## 3 Results and Discussion
### Protein Hydrolysis  
#### 3.2.1 Influence of pH and Temperature  
The _Eo/So_ ratio used in this study was 0.16 AU/g (5.7% g of Alcalase/g proteins in meat waste) according to [PERSON] and [PERSON] [62], who used the ratios from 2.9 to 9.1% for meat hydrolysis with Alcalase. The tests were performed under different conditions with respect to temperature (40-60 \({}^{\circ}\)C at a pH of 8.0) and pH (7.0-9.0 at a temperature of 50 \({}^{\circ}\)C). The pH and temperature were selected in accordance with the optimal values for Alcalase (see Section 2.1). The proteins were recovered as peptides in the forms of hydrolysates and collagen. The results obtained show how high pHs and low temperatures favor hydrolysis, producing hydrolysates with a higher percentage of hydrolyzed protein and a lower percentage of unhydrolyzed collagen (Tables 2 and 3). The effect of pH on protein hydrolysis is justified by the increase of enzymatic activity with increasing the pH and the stability of the enzyme in the pH range of 7-10 [63]. At first glance, it might seem that the effect of temperature on protein hydrolysis disagrees with the literature where a higher enzymatic activity is achieved when the temperature rises [63]. However, the Alcalase inactivation over time with an increase of temperature is so high that the efficiency of the process is greater at 40 \({}^{\circ}\)C for 240 min of reaction time [63]. According to the results obtained, within the analyzed pH and temperature ranges, lower pHs or higher temperatures are recommended to recover unhydrolyzed collagen, while higher pHs or lower temperatures are more adequate to obtain protein hydrolysates. The selection of the operating conditions of pH and temperature will depend on the size of the peptides whose obtainment is desired in the hydrolysates. Table 4 shows the average peptide chain lengths and the average molecular weights of the peptides present in the hydrolysates obtained under different operating conditions. \begin{table}
\begin{tabular}{c c c} \hline
**pH** & **Hydrolyzed Protein (\%)** & **Unhydrolyzed Collagen (\%)** \\ \hline
7.0 & 40.30 \(\pm\) 0.14 & 48.18 \(\pm\) 0.71 \\
7.5 & 41.05 \(\pm\) 0.11 & 45.41 \(\pm\) 0.94 \\
8.0 & 45.03 \(\pm\) 0.20 & 43.10 \(\pm\) 0.10 \\
8.5 & 58.65 \(\pm\) 0.38 & 42.18 \(\pm\) 0.45 \\
9.0 & 59.30 \(\pm\) 0.62 & 38.18 \(\pm\) 0.44 \\ \hline \end{tabular}
\end{table} 
Table 2: Recovery of proteins at different pHs. \begin{table}
\begin{tabular}{c c c} \hline
**T (\({}^{\circ}\)C)** & **Hydrolyzed Protein (\%)** & **Unhydrolyzed Collagen (\%)** \\ \hline
40 & 46.76 \(\pm\) 0.20 & 35.94 \(\pm\) 0.70 \\
45 & 45.82 \(\pm\) 0.27 & 39.23 \(\pm\) 0.08 \\
50 & 45.03 \(\pm\) 0.20 & 43.10 \(\pm\) 0.10 \\
55 & 41.62 \(\pm\) 0.38 & 43.17 \(\pm\) 0.34 \\
60 & 40.83 \(\pm\) 0.62 & 47.47 \(\pm\) 0.73 \\ \hline \end{tabular}
\end{table} 
Table 3: Recovery of proteins at different temperatures. To use protein hydrolysates in the food industry, they must be easily digestible and palatable, which is only reached when the molecular weight of peptides makes up the hydrolysate ranges in a certain interval because:  
1. Peptides with a high molecular weight (over 6000 Dalton) can cause allergies [64]. 2. Peptides with a molecular weight range from 1000 to 6000 Dalton have bitter taste [65]. 3. Free amino acids (molecular weight under 200 Dalton) are hyperosmotic and cause diarrhea [66]. So, to avoid the problems of allergenicity, bitterness, and hyperosmoticity, the hydrolysates should mainly consist of di- and tripeptides and have a range of molecular weight distribution as narrow and as feasible with an average molecular weight near 500 Dalton and, in any case, between 200 and 1000 Dalton [16]. Taking this into account, the most appropriate operating conditions, according to our study, should be between 7.5 and 8.5 for pH and 45 and 55 \({}^{\circ}\)C for temperature because the obtained hydrolysates have average molecular weights closest to 500 Dalton, as shown by the lower relative deviation (see Table S1 in Supplementary Materials).