6890ngc injection module

SCFAs levels were determined in the fecal supernatants by means of gas chromatography, as described by Moris et al. [36 (link)]. Briefly, cell free-supernatants (250 μl) from fecal homogenates, prepared as indicated above, were mixed with 100 μl methanol (Merck, Darmstadt, Germany), 50 μl internal standard solution (2-ethylbutyric 1.05 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA), and 50 μl of 20% v/v formic acid (Sigma-Aldrich, St. Louis, MO, USA). The mix was then centrifuged, and the supernatant was used to quantify SCFAs in a system composed of a 6890NGC injection module connected to a flame injection detector (FID) and a mass spectrometry detector (MS, 5973N) (Agilent Technologies, Madrid, Spain).

The SCFAs acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate were determined in faeces through gas chromatography (GC), using a system composed of a 6890NGC injection module (Agilent Technologies Inc., Palo Alto, CA, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as previously described. Briefly, cell-free supernatants (100 μL) from faecal homogenates were mixed with 450 μL methanol, 50 μL internal standard solution (2-ethylbutyric 1.05 mg/mL), and 50 μL 20% v/v formic acid. Following centrifugation, the supernatants of this mixture were used for SCFA quantification by GC as previously described [18 (link)].

The analysis of short-chain fatty acids (SCFAs) was performed by gas chromatography (GC) in the fecal culture supernatants to quantify acetic, propionic, butyric acid (major SCFAs), as well as isobutyric and isovaleric acids, as described in Nogacka et al. [7 (link)]. Briefly, 250 µL of culture supernatants collected at time 0 and 24 h of incubation were mixed with 0.3 mL methanol, 0.05 mL of the internal standard solution (2-ethylbutyric acid 1.05 mg/mL), and 0.05 mL of 20% (v/v) formic acid. The mixture was centrifuged, and the supernatant was collected for SCFA quantification in a system composed of a 6890N GC injection module (Agilent Technologies Inc., Palo Alto, CA, USA) connected to a flame injection detector (FID) (Agilent). Samples were analyzed in triplicate, and results were expressed in µg/mL. Increments (∆) in the levels of these compounds at 24 h of incubation with respect to the basal conditions (time 0) were calculated for each fermentation batch with the different combinations of probiotics and 2′FL.

The analysis of SCFAs was performed by Gas Chromatography (GC) in the fecal culture supernatants to quantify acetic, propionic, butyric acid (major SCFAs), as well as isobutyric and isovaleric acid (branched chain fatty acids: BCFAs). 250 µL of culture supernatants collected at time 0 and 24 h of incubation were mixed with 0.3 mL methanol, 0.05 mL of the internal standard solution (2-ethylbutyric acid 1.05 mg/mL), and 0.05 mL of 20% (v/v) formic acid. The mixture was centrifuged, and the supernatant was collected for SCFA quantification in a system composed of a 6890N GC injection module (Agilent Technologies Inc., Palo Alto, Ca, USA) connected to a flame injection detector (FID) and a mass spectrometry (MS) 5973N detector (Agilent), as described previously [24 (link),25 (link)]. Samples were analyzed in triplicate and results were expressed in µg/mL. Increments (∆) in the levels of these compounds at 24 h of incubation with respect to the basal conditions (time 0) were calculated for each fermentation batch with the different 2′FL preparations tested.

SCFA and BCFA levels were determined in the fecal supernatants by means of gas chromatography, as described by Moris et al.^{77 (link)} Briefly, 250 μl of cell free-supernatants were mixed with 100 μl methanol, 50 μl internal standard solution (2-ethylbutyric 1.05 mg/ml), and 50 μl of 20% v/v formic acid. The mix was then centrifuged, and the supernatant was injected into a system composed of a 6890NGC injection module (Agilent Technologies Inc., USA) connected to a flame injection detector (FID) and a mass spectrometry detector (MS, 5973 N) (Agilent) for quantification of both SCFA and BCFA.

SCFA, including acetic, propionic, butyric, isovaleric, isobutyric, valeric and caproic acids were detected and quantified by Gas Chromatography (GC) as described elsewhere^{78 (link)}. Briefly, cell free supernatants obtained from the homogenized feces (0.250 mL) were mixed with 0.3 mL methanol, 0.05 mL of 20% formic acid (v/v) and 0.05 mL of an internal standard solution (2-ethylbutyric 1.05 mg/mL). This mixture was centrifuged, and the supernatant was used for quantification of SCFA by GC using a system composed of a 6890NGC injection module (Agilent Technologies Inc., Palo Alto, Ca, USA) connected to a flame injection detector (FID) (Agilent). SCFA concentrations are given as mM. Molar proportions were calculated in some cases by referring the concentration of each SCFA to the total of concentration of all SCFA, considered this as 100%.