1H NMR spectra were acquired on the collected supernatants, with no further treatments, at 300 K on a Mercury-plus NMR spectrometer from Varian, operating at a proton frequency of 400 MHz. Residual water signal was suppressed by means of presaturation. 1H NMR spectra were processed by means of VNMRJ 6.1 software from Varian. To minimize the signals overlap in crowded regions, all free induction decays (FID) were multiplied by an exponential function equivalent to a -0.5 line-broadening

factor and by a gaussian function with a factor of 1. After manual adjustments of phase CYC202 price and baseline, the spectra were scaled to the same total area, in order to compare the LB-100 clinical trial results from samples of different weight and water and fiber content. The spectra

were referenced to the TSP peak, then digitized over the range of 0.5 – 10 ppm. By means of R scripts developed in-house the residual water signal region, 4.5 – 5.5 ppm, was excluded from the following computations [58]. To compensate for chemical-shift perturbations, the remaining original data points were reduced to 218 by integrating the spectra over ‘bins’, spectral areas with a uniform size of 0.036 ppm. A 34 × 218 bins table was thus obtained for statistical analysis. As some parts of the spectra are very crowded, some bins may contain peaks pertaining to different molecules. In order selleck chemical to consider this potential source of error the bins containing peaks ascribed to the same molecules were not summed up [33]. Statistical analysis All data coming from culture-dependent analysis and metabolomic analysis were obtained at least in triplicates. The analysis of variance (ANOVA) on culture-dependent analysis, GC-MS/SPME and 1H-NMR analysis, was carried out on transformed

data followed by separation of means with Tukey’s HSD, using a statistical software Statistica for Windows (Statistica 6.0 per Windows 1998, (StatSoft, Vigonza, Italia). Letters indicate significant different groups (P < 0.05) by Tukey's test. Canonical discriminant Analysis of Principal coordinates (CAP) analysis was carried out for GC-MS/SPME data [33]. This was preferred to the more common Canonical Discriminant Analysis (CDA), because it Cobimetinib purchase does not assume any specific distribution of the data, thus giving more robust results in the case of reduced number of samples. The CAP constrained ordination procedure that was carried out is summarized as follows: (i) data were reduced by performing a Principal Coordinate analysis (PCO) of the parameters, using the dissimilarity measure calculated on euclidean distances; (ii) an appropriate number of PCO was chosen non-arbitrarily, which maximizes the number of observations correctly classified; (iii) the power of classification was tested through a leave-one-out procedure; and (iv), finally, a traditional canonical analysis on the first PCO was carried out.