In vitro digestion, absorption and colonic batch fermentation model

The feed undergoes an in vitro enzymatic hydrolysis simulating the digestion in the upper digestive tract followed by a dialysis mimicking the intestinal absorption. Finally, an in vitro batch fermentation is performed and simulates the fermentation occurring in the hindgut. The ingredients, ground to a 1 mm-mesh screen, are first hydrolysed with porcine pepsin in a phosphate buffer supplemented with chloramphenicol (pH 2, 39°C, 2 hours) under gentle agitation in a water bath and then with pancreatin (pH 6.8, 39°C, 4 hours) in the same environment. Then, the hydrolysed samples are dialysed in standard regenerated cellulose membranes (6-8 kD) during 24 hours under agitation and water renewal to optimise the dialysis and are thereafter lyophilized. A faecal inoculum is prepared with a buffer solution (pH 6.8) composed of salts and minerals and frozen pig faeces (2.5%). An alternative to frozen faeces is to collect the faeces directly in syringes, to remove all the air and to place the syringes in a water bath at 39°C for transportation to the laboratory. The faeces and buffer solution need to be subjected to mechanical pummelling using a stomacher and then filtered through a 250 µm-mesh screen. The fermentation starts by adding 15 ml of faecal inoculum to 0.1 g of dried hydrolysed sample in 60-ml glass bottles filled with 3 mucin carriers making up approximately 1 g of mucus-agar and closed with a rubber stopper and crimp-caps to ensure steady and continuous anaerobic conditions. These steps are performed in an anaerobic chamber. The sealed vials are then placed in a water bath at 39°C. The released gas volumes are repeatedly recorded over 72 hours of incubation with a manometer measuring bottles’ inner pressure or with pressure sensors and connected to a computer. Gas production recordings are fitted to a mathematical monophasic model. The fermentation broth is collected and stored at -20°C for metabolomics analyses and at -80°C for microbial genomic DNA analysis. The concentrations of short-chain fatty acids, branched chain fatty acids and lactic acid can be determined by HPLC or the whole metabolomics profile can be obtained by GC-MS. Bacterial communities can be determined by qPCR or 16S sequencing.