Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

Abstract

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified ␤-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the ␤-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the ␤-casein hydrolysate as the substrate at pH 5.5 and 24°C (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 ␤-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the ␤-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species. Thermophilic lactobacilli such as Lactobacillus helveticus orLactobacillus delbrueckii along with Streptococcus thermophilus constitute essential lactic starters in Swiss-type cheese (400,000 tons of Emmentaler produced per year worldwide). The three species have a key role in the acidification step and in cheese proteolysis (13). Adequate proteolysis is essential for acceptable quality of the ripened cheese. Whole caseins are first hydrolyzed by milk protease (plasmin) and/or rennet (chymosin and pepsin) into large and intermediate-sized peptides. The proteinases and peptidases from starters subsequently hydrolyze them in small peptides and amino acids, which are known to be aroma precursors. In the three species considered, peptidases are intracellular enzymes released upon lysis in the curd, where they remain active for several weeks (14,40,41). Because of their predominant role in proteolysis, studies on peptidases of thermophilic lactobacilli and S. thermophilus have expanded greatly in recent years (5,18,35,36). Several aminopeptidases, dipeptidases, and peptidases specific to proline-containing peptides (prolidase, prolinase, X-prolyldipeptidyl aminopeptidase, and prolyl aminopeptidase) were isolated, cloned, and sequenced (20); only the general aminopeptidase(s) and the X-prolyl-dipeptidyl-aminopeptidase of thermophilic lactobacilli were shown to be...

Simultaneous Presence of PrtH and PrtH2 Proteinases in Lactobacillus helveticus Strains Improves Breakdown of the Pure α s1 -Casein

“…For both caseins, regions containing a cluster of three phosphoserine residues were resistant to hydrolysis, in agreement with the previous results of Deutsch et al (6).…”

“…In milk, only some regions in the middle of the ␣ s2 -casein sequence were hydrolyzed. The phosphorylated regions of the caseins were poorly hydrolyzed, in agreement with previous studies (6). When strains were grown in milk, a drastic reduction in the number of peptides was observed, highlighting the parts of the casein sequence that were more resistant to proteolysis when ␣ s1 -, ␣ s2 ,-and ␤-caseins were assembled into micelles.…”

Application of proteomics to the characterisation of milk and dairy products

“…The use of b-casein or peptide substrates similar to those encountered in cheese and the analysis of the peptide mixtures obtained by MS has been very useful for the characterisation and determination of the specificity of proteinases from lactic acid and propionic acid bacteria (Deutsch et al, 2000;Kunji et al, 1998;Gagnaire, Molle´, Sorhaug, & Le´onil, 1999). Kunji et al (1998) deleted one or more genes encoding key enzymes of the proteolytic system in order to elucidate the various steps involved in the b-casein proteolytic pathway of L. lactis.…”

Bioactive Peptides Derived from Casein and Whey Proteins

“…; Deutsch et al 2000 ). Several CPP, consisting of a mixture of components derived from three parent peptides, β -casein f7 -284P, α s1 -casein f61 -794P, and α s2 -casein f7 -214P, have been identifi ed in a water -soluble fraction of Grana Padano cheese (Ferranti et al 1997 ).…”

“…Furthermore, some phosphopeptides were identifi ed in the diafi ltration retentate of a water -soluble fraction of Cheddar cheese (Singh et al 1997 ), while nine phosphopeptides formed by the hydrolysis of β -casein were characterized in a soluble fraction of a ParmigianoReggiano cheese (Addeo et al 1994 ). Phosphorylated peptides were more resistant than other peptides to hydrolysis by peptidases from thermophilic LAB, in particular to the aminopeptidases (Deutsch et al 2000 ). Recently, mass -spectrometric screening of the main peptides generated by the action of the Lact.…”