2.5. Probiotic Strains and DNA Extraction

The synbiotic formulation was set up using probiotic bacteria including two Lactobacillus spp. strains and one Bifidobacterium strain, supplied by Roelmi HPC (Origgio, Italy) private strain collection. Table S1 describes the characteristics of each strain, in terms of antimicrobial activity and growth capacity on the selected FOS and inulin-type compounds.

Microbial cultures were normally cultivated in De Man, Rogosa and Sharp medium (MRS) (Conda Lab, Madrid, Spain) and incubated at 37 °C for 24 h under anaerobic conditions obtained by anaerobic atmosphere generation bags for microbiology (Sigma-Aldrich, Milan, Italy). B. animalis spp. lactis culture was supplemented with 0.3 g/L L-cysteine hydrochloride monohydrate (Sigma-Aldrich, Italy).

Total DNA was extracted from each pure probiotic culture (with a title of 10⁹ CFU/mL) using Ultraclean Microbial DNA Isolation Kit (MoBIO Laboratories, Milan, Italy) to obtain standard curves useful to quantify the presence of these probiotics in stool samples by qPCR analyses using ten-fold dilutions ranging from 10⁸ CFU/mL to 10 CFU/mL [27].

Total DNA was extracted from 400 µL of stool samples conserved in preservation tubes, using Stool Nucleic Acid Isolation Kit (Norgen Biotek Corp., Thorold, ON, Canada) following the protocol provided by the manufacturer with some modifications [34]. Briefly, 400 µL of the sample dissolved in the preservation buffer were added to 600 µL of Lysis Buffer, vortexed for 5 min, and centrifuged for 4 min at room temperature. For the final elution of the nucleic acid sample, 50 µL and then other 50 µL (total volume of 100 µL of final sample) of Elution Buffer were used.

DNA from stool samples was also used for the microbiome sequencing through Illumina MiSeq platform [35].