## 3 Results and Discussion
### Protein Hydrolysis
#### 3.2.2 Influence of Protease/Proteinic Substrate Ratio (_Eo_/_So_)  
To check the influence of the _Eo_/_So_ ratio on product recovery, experiments were carried out at different ratios, from 0.09 to 0.27 AU/g (3.2-7.5% g of Alcalase/g proteins in meat waste). This range was chosen based on literature [62] where, as previously said, the ratios from 2.9 to 9.1% are used for meat hydrolysis with Alcalase. The temperature (50 \({}^{\circ}\)C) and pH (8.0) were selected at the midpoint of the optimal values obtained previously (see Section 3.2.1). Again, the proteins were recovered as peptides in the forms of hydrolysate and collagen.  
As expected, the results obtained indicate that an increase in the protease/proteinic substrate ratio causes a higher percentage of hydrolyzed protein and a lower percentage of unhydrolyzed collagen (Table 5).  
The results in Table 5 show that a greater amount of protease in the reactor for the same concentration of proteinic substrate (higher _Eo_/_So_) allows the increase in the number of enzyme-substrate bonds formed, promoting a high number of broken pep  
\begin{table}
\begin{tabular}{c c c c} \hline
**pH** & **T** (\({}^{\circ}\)**C**) & **PCL** & **MM** (Daltons) \\ \hline
7.0 & 50 & 4.74 & 616.55 \\
7.5 & 50 & 3.90 & 506.43 \\
8.0 & 50 & 3.63 & 471.95 \\
8.5 & 50 & 3.34 & 434.13 \\
9.0 & 50 & 2.82 & 366.77 \\
8.0 & 40 & 2.96 & 385.13 \\
8.0 & 45 & 3.25 & 422.28 \\
8.0 & 50 & 3.63 & 471.95 \\
8.0 & 55 & 3.86 & 501.16 \\
8.0 & 60 & 4.51 & 586.77 \\ \hline \end{tabular}
\end{table}
Table 4: Average peptide chain length (_PCL_) and molecular weight (_MM_) of the hydrolysates obtained under different pHs and temperatures (T).  
\begin{table}
\begin{tabular}{c c c} \hline
**Eo/_So_ (**AU/g**) & **Hydrolyzed Protein (\%)** & **Unhydrolyzed Collagen (\%)** \\ \hline
0.09 & 29.79 \(\pm\) 0.17 & 59.37 \(\pm\) 0.11 \\
0.11 & 35.42 \(\pm\) 0.40 & 53.85 \(\pm\) 0.06 \\
0.16 & 45.03 \(\pm\) 0.16 & 43.26 \(\pm\) 0.11 \\
0.21 & 49.93 \(\pm\) 0.26 & 34.39 \(\pm\) 0.05 \\
0.27 & 64.42 \(\pm\) 0.24 & 31.54 \(\pm\) 0.06 \\ \hline \end{tabular}
\end{table}
Table 5: Recovery of proteins at different protease/proteinic substrate ratios (_Eo_/_So_).  
tide bonds, favoring the hydrolysis, and generating an increase in the amount of the hydrolysates produced.  
However, collagen is a fibrous-type protein whose behavior against hydrolysis differs from that of globular-type proteins. Whereas globular-type proteins are more easily hydrolyzed given their characteristics, collagen is less soluble and more difficult to hydrolyze in its native state, which means that a higher concentration of protease is necessary for its hydrolysis [67]. Therefore, for a greater recovery of collagen for industrial applications, it would be convenient to work with near zero initial concentrations of Alcalase.  
The choice of the _Eo/So_ operating conditions will rely on the required size of the peptides in the hydrolysates. Table 6 shows the average length of the peptide chain and the average molar mass for the hydrolysates obtained using different _Eo/So_ ratios.  
As previously said, to use protein hydrolysates in the food industry, the molecular weight of the hydrolysates should be of the order of 500 Dalton and, in any case, between 200 and 1000 Dalton [16]. Bearing this in mind, the best quality hydrolysates would be those obtained for the ratios of protease/protein substrate concentrations between 0.11 and 0.21 AU/g since, with these ratios, the average molar mass of the final products is closest to 500 Dalton, as shown by the lower relative deviation (see Table S2 in Supplementary Materials).