Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening, To compare their pe ptidase activities toward a dairy substrate, a tryptic/chymotryptic hydroly sate of purified p-casein was used, Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the p-casein hy drolysate, as shown by tandem mass spectrometry, Cell extracts prepared fro m Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus de lbrueckii subsp, lactis ITG LL14 and ITG LL51, L. delbrueckii subsp, bulgar icus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 1 02303, and TA 060 were standardized in protein. The peptidase activities we re assessed with the p-casein hydrolysate as the substrate at pH 5.5 and 24 degreesC (conditions of warm room ripening) by (i) free amino acid release , (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry, Regardless of strain, L, helveticus was the most efficient in hydrolyzing p-casein peptides, Interestingly, cell extra cts of S. thermophilus were not able to release a significant level of free proline from the p-casein hydrolysate, which was consistent with the ident ification of numerous dipeptides containing proline, With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or we akly hydrolyzed indicating their high intrinsic resistance to peptidase act ivities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypept idase in the cell extracts of these species.