## 3 Results and Discussion
### Proteins and Peptides Characterization  
Fourier-transform infrared (FTIR) spectroscopy is a method widely used for the characterization of proteins and peptides [57]. It has been reported that proteins and peptides generate nine distinct infrared absorption bands (i.e., amide A, amide B, and amide I-VII) [58; 59]. The most predominant bands are recorded at approximately 1650 cm\({}^{-1}\) and at 1550 cm\({}^{-1}\), corresponding to the amide I and amide II bands.  
Figure 3 represents FTIR spectra of proteins and peptides samples. The characteristic bands of amide I, amide II, and amide III, as well as the C-C and C=O stretching vibration are detectable in the obtained FTIR spectra.  
Figure 2: Comparison of different cell lysis methods for the recovery of intracellular proteins from _Rhodomonas_ sp.  
The main peaks associated with crude protein preparation were observed at 3274 cm\({}^{-1}\), 2927 cm\({}^{-1}\), 1636 cm\({}^{-1}\), 1527 cm\({}^{-1}\), and 1044 cm\({}^{-1}\), corresponding to N-H stretch, O-H stretch, C=O stretch at amide I; N-H deformation/C-N stretch at amide II, and C-O stretch, respectively (Table 2). After the digestion of proteases, the spectra of the produced peptides revealed the appearance and shift of several peaks compared to the nonhydrolyzed control (Figure 3). In addition, peaks related to the amide bands, which does not change location, showed a decreasing amplitude in the peptides' spectra. The shifted and/or appeared peaks in the peptides spectra were recorded in various regions, mainly at 1500-1400 cm\({}^{-1}\), associated with C-terminal groups. Protein digestion using proteases generates COO\({}^{-}\) groups.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**Alcalase Hydolyasate** & \multicolumn{2}{c}{**Pepsin Hydolyasate**} & \multicolumn{2}{c}{**Proteins**} & \multicolumn{2}{c}{**Wave Number**} & \multicolumn{1}{c}{**Functional Groups**} \\ \hline
**Wave Number** **(cm\({}^{-1}\))** & **Area** & **Wave Number** **(cm\({}^{-1}\))** & **Area** & **Wave Number** **(cm\({}^{-1}\))** & **Area** & **Range (cm\({}^{-1}\))** & **Functional Groups** [57,60,611] \\ \hline
3181 & \(-\)365.280 & & & 3274 & \(-\)5.315 & & \\
3102 & \(-\)84.440 & & & & & \\
2972 & \(-\)93.397 & & & & & \\
2942 & \(-\)21.223 & & & & & \\ \hline
2806 & \(-\)37.558 & & & & & \\
2658 & \(-\)29.335 & & & & & \\
2598 & \(-\)39.612 & & & & & \\
2468 & \(-\)8.542 & & & & & \\
2094 & \(-\)55.081 & & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 2: Summary of wave numbers and corresponding functional groups.  
Figure 3: FTIR spectra of _Rhodomonas_ sp. crude protein and peptides produced after alcalase and pepsin hydrolysis.  
Recently, FTIR spectroscopy has been considered an efficient approach for the structural characterization of proteins and peptides [57]. It could provide information on peptides and proteins size as well as on the secondary structure of protein [60]. It is also a useful technique for the investigation of protein-related parameters, mainly the effects of solvent, temperature, and pH on proteins [62; 63]. [PERSON] et al. [57] proved the potential of using FTIR for monitoring protein sizes during enzymatic digestion.