# Tannin Extraction from Chestnut Wood Waste: From Lab Scale to Semi-Industrial Plant
## 3 Results and Discussions  
### Maswe  
Sequential Extraction Screening  
The CWS were extracted in subcritical water (see Section 2.3), adopting a sequential step approach, investigating the possibility of enhancing the global efficiency of the process. According to that, the first step could act at the same time as the extraction and pre-treatment step, allowing us to improve the recovery of active compounds, achieving an efficient extraction performance. In addition, this approach would be investigated in terms of \"pre-fractionation\" of the final product achieved, hence exploiting the different solubilities and extraction thermodynamics/kinetics of metabolites, according to their mass weight, dimensions, and chemical features (i.e., flavonoids versus tannins)  
In the starting extraction set, the study considered a quick first step at 120 \({}^{\circ}\)C for 5 min, followed by a subsequent step at 150 \({}^{\circ}\)C for 15 min. In order to monitor the extraction process, the TPC was followed, as a key metric for polyphenol recovery. As reported in Table 2, the first step led to the highest yield (12.15 versus 5.48%) with concomitant TPC selectivity values (646.36 versus 242.77 mg GAE/gext). The polyphenols' yield, expressed on the dry matrix (DM) treated, follows the trend and shows advantageous results for the former step (78.53 versus 13.30 mg GAE/gDM). To track the process outcomes, the cumulative yield of the protocol was also calculated, both as the dry product yield (16.97%) and polyphenol extraction total yield from the matrix (90.22 mg GAE/gDM); see Figure 1.  
With the aim to enhance the overall selectivity of the protocol, a dedicated pre-treatment was implemented in the cascade. Thus, the biomass was treated at room temperature (RT) for 2 h to verify the removal of non-active co-extracts (Table 3). \begin{table}
\begin{tabular}{c c c c c} \hline \multirow{2}{*}{**T (\({}^{\circ}\)C)**} & \multirow{2}{*}{**t (min)**} & \multirow{2}{*}{**Yield (\%)**} & \multicolumn{2}{c}{**TPC**} \\ \cline{3-4}  & & & **Selectivity** & **Yield** \\  & & & **(mg GAE/gext)** & **(mg GAE/gDM)** \\ \hline
120 & 5 & 12.15 & 646.36 \(\pm\) 10.45 & 78.53 \(\pm\) 1.27 \\
150 & 15 & 5.48 & 242.77 \(\pm\) 5.72 & 13.30 \(\pm\) 0.31 \\ \hline \end{tabular}
\end{table} 
Table 2: Extraction conditions, yield, and TPC of the sample obtained with the process performed at 120 \({}^{\circ}\)C and 150 \({}^{\circ}\)C. \begin{table}
\begin{tabular}{c c c c c} \hline \multirow{2}{*}{**T (\({}^{\circ}\)C)**} & \multirow{2}{*}{**t (min)**} & \multirow{2}{*}{**Yield (\%)**} & \multicolumn{2}{c}{**Selectivity**} & \multicolumn{2}{c}{**Yield**} \\  & & & **(mg GAE/gext)** & **(mg GAE/gDM)** \\ \hline RT & 120 & 3.25 & 266.16 \(\pm\) 24.43 & 8.65 \(\pm\) 0.79 \\
120 & 5 & 6.52 & 713.35 \(\pm\) 2.30 & 46.51 \(\pm\) 0.15 \\
150 & 15 & 5.82 & 288.13 \(\pm\) 3.57 & 16.77 \(\pm\) 0.21 \\ \hline \end{tabular}
\end{table} 
Table 3: Extraction conditions, yield, and TPC of the samples obtained with the process performed at RT, 120 \({}^{\circ}\)C, and 150 \({}^{\circ}\)C.  
The pre-treatment led to a low yield (_approx_. 3%), not affecting in a positive way the following extraction step. The overall yield of the process was 14.83% (instead of 16.97%, in the first condition set; see Table 2 and Figure 1). Despite the outcomes not being encouraging in terms of yield, the pre-treatment enhanced the selectivity of the following step (120 \({}^{\circ}\)C: increasing _approx_. 10%, 713.35 versus 646.36 mg GAE/gext; 150 \({}^{\circ}\)C: increasing approx. 15%, 288.13 versus 242.77 mg GAE/gext). This demonstrates the effectiveness of the pre-treatment, probably due to the swelling effect and the partial removal of non-active compounds (such as salts and saccharides). However, the resulting total TPC yield is moderate: from 1 g of the matrix, only 68.81 mg GAE can be recovered. In order to promote a fraction enrichment, a flash pre-treatment at a higher temperature was tested (100 \({}^{\circ}\)C, 5 min; see Table 4).