Breakdown of peptides from a casein hydrolysate by rumen bacteria. Simultaneous study of enzyme activities and physicochemical parameters.

The breakdown of a pancreatic hydrolysate of casein (tryptone) by an inoculum of ruminal mixed bacteria was studied in vitro. Peptides were degradated at 33% after 5 h. The dipeptidyl aminopeptidase type 1 (DAP-1), exoaminopeptidase and leucine aminopeptidase (LAP) like activities were measured using, respectively, Gly-Arg-MNA, Ala-pNa and Leu-pNA as substrates. While the total proteolytic activity remained stable throughout peptide breakdown, the DAP-1 already present at the beginning of fermentation increased until two times its early activity. This peptidase activity is a major index for the presence of Prevotella ruminicola. A peak of exoaminopeptidase and LAP activities were observed at 3 h incubation. This latter activity, which was associated with the production of lactate after 3 h, suggested that Streptococcus bovis was present in the environment. With the use of high-performance size exclusion chromatography (HPSEC), the size of the peptides during their breakdown could be measured, showing that high molecular weight peptides (4 to 2 kDa) were apparently more rapidly broken down to the profit of small-sized peptides (< 0.5 kDa), which increased. These degradation abilities were closely linked to DAP-1 activity. Separation of tryptone peptides by reverse-phase high-performance liquid chromatography (HPLC) showed that more hydrophilic peptides disappeared than hydrophobic peptides. Moreover, a few peaks eluting for one in the hydrophilic area and for the others in the hydrophobic area resisted the bacterial breakdown. In contrast, amino acid profiles indicated that hydrophobic amino acids were not taken up significantly faster than the hydrophobic amino acids by mixed ruminal bacterial.