Microbial Bioprospecting and Enzymatic Bioprocesses as Strategies for Exploring Bioactive Peptide Potential

Bioprospecting of microorganisms from environmentally diverse and underexplored habitats is an important strategy for discovering microbial enzymes with biotechnological potential. Among these microorganisms, bacteria of the genus Bacillus are widely distributed in the environment and recognized for their ability to secrete extracellular enzymes, particularly peptidases, which can be exploited to generate bioactive peptides through protein hydrolysis. This study aimed to investigate the antioxidant and antimicrobial potential of peptides obtained by enzymatic hydrolysis in bioprocesses using casein as a protein substrate. An environmental sample was collected from the Araçá region (São Sebastião/SP/Brazil). For bacterial isolation, the sample was inoculated onto tryptic soy agar (TSA), incubated at 30 °C for 24 h, and isolates exhibiting morphological characteristics compatible with members of the genus Bacillus were selected for further bioprocessing assays. Microorganisms were inoculated into a standard medium (3 g/L TSB, 1 g/L K₂HPO₄, 0.5 g/L MgSO₄·7H₂O, 5 g/L NaCl, and 1 g/L (NH₄)₂SO₄) as the control condition or into the same medium supplemented with 1% casein. Cultures were incubated at 30 °C and 180 rpm. After 20 and 40 h, samples were collected and processed to determine pH, peptidase activity, and the presence of bioactive peptides, which were subsequently evaluated for their antioxidant and antimicrobial potential. Regarding pH, the presence of casein delayed the initial alkalinization but resulted in a greater overall pH variation at the end of the bioprocess, probably as a consequence of the proteolytic degradation of casein. Peptidase activity indicated that the microorganism was an efficient protease producer even in the absence of an inducer. The highest activity was observed in the control group at 20 h (0.31 U/mL), whereas the casein-supplemented group reached its maximum activity at 40 h (0.15 U/mL). Despite the lower proteolytic activity, antioxidant activity was significantly higher in the casein-supplemented group, which exhibited DPPH radical-scavenging activity of 14.5% and 7.9% after 20 and 40 h of bioprocessing, respectively, compared with 0% and 0.7% in the control group. These results suggest that peptides with antioxidant potential were generated predominantly during the early stages of the bioprocess. Antimicrobial activity was evaluated using an agar diffusion assay against Escherichia coli and Staphylococcus aureus. Regarding antimicrobial activity, under the tested conditions, no inhibition zones were observed for the peptides, in contrast to the kanamycin control. However, this does not exclude antimicrobial potential, as agar diffusion has limited sensitivity for peptides that interact with the medium and diffuse poorly through the gel. Overall, these results indicate that the Bacillus-like isolate obtained from the Araçá region is an efficient peptidase producer and that the resulting hydrolysates possess antioxidant properties. 

The authors gratefully acknowledge PRPI/USP and FAPESP (2024/01176-0) for their financial support.