Study on Rapeseed Albumin Hydrolysis by PrtS Protease from Streptococcus thermophilus and Bioactivity Characterization of Resulting Hydrolysates

Abstract

Lactic acid bacteria are well known for hydrolyzing milk proteins, but their application to plant proteins remains limited. This study evaluated the ability of the cell-wall-anchored PrtS protease from two Streptococcus thermophilus strains to hydrolyze rapeseed albumins (RAs), aiming to generate bioactive peptides with potential food functionality. The specific activity of PrtS was first determined using a chromogenic substrate. RAs were then hydrolyzed using 10X- and 100X-concentrated cell pellets of each strain to assess the hydrolysis kinetics and the enzymatic mechanism. The results showed concentration-dependent hydrolysis, with protein conversion and the degree of hydrolysis increasing threefold at 100X for both strains. Despite the increased hydrolysis, the peptides produced had similar average sizes, averaging at five amino acids, indicating a consistent "one-by-one" cleavage mechanism. The in vitro testing of the RA hydrolysates produced with 100X PrtS from S. thermophilus LMD-9 revealed dose-dependent antioxidant activity comparable to native RAs. Importantly, unlike native RAs, these hydrolysates did not induce increased secretion of the pro-inflammatory mediator IL-8 in inflamed HT-29 cells, suggesting a reduced pro-inflammatory potential. These findings demonstrate that PrtS protease from S. thermophilus can effectively hydrolyze rapeseed proteins to produce functional hydrolysates with improved bioactivity profiles. Such hydrolysates have promising applications as functional ingredients in plant-based food products, contributing both to health benefits and potential food preservation through antioxidant activity.

Keywords: PrtS protease; Streptococcus thermophilus; bioactivity; enzymatic hydrolysis; lactic acid bacteria; rapeseed albumins.

Figure 1

Growth in LM17 broth (a) and specific activity of PrtS protease (b) of Streptococcus thermophilus LMD-9 (●) and 4F44 (○) using Suc-Ala-Ala-Pro-Phe-Lys-pNA as substrate. The synthetic substrate was prepared at 1.24 g/L in 1 vol of N, N-dimethyl formamide and 9 vol of 50 mM Tris HCl buffer (pH 8), supplemented with 5 mM CaCl₂. The specific activity was defined as µmol of the pNa released per unit time per OD under experimental conditions. Results are presented as means of 3 observations.

Figure 2

Size-exclusion chromatograms at 214 nm of samples drawn during hydrolysis of rapeseed albumins by the PrtS protease from cells concentrated 10X (a,b) and 100X (a’,b’) of S. thermophilus LMD-9 (a,a’) and 4F44 (b,b’) strains. Both strains were grown to an OD_650nm of 1 in LM17 broth. Followed by washing with 100 mM Tris-HCl buffer (pH 7.5), supplemented with 5 mM CaCl₂. The cells of each strain were concentrated 10x and 100x to increase the PrtS protease quantity and incubated with a 0,1% rapeseed albumin solution for up to 24 h at 37 °C. Samples were drawn at the beginning (T0) and after 1 (T1), 2 (T2), 4 (T4), and 24 h (T24) of hydrolysis.

Figure 3

Hydrolysis kinetics, i.e., protein conversion rates (Xp; %, squares), degrees of hydrolysis (DH; %, triangles), and mean numbers of residues per peptide (Naa; round) for the hydrolysis of rapeseed albumins with PrtS protease from S. thermophilus strains (filled marks: LMD9; empty marks: 4F44) over 24 h for both cell concentrations 10X (a) and 100X (b).

Figure 4

Protein conversion rate (Xp; %, squares) and mean number of residues per peptide (Naa; circles) for the hydrolysis of rapeseed albumins by PrtS protease from S. thermophilus strains (filled markers for LMD-9 and empty markers for 4F44), as a function of degree of hydrolysis (DH, %), determined by size-exclusion chromatography, at cell concentrations of 10X (a) and 100X (b).

Figure 5

Relationship between concentration and radical-scavenging activity of antioxidants Trolox and gallic acid, and rapeseed albumins (RAs) and hydrolysates of these proteins (H4 and H24), which were generated by PrtS protease from S. thermophilus LMD-9 strain after 4h or 24h. The activity was measured by the ABTS assay at 740 nm using a microplate reader. Experiments were performed in triplicate. Error bars show standard deviations. ABTS RSA: ABTS radical-scavenging activity.

Figure 6

Impact of rapeseed albumins (RAs) and their hydrolysates (H4 and H24) on IL-8 secretion in LPS-stimulated HT-29 cells. HT-29 cells were incubated for 3 h with LPS (50 ng/mL) and with RA or its hydrolysates. IL-8 secretion in the culture medium was measured by ELISA after 3 h of treatment. The negative control (LPS−) represents untreated cells, and dexamethasone (Dex) was used as a positive control at 25 µM. IL-8 secretion, IL-8 (%), is expressed as a percentage of the IL-8 released by cells compared with its release by cells treated with LPS alone. Data are presented as the means ± SEMs of three independent experiments (n = 3). ** p < 0.01.