Despite hydrolysis with commercial enzymes like trypsin
Despite hydrolysis with commercial enzymes like trypsin, pepsin, Alcalase, Flavourzyme and Thermolysin have been used to digest parent proteins and generate peptides, microbial fermentation by proteolytic species has also proved to be a successful strategy (Fakhfakh et al., 2013, Jemil et al., 2016, Niu et al., 2013, Yang et al., 2016). In fact, the simultaneous action of different proteases showing diverse specificities in the fermentation medium enhances the proteolytic activity and increases the chance of small peptide generation. Among the microbial species, different species of the Bacillus genus may have a unique place due to their proteolytic properties. In this respect, Jemil, Mora, et al. (2016b) tested fermentation products of Sardinelle protein with Bacillus subtilis A26 and Bacillus amyloliquefaciens An6 to produce antioxidant and ACE-inhibitory peptides whereas Zhang et al. (2014) used B. subtilis to hydrolyse peanut proteins. In another study, Jemil, Abdelhedi, et al. (2016a) prepared antibacterial peptides from zebra blenny muscle proteins after fermentation with B. mojavensis A21. Fakhfakh et al. (2013) also revalorised wool waste by generating bioactive peptides using B. pumilus fermentation. The use of B. subtilis fermentation has given good results in terms of bioactive peptide generation, mainly due to the intense proteolytic activity produced by this specie that results in a better chance to obtain small bioactive peptides.
A considerable amount of protein-rich by-products could be generated from the tomato processing industries all over the world as protein constitutes 28% of tomato seed weight. In order to valorize such protein source, fermentation conditions to produce bioactive peptides from tomato waste proteins by using B. subtilis A14h have been tested (Moayedi, Hashemi, & Safari, 2015). The mentioned strain showed higher ability to release peptides from tomato waste proteins than did other B. subtilis isolates, e.g. K46b and H13h (data not shown). The effects of amino gpr119 agonist composition and molecular weight distribution on antioxidant and ACE-inhibitory activity of resulting peptides have been reported (Moayedi et al., 2017). In this study, isolation and identification of the most active peptide sequences obtained from the fermentation of tomato seeds with B. subtilis has been achieved through the use of chromatography, mass spectrometry, and bioinformatic tools. The in vitro activities of synthesized peptide sequences have also been evaluated.
Materials and methods
Results and discussion
Conclusion In the current study, B. subtilis fermentation was used to generate bioactive peptides that were encrypted in proteins from tomato seeds. The resulting peptide mixture was fractionated according to peptide size and relative hydrophobicity. After each purification step, ACE-inhibitory and antioxidant activities were measured. Peptide identification, with mass spectrometry in tandem, revealed that many of the peptides contained in the most active fractions were below 600 Da, and 5–6 amino acid residues in length. There were six peptides with IC50 below 33 μM, DGVVYY (IC50 = 2 µM) being the most promising peptide for inhibiting ACE activity. On the other hand, GQVPP showed excellent antioxidant activity (97% DPPH scavenging activity at 0.4 mM). The results revealed that proteolytic enzymes from B. subtilis effectively cleaved parent proteins of tomato seeds into short peptides showing good antioxidant and ACE inhibitory activities. However, although amino acid composition and sequence in the peptide structure greatly influence the peptide activity, they do not guarantee its expected bioactivity.
Acknowledgement This work was funded by Grant of Ministry of Science, Research and Technology of Iran, Research Council of University of Tehran (Tehran, Iran) and Emerging Research Group Grant from Generalitat Valenciana (Valencia, Spain) (GV/2015/138). Also a Juan de la Cierva de Incorporación postdoctoral contract to Dr. Mora is acknowledged. Q-ToF nLC-MS/MS was carried out in the SCSIE University of Valencia Proteomics Unit (Spain), a member of ISCIII ProteoRed Proteomics Platform. Authors would like to thank Carolina Diaz Noriega for her technical support.