## 3 Results and Discussion
### Structural Analysis by ATR-FTIR  
The structural conformation of proteins affects the digestibility and functional properties of the SPI, thus playing a crucial role in the application of the isolate. FTIR spectroscopy determines the functional groups present in an organic sample and has been recently used to determine secondary structures in protein samples [52; 53]. In addition, the number of  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Factors**} & **Predicted** & **Predicted Protein** & **Confidence** & **Experimental Protein** \\  & **Optimized Level** & **Yield (\%)** & **Interval (95\%)** & **Yield (\%)** \\ \hline Temperature (\({}^{\circ}\)C) & 70 & 49.79 & (46.66–52.93) & 48.30 \(\pm\) 0.21 \\ Time (min) & 44.7 & - & - & - \\ Alkaline & 3 & - & - & - \\ solution (coded) & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 6: Multiple response prediction.  
Figure 4: Electrophoretic profile of soy protein isolates. REF: standard; COM: commercial soy protein isolate; SPI: soy protein isolate extracted at optimal conditions (alkaline solution 3, for 45 min and 70 \({}^{\circ}\)C).  
\(\beta\)-conformations (\(\beta\)-sheet and \(\beta\)-turn) and \(\alpha\)-helix determines the digestibility and degree of hydrolysis of the protein [51].  
Figure 5A depicts the FTIR spectra of the soybean flour, commercial SPI, and obtained SPI. The three samples showed the characteristic spectra of soybean: at \(\sim\)3400 cm\({}^{-1}\), the -NH and -OH stretching' amide I between 1700 and 1600 cm\({}^{-1}\); and amide II between 1600 and 1500 cm\({}^{-1}\)[37]. Figure 5B-D present the deconvolution of the amide I in the obtained SPI, commercial SPI, and soybean flour, respectively. [PERSON] et al. (2015) and [PERSON] et al. (2008) reported that the range of wavenumber (cm\({}^{-1}\)) within each secondary structure is present [37; 38]. Then, knowing the number of peaks corresponding to \(\beta\)-conformations, \(\alpha\)-helix, and random coil, the relative percentage was calculated.  
The calculated percentages of secondary structures in the samples are reported in Table 7. This study shows that SPI samples have a higher ratio of \(\beta\)-conformations and \(\alpha\)-helix (\(\beta\):\(\alpha\) ratio) than the soybean flour sample, which is consistent with [PERSON] et al. (2015), who found that a higher \(\beta\)-conformations number was correlated with more heat treatment on the sample [53]. Soybean flour results were comparable to [PERSON] et al. (2016), who obtained a 26% higher \(\beta\):\(\alpha\) ratio that can be explained due to differences in soybean treatment [54]. Our SPI sample had the highest \(\beta\):\(\alpha\) ratio, followed by commercial SPI and soybean flour. [PERSON] et al. (2012) and [PERSON] et al. (2016) found a negative correlation between \(\beta\)-conformations presence and digestibility. However, this decrease in digestibility is necessary to avoid antinutritional factors such as urease activity (UA), which will be discussed next [55; 51].  
Figure 5: FTIR spectra of soybean flour, commercial SPI, and obtained SPI (**A**); deconvolution of obtained SPI (**B**), deconvolution of commercial SPI (**C**), and deconvolution of soybean flour sample (**D**).