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

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

S M Deutsch et al. Appl Environ Microbiol. 2000 Dec.

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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 beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-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 beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees 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 beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-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.

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Figures

FIG. 1

FIG. 1

β-Casein sequence (209 amino acids; one-letter amino acid code). Phosphorylated serines are indicated by the letter “U” and highlighted in grey; every 10th amino acid is framed in bold. The 34 initial peptides of the hydrolysate are represented in black rectangles above the β-casein sequence. After 72 h of hydrolysis, nonhydrolyzed peptides were identified by MS-MS and are presented below the sequence with different patterns, depending on the cell extract used: ▥ for L. helveticus ITG LH1, ▧ for L. delbrueckii subsp. lactis ITG LL14, and formula image for S. thermophilus TA 060.

FIG. 2

FIG. 2

Time course of hydrolysis of the β-casein hydrolysate by cell extracts of the following strains: (a) _L. helveticus_ITG LH1 (—●—), ITG LH77 (○), and CNRZ 32 (---formula image---), (b)L. delbrueckii subsp. lactis ITG LL14 (—■—) and ITG LL51 (□), and L. delbrueckii subsp.bulgaricus NCDO 1489 (▴) and CNRZ 397 (▵); and (c)S. thermophilus CNRZ 385 (—⧫—), TA 060 (◊), and CIP (Pasteur Institut Collection, Paris, France) 102303 (---formula image---). The enzyme reactions were performed for 72 h at 24°C in 20 mM acetate ammonium buffer (pH 5.5) with 137.5 μg of cell extract proteins per ml for 0.7 mg of lyophilized β-casein hydrolysate per ml. Blanks (⋯●⋯, ⋯⧫⋯, and ⋯▴⋯) were assayed with the same conditions but with boiled cell extracts.

FIG. 3

FIG. 3

Hydrolysis of the β-casein hydrolysate by cell extracts (▴) of L. helveticus ITG LH77 (a) and_S. thermophilus_ CIP 102303 (b): effect of the addition at 72 h of fresh β-casein hydrolysate (■) or fresh cell extracts (●).

FIG. 4

FIG. 4

Free amino acids released after 24 h by hydrolysis of β-casein peptides by cell extracts of the following strains: (a)L. helveticus ITG LH1 (&cjs3744;), CNRZ 32 (formula image), and ITG LH77 (▯); (b) L. delbrueckii subsp. bulgaricus NCDO 1489 (&cjs3744;), and CNRZ 397 (formula image and L. delbrueckii subsp. lactis ITG LL14 (▯), and ITG LL51 (formula image); and; (c)S. thermophilus CNRZ 385 (&cjs3744;), TA 060 (formula image), and CIP 102303 (▯). Curves indicate values obtained after 72 h of incubation for L. helveticus ITG LH1 (a; ●), L. delbrueckii subsp.lactis ITG LL14 (b; ▪), and S. thermophilus CNRZ 385 (♦), and TA 060 (formula image) (c).

FIG. 5

FIG. 5

RP-HPLC profiles of β-casein peptides at time zero and after incubation for 24 h at 24°C with cell extracts of_L. helveticus_ ITG LH1, L. delbrueckii subsp.lactis ITG LL14, and S. thermophilus TA 060.

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