Production of bioactive peptides by using the proteases obtained from Bacillus strains and investigation of some properties of the bioactive peptides

In this study, bioactive peptide (BAP) production from plant-based proteins was carried out through enzymatic hydrolysis. In the first part of the study, the potential use of six different plant products [einkorn flour, dry bean flour, yellow lentil flour, buckwheat, white quinoa, and Galia melon (seed part)] as protein precursors for producing bioactive hydrolysates was determined. Plant protein isolates were extracted using the alkaline solvent method and hydrolyzed under suitable proteolytic reaction conditions (50 °C, pH 7) with four different commercial proteases (Alcalase, Flavourzyme, Neutrase, rTrypsin) at a 5% E/S ratio (v/v, 2 h). The antioxidant activity of the protein hydrolysates was determined by DPPH scavenging. The protein content of the isolates was found to range from 24.7% (buckwheat) to 76.7% (yellow lentil), and the extraction yield ranged from 3.6% (buckwheat) to 36.2% (yellow lentil). The highest DPPH scavenging was obtained with Alcalase and Flavourzyme, and among the six different plant protein sources, einkorn protein hydrolysates exhibited the highest activity (30-73%). Based on these results, einkorn flour was chosen as the protein source for BAP production in the continuation of the study. To increase the extraction yield of einkorn proteins, the effect of pH (8-10) and solid-to-solvent ratio (%10-30 w/v) was investigated. Accordingly, pH 10 and 10% solid content were determined as the optimal conditions (extraction yield: ~23%). Additionally, BAP was produced using bacterial crude protease. A microorganism screening study was conducted to select the protease-producing bacterial isolate, and the protease production ability of the isolates was examined. For this, the proteolytic activity of eight different Bacillus strains was evaluated on a solid medium containing milk powder. The highest proteolytic activity was observed with crude protease obtained from Bacillus sp. EBTA7 strain. The protease activity conditions for this selected strain were optimized, and the activity was tested under different pH (7-11) and temperature (50-80 °C) values. The optimum conditions were determined to be 60 °C and pH 9 (2056 U/mL). The selected Bacillus sp. strain was identified as Bacillus mojavensis based on molecular identification, with 99.86% similarity. In the continuation of the study, einkorn flour was hydrolyzed with commercial proteases (Alcalase, Flavourzyme, Neutrase, rTrypsin) and Bacillus mojavensis sp. EBTA7 crude protease. Hydrolysis was performed for all proteases at two different E/S ratios (w/v) (commercial proteases: 0.25% and 1%; crude protease: 50% and 100%). The degree of hydrolysis (DH) was determined to range from 6.0% to 35.6%, and it was found that increasing the E/S ratio for crude enzyme improved the DH. The SDS-PAGE profile of the proteins generally matched the DH findings. The bioactivity of the hydrolysates was evaluated by measuring the scavenging of free radicals (DPPH and ABTS), iron (II) ion chelation activity, and total phenolic content (TPC). The DPPH scavenging activity of the hydrolysates ranged from 17.7 to 33.0 μmol Trolox equivalent (TE)/g, and ABTS scavenging activity ranged from 107 to 190 μmol TE/g. The iron chelation activity was between 0.09 and 3.08 mg EDTA/g, and the TPC was found to be between 6.94 and 14.98 mg gallic acid equivalent/g. Additionally, some techno-functional properties of the hydrolysates were examined, including emulsion activity index (EAI), emulsion stability index (ESI), oil binding capacity (OBC), and foaming properties. The EAI ranged from 28 to 61 m²/g, ESI from 11 to 227 minutes, OBC from 0.70 to 2.94 g/g, foam expansion from 27% to 84%, and foam stability from 22% to 94%. FTIR spectroscopy indicated that the secondary structure of the commercial protease hydrolysates was predominantly β-sheet, while the crude protease hydrolysates exhibited an α-helix structure. The zeta potential of the hydrolysates ranged from -20.0 mV to -3.19 mV, with the crude protease hydrolysates having the highest zeta potential (-20 mV). All hydrolysates were found to have high antioxidant capacity, with Bacillus mojavensis sp. EBTA7 hydrolysates demonstrating significantly higher OBC, emulsion, and foam stability. In the final part of the study, einkorn protein isolate (EPI) and crude protease were produced under