**2.2.1 Microbiological analysis**

For microbial analysis, about 1 g of samples (jejunum, ileum, caecum) was placed in a sterile polyethylene stomacher Lab Blender bag (Seward Medical Limited, London, UK) with 9 ml of sterile anaerobic diluent (0.4 g Na HCO3, 0.05g L-cysteine HCl, 1 ml resazurine 0.1%), 7.5 ml mineral solution I (0.6% K2HPO4), 7.5 ml mineral solution II (1.2% NaCl, 1.2% (NH4)2SO4, 0.6% KH2PO4, 0.12% CaCl2, 0.25% MgSO4 and 84 ml distilled water, pH 6.8) and stomached (Stomacher Lab Blender 80, Seward Medical Limited, London, UK) for 5 min under a CO2 atmosphere. A series of 10-fold dilutions (10-2 to 10-8) were made in the same diluents. From appropriate dilutions, 0.1 ml aliquots were spread onto one non-selective agar plate: trypticase soy blood agar with 10% sheep blood (BBL, Microbiology systems, Cockeysville, USA) for aerobes. Aliquots (0.1 ml) were also spread on 5 selective agar media as follows: Beerens medium (Beerens, 1990) for *Bifidobacterium,* Rogosa agar (Imuna, Šarišské Michaľany, Slovak Republic) for *Lactobacillus,* Enterococcosel agar (BBL) for *Enterococcus*, MacConkey agar (Imuna) for *Coliforms* and Endo agar (Imuna) for *Enterobacteriaceae.* Plates for the enumeration of *aerobic* bacteria were incubated for 2 days at 37oC. Colonies were counted and bacteria were Gram stained and visualized under a microscope for morphological characterization. The viable counts are expressed as the log 10 of colony forming units (cfu)g-1 of sample.
