## 3 Results and Discussion
### Characterization of Eggshell-Waste-Derived Eggshell Membranes  
ESW-derived ESMs obtained through the transformation of ESW halves and pieces using 10% (\(w/v\)) acetic and 15% (\(w/v\)) \(o\)-phosphoric acid are shown in Figure 4a,c, and milled ones in Figure 4b,d. ESW-derived EMSs differentiated in their color regarding the acid used. Those obtained using 15% (\(w/v\)) \(o\)-phosphoric acid treatment of ESW showed a somewhat whiter intensity than those obtained using 10% (\(w/v\)) acetic acid treatment (Figure 4b,d), indicating possible differences in the ESMs chemical composition. The basic chemical composition of acid-derived ESW eggshell membranes and the raw ones is shown in Table 2. ESM obtained through ESW transformation using 10% (\(w/v\)) acetic acid had a somewhat higher amount of total proteins (96.48 \(\pm\) 1.75 g per 100 g of ESW per dry weight basis) than those produced using 15% (\(w/v\)) \(o\)-phosphoric acid (93.36 \(\pm\) 1.69 g per 100 g of ESW per dry weight basis), but both had a much higher protein content than the raw one (84.26 \(\pm\) 0.77 per 100 g of ESW per dry weight basis). On the other hand, the lipid content in ESMs obtained via acid transformation were slightly lower than those observed in raw EMS (Table 2). However, it should be noted that lipids were present in an amount lesser that 1%. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Content**} & \multirow{2}{*}{**Raw ESM**} & **10\% Actic-Acid-Derived** & **15\% \(o\)-Phosphoric-Acid-Derived** \\  & & **ESM** \\ \hline Dry matter [g/100 g] & 91.45 \(\pm\) 0.36 & 90.85 \(\pm\) 0.12 & 90.45 \(\pm\) 0.10 \\ Proteins [g/100 g\({}_{\text{d.w.}}\)].2 & 84.26 \(\pm\) 0.77 & 96.48 \(\pm\) 1.75 & 93.36 \(\pm\) 1.69 \\ Lipids [g/100 g\({}_{\text{d.w.}}\)]. & 0.57 \(\pm\) 0.06 & 0.42 \(\pm\) 0.11 & 0.26 \(\pm\) 0.09 \\ \hline \hline \end{tabular}  
* result present mean value \(\pm\) standard deviation of five independent determinations. 2 d.w.b.—dry weight basis.  
\end{table} 
Table 2: Chemical Composition of Obtained Eggshell Membranes 1. Figure 4: Eggshell Waste Derived Eggshell Membranes after Drying (**a,c**) and Milling (**b,d**). Legend: (**a,b**)—10% (\(w/v\)) acetic-acid-derived ESM; (**c,d**)—15% (\(w/v\)) \(o\)-phosphoric-acid-derived ESM. Since the basic chemical analysis of the amount of total proteins and lipids of obtained ESMs (Table 2) indicated some differences in chemical composition, it was necessary to elucidate which components of raw ESM has been extracted using 10% (\(w/v\)) acetic and 15% (\(w/v\)) \(o\)-phosphoric acid. This was performed via FTIR-ATR analysis as shown in Figure 5.  
FTIR-ATR analysis (Figure 5) revealed that raw eggshell membranes exhibited a higher intensity for all detected absorbance peaks than ESMs obtained using a 10% (\(w/v\)) acetic and 15% (\(w/v\)) \(o\)-phosphoric acid ESW treatment. The observed differences in the amount of lipids between raw and 10% (\(w/v\)) acetic and 15% (\(w/v\)) \(o\)-phosphoric-acid-derived ESMs (Table 2) were proven via decreased peak intensity with a wavenumber of 2919 cm\({}^{-1}\) (Figure 5) attributable to the C-H stretching vibration of endogenous lipids [58]. Strikingly, much higher amounts of total proteins were detected in EMS obtained using 10% (\(w/v\)) acetic and 15% (\(w/v\)) \(o\)-phosphoric acid treatment than those in raw ESM (Table 2), which was apparently opposite to the observed FTIR-ATR absorbance intensity of peaks attributable to the protein presence in examined ESM samples (Figure 5).