Characterization of the pattern of alphas1- and beta-casein breakdown and release of a bioactive peptide by a cell envelope proteinase from Lactobacillus delbrueckii subsp. lactis CRL 581

Abstract

The cell envelope-associated proteinases (CEPs) of the lactobacilli have key roles in bacterial nutrition and contribute to the development of the organoleptic properties of fermented milk products as well, as they can release bioactive health-beneficial peptides from milk proteins. The influence of the peptide supply, carbohydrate source, and osmolites on the CEP activity of the cheese starter Lactobacillus delbrueckii subsp. lactis CRL 581 was investigated. The CEP activity levels were controlled by the peptide content of the growth medium. The maximum activity was observed in a basal minimal defined medium, whereas in the presence of Casitone, Casamino Acids, or yeast extract, the synthesis of CEP was inhibited 99-, 70-, and 68-fold, respectively. The addition of specific di- or tripeptides containing branched-chain amino acids, such as leucylleucine, prolylleucine, leucylglycylglycine, or leucylproline, to the growth medium negatively affected CEP activity, whereas dipeptides without branched-chain amino acids had no effect on the enzyme's production. The carbon source and osmolites did not affect CEP activity. The CEP of L. delbrueckii subsp. lactis CRL 581 exhibited a mixed-type CEP(I/III) variant caseinolytic specificity. Mass-spectrometric screening of the main peptide peaks isolated by reverse-phase high-pressure liquid chromatography allowed the identification of 33 and 32 peptides in the alpha(s1)- and beta-casein hydrolysates, respectively. By characterizing the peptide sequence in these hydrolysates, a pattern of alpha(s1)- and beta-casein breakdown was defined and is reported herein, this being the first report for a CEP of L. delbrueckii subsp. lactis. In this pattern, a series of potentially bioactive peptides (antihypertensive and phosphopeptides) which are encrypted within the precursor protein could be visualized.

FIG. 1.

Growth (OD₅₆₀), pH, and CEP specific activity of L. delbrueckii subsp. lactis CRL 581 grown in MDM. The LDH activity determined in the whole-cell suspensions was less than 1% of the total cell LDH activity, indicating that the proteolytic activity detected was due to the action of CEP. Error bars show standard deviations.

FIG. 2.

Time course of hydrolysis of α_s1-casein (A) and β-casein (B) by L. delbrueckii subsp. lactis CRL 581 after growth in MDM medium. Purified α_s1-casein and β-casein were added to washed-cell suspensions of L. delbrueckii subsp. lactis CRL 581, and samples were taken immediately after addition (0 h, lanes 1) and at 15 min (lanes 2), 30 min (lanes 3), 1 h (lanes 4), 2 h (lanes 5), and 4 h (lanes 6) of incubation at 40°C. Cells were removed by centrifugation, and the supernatants were analyzed by SDS-PAGE.

FIG. 3.

RP-HPLC profiles of the peptides from the 1%-TFA-soluble fraction obtained from the control (0 h, dotted line) and after 4 h (solid line) of hydrolysis of α_s1-casein (A) and β-casein (B) by the action of CEP from L. delbrueckii subsp. lactis CRL 581. AU, absorbance units.

FIG. 4.

Locations of the main peptides (double-ended arrows) identified in the primary sequences of α_s1-casein (A) and β-casein (B) and released by CEP from L. delbrueckii subsp. lactis CRL 581. Zs are phosphoserines. The phosphopeptides are indicated with dashed-line double-ended arrows.

FIG. 5.

Specificity of CEPs of LAB, including CEP from L. delbrueckii subsp. lactis CRL 581, toward the α_s1-casein fragment comprising residues 1 to 23. The cleavage sites are indicated by arrows.