylvaleric acid (AAA1540506) from Fisher Scientific (Hampton, NH).