3.7. Microbial Analysis

Although there are accurate molecular methods such as quantitative PCR and fluorescence in situ hybridization (FISH) with ribosomal RNA-targeted oligonucleotide probes, in the present study bacterial count methods were employed. Thus, samples (5 g fw of slurry) from the three colon vessels (AC, TC, and DC) during the stabilization (at 0, 6, 8, and 12 days) and intake period (at 0, 2, 5, 7, 9, and 13 days) were aseptically transferred to tubes containing Anaerobe Basal Broth (Oxoid, Thermo Scientific, Basingstoke, UK) transported immediately to the microbiology laboratory of AINIA (Valencia, Spain), and analyzed within two hours [78]. The isolation and enumeration of specific groups of bacteria from the fermentation products were done using selective and differential growth media and suitable incubation conditions [79]: VRBD Agar (Merck-Millipore, Burlington, MA, USA) for Enterobacteriaceae; VRBL Agar (Merck-Millipore) for total coliforms; TSC Agar (Oxoid) for Clostridium spp.; Tos-propionate Agar (Merck-Millipore) for Bififobacterium spp.; Schaedler Anaerobe Agar (Oxoid) for total anaerobes; and MRS Agar (Oxoid) for Lactobacillus spp. All plates were incubated at 37 °C in anaerobic conditions for 24 h (Enterobacteriaceae, Clostridium spp., and total coliforms), 48 h (Bififobacterium spp. and Lactobacillus spp.), and 72 h (total anaerobes). The identification of Lactobacillus microorganisms from MRS-agar plates were carried out by MALDI-TOF mass spectrometry [80]. Anaerobic bacteria and Enterobacteriaceae were analyzed as indicators of total colonic microbiota, total coliforms, and Clostridium spp. as a part of regular microbiota composition. Although they are associated with potential harmful effects, Bifidobacterium spp. and Lactobacillus spp. were associated with the beneficial microbiota of the colon. Analysis was performed in triplicate (n = 3).