# Extraction of Novel Bioactive Peptides from Fish Protein Hydrolysates by Enzymatic Reactions
## 3 Application of Enzymatic Hydrolysis for Fish Protein Isolation  
Large amounts of fish and fish byproducts are currently being discarded and utilized for low-quality and low-value products. Fish byproducts contain high-value bioactive molecules, found in the fish muscle. In addition, solid and liquid byproducts from fish are a good alternative source to utilize and recover potent bioactive molecules that may be present. One way to valorize this biomass is the use of enzyme technology, which is a widely practiced way to produce a broad spectrum of food ingredients with a wide range of applications. The hydrolyzation of proteins from any source can be carried out via chemical (acid or alkali) treatment or the application of biochemical methods ([PERSON] et al., 2015), and is achieved via proteolytic enzymes that are naturally occurring in fish muscle tissues. In addition, the treatment of commercial enzymes accelerates the process (enzymatic hydrolysis) and further breaks down larger-sized peptides into amino acids. Many studies in the literature on novel methods of fish protein hydrolysis have been published in scientific journals. A recent study was conducted by [PERSON] et al. (2018) on hybridizing plasma functionalized water and ultrasound pretreatment for enzymatic hydrolysis (HPUEH). The authors indicated that the optimized method accelerated the hydrolysis and revealed small-sized peptides with increased surface area and improved bioactivities. However, the use of these novel method needs further evaluation before they can be utilized for industrial-scale production. Another method developed by [PERSON] et al. (2018) utilized enzymatic hydrolysis by using commercial enzymes for fucoidan extraction from Gagome kelp and used \(\beta\)-glucosidase for the efficient degradation of cellulose, hindering the fucoidan release from the cell wall. However, both methods--as well as other novel methods available--have one major purpose: the extraction of potent bioactive compounds and further separation for improved functionality and bioactivities. In the hydrolysis of proteins, degradation or breakdown converts these molecules into smaller molecular structures, peptides, and amino acids, and smaller-molecular-mass peptides can be absorbed by the body of an organism faster than proteins ([PERSON] et al., 2018). Currently, many approaches to the enzymatic hydrolysis of proteins have been developed. Most methods follow the same principle with modifications ([PERSON] et al., 2018). Generally, the principle of hydrolysis involves the solubilization of a solid (fish) with water, mixed 1:1. This process needs further evaluation as to the ratio of water added to the solid, to ensure the suitable extent of protein breakdown and to avoid extra costs on drying unnecessary  
Figure 2: Schematic diagram of fish waste generation from fish harvesting and processing. water fractions from FPH in the drying process [15]. The extent of hydrolyzation is evaluated using the parameter named \"degree of hydrolysis\" (DH), which is commonly used and is an important factor related to the hydrolysis yield [37]. DH is measured and expressed as the ratio of broken peptide bonds to the total peptide bonds in the mixture per unit weight (Equation (1)): where DH is the degree of hydrolysis expressed in percent; \(Pbr\) is the number of broken peptide bonds; and \(Ptot\) is the total number of peptide bonds in the mixture. \[\text{DH }=\frac{Pbr}{Ptot}\times 100\% \tag{1}\]  
The enzymatic hydrolysis of proteins has been extensively investigated in recent years, and products of this process have been utilized for industrial and nutraceutical purposes [7]. Marine products and byproducts, for example, have been studied and were found to have high contents of functional peptides and amino acids. In the process of enzymatic protein hydrolysis (EPH), endogenous or added enzymes can be used as catalysts to break down peptide bonds between amino acids [15].