the optimum conditions identified in the first part and in high capacity. BAP production was carried out with crude protease, and EPI with 67% protein content was prepared at a 5% concentration in a buffer solution (100 mM, pH 9). Prior to hydrolysis, the lyophilized crude protease was adjusted to an activity value of 2056 U/mL, and hydrolysis was performed at three different E/S ratios (30%, 40%, and 50%) at 60°C for 5 hours. The hydrolytic reaction mixture was centrifuged, and the supernatant was collected, lyophilized, and the DH value of the dried hydrolysate was determined. The desired DH value of 19.9% was obtained at the 50% E/S ratio, and in the continuation of the study, hydrolysis was performed at this ratio to obtain the einkorn protein hydrolysate (EPH) in the same manner. The hydrolysate was separated into four peptide fractions (≤ 1 kDa, 1-5 kDa, 5-10 kDa, and > 10 kDa) using an ultrafiltration system with membranes of different pore sizes (10 kDa, 5 kDa, 1 kDa). The antioxidant and anti-diabetic properties of the molecular weight (MW) fractions were determined through in vitro spectrophotometric analyses, and the 50% inhibitory concentration (IC50) was calculated. Additionally, the techno-functional properties of the fractions were evaluated. The primary structure was determined through surface hydrophobicity (H0) and amino acid composition, while the secondary structure was determined through FTIR and zeta potential analyses. Finally, the MW > 10 kDa fraction was further purified by hydrophobic interaction chromatography (HIC). The peptide fractions obtained through ultrafiltration (UF) had protein contents ranging from 43.4% to 81.7%. The IC50 values for DPPH scavenging ranged from 15.0 to 88.3 mg/mL, for ABTS from 3.9 to 4.5 mg/mL, and for iron (II) chelation from 0.17 to 0.34 mg/mL. A positive correlation was found between the activity and peptide MW for DPPH scavenging, with the highest activity observed for the MW > 10 kDa fraction. For ABTS scavenging, UF application did not significantly affect the activity. It was found that smaller peptide fractions (≤ 1 kDa and 1-5 kDa) had higher iron (II) chelation capacities. The reducing antioxidant power for iron ions increased linearly with peptide concentration and weakened after hydrolysis, but significantly improved with UF application (> 10 kDa). The hydrolysis process enriched the TPC content of the proteins, with similar levels of TPC found across all fractions. The anti-diabetic properties of the fractions were investigated through α-amylase inhibition, with no significant inhibitory activity observed. The EAI for the fractions ranged from 5.3 to 29.3 m²/g, ESI from 15.2 to 27.0 minutes, OBC from 0.32 to 9.21 g/g, foam expansion from 11% to 77%, and foam stability from 1% to 87%. The MW > 10 kDa fraction was identified as the best fraction in terms of EAI, OBC, foam expansion, and stability. The H0 value of the MW > 10 kDa fraction was significantly higher than the other fractions, which was confirmed by its rich hydrophobic amino acid composition. FTIR spectra showed that the secondary structure of einkorn proteins was maintained after hydrolysis, with the MW > 10 kDa fraction exhibiting α-helix and the < 5 kDa fractions exhibiting β-sheet structures. The zeta potential of the peptide fractions ranged from -14.1 to -27.7 mV. Analysis of the total amino acid composition revealed that after hydrolysis and UF treatment, the number of hydrophilic and hydrophobic amino acids increased, with more hydrophilic amino acids being exposed. The MW > 10 kDa fraction was found to contain all essential amino acids except methionine. Further purification of the MW > 10 kDa fraction by HIC resulted in two advanced fractions with distinct hydrophobic characteristics. Mass spectrometry analysis of these advanced fractions revealed high molecular weight glutenin (88 kDa) proteins. In summary, the second part of the study demonstrated the feasibility of using crude protease produced in the laboratory for enzymatic hydrolysis in BAP production. The peptide fractions obtained were generally found to exhibit enhanced antioxidant properties. Furthermore, ultrafiltration was shown to improve the antioxidant and functional properties of the peptides. In conclusion, this study successfully produced BAPs from various plant-based proteins and determined that the enzymatic hydrolysis of einkorn proteins significantly improved their bioactive and techno-functional properties.