# Collagen-Based Bioactive Bromelain Hydrolysate from Salt-Cured Cod Skin
## 4 Discussion  
This study aimed to develop a new integrated methodology to produce a collagen-rich enzymatic hydrolysate from SCCS sustainably and simply. On the one hand, enzymatic hydrolysis is the best way to hydrolyse fish skin without losing nutritional and healthy values [35]. On the other hand, the increased fish processing sector results in massive production of waste, which makes it a challenge to dispose of these materials to optimise value and reduce environmental impact [36]. So, in the first step, we compared the BR hydrolysis capacity to CHC, a specific protease for collagen. BR is a vegetal cysteine endopeptidase with broad specificity used for many industrial applications [12] and was already proved to be effective in hydrolysed collagens [37]. BR was also successfully applied to collagen extraction from the skin of bigeye tuna (_Thumus obesus_). It showed the highest collagen yield between other proteases (papain, pepsin, and trypsin) with excellent chemical properties and antioxidant activity [38]. Figure 5: Metabolic inhibition of optimal hydrolysate with a concentration of 1 mg/mL for 24 h against two mammalian cell lines, namely: human keratinocyte HaCaT cells; and mouse fibroblast L929 cells. Each boxplot represents \(n=6\). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Factor \({}^{\dagger}\)** & **Optimal** & \\ \hline \(\mathrm{X_{A}}\) & & 4.0 \\ \(\mathrm{X_{B}}\) & & 29.8 \\ \(\mathrm{X_{C}}\) & & 30.0 \\ \multicolumn{3}{c}{Optimal desirability: 0.516} \\ \hline \hline  & **Model validation** & \\
**Response \({}^{\ddagger}\)** & **Predicted \({}^{\ast}\)** & **Observed** \\ \hline Protein concentration\(\pm\) & 3.3 \(\pm\) 0.7 & 2.2 \\ ORAC & 517 \(\pm\) 92 & 414 \\ iACE & 63 \(\pm\) 11 & 59 \\ \hline \hline \end{tabular} \({}^{\dagger}\) Factors: pH (\(\mathrm{X_{A}}\)); temperature (\(\mathrm{X_{B}}\)) expressed in \({}^{\circ}\)C; time (\(\mathrm{X_{C}}\)) expressed in min. \({}^{\ddagger}\) Responses: protein concentration expressed in mg/mL; ORAC expressed in umol TE/g protein; iACE expressed in % inhibition/(0.15 mg/mL protein). \({}^{\ast}\) Values expressed as mean \(\pm\) SD of three replicates.  
\end{table} 
Table 3: Derringer optimisation and model validation. Furthermore, [PERSON] et al. [11] found that BR (10.62%) had the second-best DH (%) of water-washed catla meat in the optimum conditions after the commercial enzyme protamex (12.61%). In another case, [PERSON] et al. [39] found that the BR was between the best enzymes for the hydrolysis (50.59% DH) of gelatine extracted from Nile tilapia skin. [PERSON] et al. also investigated the hydrolysis of stonefish protein with BR, obtaining 54.62% of DH's optimum conditions [7]. In our case, although the DH did differ between the enzymes, with BR, proper digestion of the protein material is still obtained. In addition, the electrophoresis for both enzymes showed that BR is an excellent enzyme to obtain low molecular weight peptides from the collagen of cod skin. Low molecular weight collagen hydrolysates are generally thought to exert better bioactivities. However, many factors affect their preparation, such as the long-lived protein property, the strong and tough cross-links, and the presence of Hydroxyproline (Hyp) in the structure that plays a key role in collagen stability [13]. Possibly, since BR is a protease that acts by making random cuts, this property helps to increase the probability of obtaining low molecular weight hydrolysates with greater bioactivities. Consequently, during the optimisation process, the high value of protein extraction and high antioxidant and antihypertensive activities in the cod collagen-rich enzymatic hydrolysate were found. The optimum conditions were pH 4.0 and temperature 28.9 \({}^{\circ}\)C during 30 min of hydrolysis. Previously, collagen peptides derived from fish waste were reported to have antioxidative activity [10; 11; 40; 41] and antihypertensive activity [39; 42; 43; 44].