# Tannin Extraction from Chestnut Wood Waste: From Lab Scale to Semi-Industrial Plant
## 3 Results and Discussions
### Lab-Scale Downstream--Membrane Filtration  
The encouraging results obtained by the sequential extraction was further developed, focusing on the enhancement of the tannin-rich fraction. According to that goal, membrane filtrations (Section 2.5) were performed aiming at an additional fractionation of the extract. In detail, different filtration protocols were carried out on the liquor derived from the 100 \({}^{\circ}\)C extraction (global tannin content of 88%). The first approach consisted of a 1000 Da UF, leading to the recovery of a concentrated permeate (PUF) and a diluted retentate (RUF). This process led to a fractionation of the polyphenol's population, mainly caused by the retention of tannins (characterized by a higher weight and larger dimensions) and the permeation of the smaller molecules (monomers such as gallic acid, ellagic acid, and others). The starting material was characterized by 88.07% of tannins, while the obtained RUF reached 92.00%, with a concentration increase of 14.60%. The RUF was successively processed investigating the application of diafiltration. The aim of this technique was to further concentrate the tannin fraction in the retentate, by adding fresh water and enhancing the separation driving force, replacing the removed volume of the permeate. Moreover, diafiltration reduces the matrix effect of the solution, improving the membrane rejection and metabolite discrimination. Two different tests were performed to investigate the impact of the water amount in diafiltration. Thus, RUF was divided into two streams: a single diafiltration (with a quarter of the removed volume) and a triple diafiltration (complete replacement of the water removed in the previous step), as described in Figure 6.  
Figure 5: Pre-treatment influence on extract population of tannins and polyphenols. The first test was carried out with only a quarter of the water removed in the first UF step, with the aim of saving water. Unfortunately, the obtained result in tannin enrichment was negligible. The small amount of water probably does not allow us to overcome the matrix effect and recover the membrane performance. On the other hand, the second experiment allowed us to achieve engaging results: the average tannin concentration reached nearly 100% at the end of the sequential process (Figure 7, brown line). Hence, it is possible to state that, as expected, monomeric polyphenols mostly crossed the 1000 Da membrane, concentrating the heavy metabolites in the retenate. Further investigations conducted showed that TPC selectivity is almost unaffected, across the successive retention fractions (Figure 7, ruby bars, RUF RD3) with small variations due to the variability of the method. It is worth noting that this last parameter refers to all active polyphenolic fractions, so its constant trend could indicate the additional removal of \"non-active\" compounds, such as salts and oligosaccharides, despite the decrease in monomeric polyphenols. Figure 6: (**a**) Single diafiltration with 1/4 of removed water; (**b**) triple diafiltration with complete water replacement. Figure 7: Tannin enrichment with the triple diafiltration (UF). As a further confirmation, the \"tannin/monomeric polyphenol\" ratio of the retentates across the process has been analysed and reported in Figure 8. It is possible to appreciate the increasing values of the tannin percentage as opposed to the polyphenol decrease, starting from the first retentate (RUF) and gradually proceeding together with the diafiltration steps (RD1, RD2, and RD3). After the UF protocol, the resulting permeates were combined and processed with an NF membrane, aiming to concentrate the metabolites and recover water at the same time. The latter can be ideally recycled within the process. After NF, the recovered fractions were a retentate (RNF) and a permeate (PNF). The feed concentration was _approx_. 0.75 mg/mL and, through this process, the RNF reached 10.06 mg/mL (a 13.5-times increase), with 752.71 mgGAE/gext (meaning _approx_. 75.3% of the monomeric polyphenol content).