# Rapid Green Extractions of C-Phycocyanin from _Arthrospira maxima_ for Functional Applications
## Materials and Methods
### Green Extraction Design for Phycobilproteins Extraction
#### 2.3.2 High-Pressure Homogenization (HPH) Design  
A GEA Niro Soavi Homogenizer (Panda PLUS model, Parma, Italy) was used as the HPH system. The ratio of _A. maxima_ biomass and extraction solvent used was 2:100 (_w/v_) with two passages. After the HPH process, the aliquots were centrifuged at 3140\(\times\)_g_ for 5 min to quantify the C-PC. The factors evaluated were the pressure applied in the homogenization and solvents. For optimizing HPH extraction conditions, a factorial experimental design was used based on two factors, considering five levels of pressure: 800, 1000, 1200, 1400 and, 1600 bar. However, in the factor solvent there were three levels: distilled water, Na-phosphate buffer (pH 7.0) 100 mM and, Na-phosphate buffer:water 1:1, (_v/v_). The effect of the factors and its interactions on specific response as C-PC content was studied. A total of 15 experiments (n = 3) as shown in Table 2 for _A. maxima_.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{4}{c}{**HPH Extraction Conditions**} \\ \hline
**Exp.** & **Power (W)** & **Time (s)** & **Solvent Type \({}^{*}\)** & **C-PC Content (mg/g)** \\ \hline
13 & 0 & 0 & \(-1\) & 134.7 \(\pm\) 2.6 \\
14 & 0 & 0 & 0 & 121.2 \(\pm\) 24.9 \\
15 & 0 & 0 & +1 & 136.9 \(\pm\) 1.8 \\
16 & 0 & +1 & \(-1\) & 153.0 \(\pm\) 5.2 \\
17 & 0 & +1 & 0 & 87.5 \(\pm\) 17.4 \\
18 & 0 & +1 & +1 & 172.9 \(\pm\) 16.1 \\
19 & +1 & (300) & \(-1\) & \(-1\) & 109.1 \(\pm\) 12.7 \\
20 & +1 & \(-1\) & 0 & 61.8 \(\pm\) 25.9 \\
21 & +1 & \(-1\) & +1 & 92.0 \(\pm\) 8.1 \\
22 & +1 & 0 & \(-1\) & 85.7 \(\pm\) 30.0 \\
23 & +1 & 0 & 0 & 46.8 \(\pm\) 14.2 \\
24 & +1 & 0 & +1 & 86.9 \(\pm\) 10.7 \\
25 & +1 & +1 & \(-1\) & 37.8 \(\pm\) 6.6 \\
26 & +1 & +1 & 0 & 35.1 \(\pm\) 5.7 \\
27 & +1 & +1 & +1 & 44.2 \(\pm\) 2.2 \\ \hline \hline \end{tabular} \({}^{*}\) Three independent extraction solvents were used: (\(-1\)) water: distilled water; (0) Buffer: Na-Phosphate buffer pH 7.0, 100 mM and (+1) Buffer:water: Na-Phosphate buffer pH 7.0, 100 mM:water 1:1 (_v/v_).  
\end{table}
Table 1: _Cont._  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{4}{c}{**HPH Extraction Conditions**} \\ \hline
**Exp.** & **Pressure (Bar)** & **Solvent \({}^{*}\)** & **C-PC Content (mg/g)** \\ \hline
1 & \(-1\) & (800) & \(-1\) & (Water) & 218.1 \(\pm\) 10.1 \\
2 & \(-1\) & & 0 & (Buffer) & 225.9 \(\pm\) 0.4 \\
3 & \(-1\) & & +1 & (Buffer:water) & 179.3 \(\pm\) 1.1 \\
4 & \(-0.5\) & (1000) & \(-1\) & & 224.0 \(\pm\) 3.3 \\
5 & \(-0.5\) & & 0 & & 233.7 \(\pm\) 5.6 \\
6 & \(-0.5\) & & +1 & & 203.4 \(\pm\) 6.9 \\
7 & 0 & (1200) & \(-1\) & & 252.5 \(\pm\) 4.1 \\
8 & 0 & & 0 & & 268.8 \(\pm\) 6.4 \\
9 & 0 & & +1 & & 212.3 \(\pm\) 9.3 \\
10 & +0.5 & (1400) & \(-1\) & & 249.1 \(\pm\) 6.1 \\
11 & +0.5 & & 0 & & 291.9 \(\pm\) 6.7 \\
12 & +0.5 & & +1 & & 210.3 \(\pm\) 1.2 \\
13 & +1 & (1600) & \(-1\) & & 224.3 \(\pm\) 6.4 \\
14 & +1 & & 0 & 257.4 \(\pm\) 7.4 \\
15 & +1 & & +1 & 177.9 \(\pm\) 5.9 \\ \hline \hline \end{tabular} \({}^{*}\) Three independent extraction solvents were used: (\(-1\)) water: distilled water; (0) Buffer: Na-Phosphate buffer pH 7.0, 100 mM and (+1) Buffer:water: Na-Phosphate buffer pH 7.0, 100 mM:water 1:1 (_v/v_).  
\end{table}
Table 2: Experimental design matrix including extraction conditions and results for C-PC content as response variable studied for the optimization of the high-pressure homogenization (HPH) of the cyanobacteria _Arthrospira maxima_. Values presented are mean \(\pm\) SD, n =3.