## 2 Results and Discussion  
### Proximate Analysis and pH Values of Extracted Gelatin and Hydrolyzed Gelatin Peptides  
The samples of G and HGPs (Figure 2) were extracted from unsorted, mixed, and dehydrated crude tuna side streams.  
The collagenous material of this biomass is mainly present in the bones and the connective tissue. Spray-drying dehydration was chosen as the most ideal drying method as it represents the most scalable process in terms of time and energy consumption. The method was compared against oven drying and lyophilizing (Figure S1). The two protein samples (of G and HGPs) were analyzed to determine their proximate compositions (residual moisture, proteins, and ashes) and pH values (Table 1). The proximate compositions of the samples were similar. In particular, the residual moisture was, in both samples, between 4 and 5% after the spray-drying dehydration treatment, which would allow one to obtain optimal and standardized results that warrant a long shelf life. The crude protein fraction of 90-93% was higher than the 82% value reported by [PERSON] et al. [12], referring to gelatin extracted from tuna bones, and it was in line with the result of 90%, obtained by [PERSON] et al., for a gelatin extracted from the bones of the skipjack tuna [13]. As for HGPs, the Gelatin Manufacturers of Europe Association (GME) states that proteins make up 97% of the total weight in collagen peptides [14]. The total contents of ashes in G and HGPs were approximately 3% and 5%, respectively. One factor contributing to the increased quantity of ashes in HGPs could be explained by the addition of sodium hydroxide to the sample during the extraction process. The percentage of ashes was lower than in the ones obtained by [PERSON] et al., where they had between 7 and 8% in gelatin extracted from tuna bones [15]. The quantity of ashes depends on the content of minerals that are naturally present in bones. In both cases, considering the results of the proteins and low percentage of ashes, the two protein samples showed high purity, which is essential for many possible applications, such as for biomaterials, in which the high content in salt can increase the film opacity since the composition can affect the transparency [16].  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Gelatin (G)** & **Hydrolyzed Gelatin Peptides (HGPs)** \\ \hline Residual moisture 1 (g/100 g) & 4.6 \(\pm\) 0.3 & 4.1 \(\pm\) 0.1 \\ Proteins 1 (g/100 g) & 92.9 \(\pm\) 0.8 & 90.0 \(\pm\) 0.7 \\ Ashes 1 (g/100 g) & 3.1 \(\pm\) 0.6 & 4.6 \(\pm\) 0.2 \\ pH 1 & 3.4 \(\pm\) 0.1 & 4.8 \(\pm\) 0.1 \\ \hline \hline \end{tabular} 1  
\end{table}
Table 1: Proximate analysis and pH of gelatin (G) and hydrolyzed gelatin peptides (HGPs).  
Figure 2: Spray-dried gelatin (G) and hydrolyzed gelatin peptides (HGPs) obtained from the proposed single-cascade flow chart extraction.  
Regarding the pH, it is strictly dependent on both the extraction conditions and the starting raw material. In this study, the pH was lower for G than HGPs, with the values being 3.4 and 4.8, respectively. [PERSON] et al. [17] observed that the pH of the gelatin extracted from the bones of the king weakfish was around 4.  
The microbiological and oxidation parameters and the presence of chemical contaminants (i.e., dioxins, PHA (benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene and chrysene, metals, and PCB) were determined using the official methods and were checked on both the starting material (pre- and post-dehydration steps) and on the extracted bioactives. Some data were reported in the first part (Part I) of this research publication [1]; nevertheless, all the levels of contaminants were below the limits reported by Commission Regulation (EU) 2023/915 of 25 April 2023 on the maximum levels for certain contaminants in food.