# Generation and Characterization of Novel Bioactive Peptides from Fish and Beef Hydrolysates
## 4 Discussion  
The rise in food-related diseases--such as heart diseases, hypertension, and obesity--has prompted consumers to seek food products that provide both nutritional and health benefits. In this study, two unreported multifunctional peptides were generated, purified, and identified from fish and beef using a Sep-Pak C18 cartridge, RP-HPLC, ESI-TOF MS, and Edman degradation. Further characterization of the peptides demonstrated that hydrolysis of proteins by enzymes, such as pepsin, can be a good strategy to obtain bioactive peptides and that peptides obtained from fish and beef are prospective sources of bioactive peptides. Amino acid sequences of F21 showed high similarity to those of GAPDH. GAPDH has a variety of biological functions, including regulation of the sixth stage of glycolysis and the prevention of cell death [21]. Two GAPDH-related peptides, YFGAP and SJGAP--obtained from fish skin--showed antibacterial properties and molecular masses of 3.4 kDa [22,23]. Each peptide was composed of 32 amino acid residues that had not undergone any post-translational modifications. The fish peptide identified in this study had a different amino acid sequence from YFGAP and SJGAP, and GAPDH can be considered a good source of bioactive peptides. Beef is also considered a potential source of bioactive peptides. In previous studies, small peptide fragments produced by acid whey digestion of beef showed various biological functions, such as immunomodulatory, ACE inhibitory, and antioxidative activities and enhancement of the cardiovascular and nervous systems [24]. Additionally, some other bioactive peptides originating from beef have been identified and characterized [25,26]. The antibacterial activity of bioactive peptides depends on the allocation of hydrophobic and charged residues in the peptide structure [27]. The existence of positively charged residues, as well as an amphip structure, allows the peptide to interact with the bacterial membrane via electrostatic intercommunication, similarly to other antimicrobial peptides [28]. Furthermore, the occurrence of aromatic residues, random coils, and helical regions in peptides is frequently linked to biological activity [29]. The fish peptide obtained in the present study lacked helix formation, whereas YFGAP and SJGAP contained one helix and two strands respectively [22,23], suggesting that F21 shows different biological activity from that of the previously reported peptides. The fish hydrolysates and F21 exhibited broad antimicrobial spectra compared to beef, which can be attributed to differences in the amino acid sequence and secondary structure. [PERSON] et al. [22] reported that the purified fish peptide (YFGAP) exhibited antimicrobial activity against some Gram-negative bacteria, _E. coli_ D31 (minimal effective concentration (MEC), 6.2 \(\mathrm{\SIUnitSymbolMicro m}\)) and Gram-positive _B. subtilis_ KCTC1021 (MEC, 13 \(\mathrm{\SIUnitSymbolMicro m}\)), which was similar to that of F21. A few studies have shown peptides extracted from meat with considerable antibacterial activity [30]. [PERSON] et al. [31] reported that certain bioactive peptides extracted from beef demonstrated antimicrobial activity against _E. coli_, _Salmonella typhimurium_, and _Pseudomonas_, which can be considered as a broader antibacterial spectrum than that of B34. However, [PERSON] and [PERSON] [32] stated that there was no proof of antimicrobial activity of peptides obtained from cow and pork on _Staphylococcus aureus_ and