# Extraction of Novel Bioactive Peptides from Fish Protein Hydrolysates by Enzymatic Reactions
## 3 Application of Enzymatic Hydrolysis for Fish Protein Isolation
### Enzymes from Microorganisms  
Exogenous enzymes are commonly utilized in the production of hydrolysates, silages, and other fish byproducts due to their capacity to increase the rate of production when added in appropriate levels [52]. Currently, endopeptidase from bacterial cultures are being produced and utilized as enzymes for starter cultures in fermentation, and in the production of fish byproducts, and these are known to release bioactive protein hydrolysates from fish [53]. Some known examples are commercially produced enzymes from _Bacillus licheniformis_ and fungi _Aspergillus oryzae_, commonly known as Alcalase(r) 2.4 L and Flavourzyme(r), respectively [54]. Additionally, enzymes such as Neutrase(r) (_Bacillus amyloidateaciens_) and Protamex(r) (_Bacillus licheniformis_ and _Bacillus amyloidateaciens_) are also synthesized from bacterial cultures and are utilized in the industry. These enzymes have been studied for their mechanism of action in various food products, resulting in products with high yields and increased functionality. [PERSON] et al. [55] investigated the properties of FPH from _Collichthys niveatus_ hydrolyzed at different conditions using the abovementioned enzymes. Results from their study revealed that the Neutrase enzyme catalyzed the hydrolysis processes most effectively compared to other enzymes. Furthermore, the enzyme Neutrase revealed high contents of sweet and umami taste from amino acids. Novel bioactive peptides were also studied by [PERSON] et al. [56], derived from the enzymatic hydrolysate of Sardinelle muscle proteins by microbial proteases. In their study, _Bacillus subtilis_ A26 proteases used as enzymes in the hydrolysis process revealed a total of 62 peptides and were found to exhibit antibacterial, antioxidant, and ACE-inhibitory activities. Some of the recent studies on novel bioactive peptides derived from the enzymatic hydrolysis of various FPHs from fish waste byproducts and discards are presented in Table 1.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Sproducts** & **Species** & **Protease** & \begin{tabular}{c} **Hydrolysis** \\ **Conditions** \\ \end{tabular} & \begin{tabular}{c} **Peptides/Amino Acid** \\ **Fractions** \\ \end{tabular} & **References** \\ \hline Head & \begin{tabular}{c} Squid \\ (_Loligo formosana_) \\ \end{tabular} & \begin{tabular}{c} Alcalase, \\ Flavourzyme \\ \end{tabular} & \begin{tabular}{c} 3\% E/S, 12.5 h, \\ unadjusted pH \\ \end{tabular} & Arg-Glu-Gly-Tyr-Phe-Lys & [57] \\ \hline Roe & \begin{tabular}{c} Tuna \\ (_Katsuwonus pelamis_) \\ \end{tabular} & Alcalase, trypsin & 1\% E/S, 4 h, 55 \({}^{\circ}\)C & Cys-Gly-Arg & [58] \\ \hline Skin & \begin{tabular}{c} Puffer fish \\ (_Takiugu flavidus_) \\ \end{tabular} & Alcalase, neutral & \begin{tabular}{c} 2000 U/g \%/E/S, \\ 5 h, pH 8, pH 7, \\ pH 2 \\ \end{tabular} & Pro-Pro-Leu-Leu-Phe-Ala- & [59] \\ \hline Mixed waste & \begin{tabular}{c} Round scad \\ (_Decapterus maruadsi_) \\ \end{tabular} & Neutrase & \begin{tabular}{c} 0.3\% E/S, 6 h, \\ pH 7, 50 \({}^{\circ}\)C \\ \end{tabular} & KGFP, FPSV, FPFP, WPDGR & [60] \\ \hline Cartilage & \
\begin{tabular}{c} Siberian sturgeon \\ (_Acipenser baerii_) \\ \end{tabular} & Alcalase & - &
\begin{tabular}{c} GPTGED, GEPGEQ, \\ GEPGEPAG, VPPQD, \\ GELEDHA, GDRGAEG, \\ PRGFRPV, GEVGFFE, \\ GFIGNG \\ \end{tabular} & [61] \\ \hline \hline \end{tabular}
\end{table}
Table 1: Fish byproducts utilized to produce novel peptides derived from enzymatically hydrolyzed fish proteins.