*3.5. Total Phenolic Content and Antioxidant Activity*

Pérez-Armada et al. (2019) [5] indicated that HS show a great potential as a source of natural antioxidants. These authors solubilized a part of the phenolics in HS by autohydrolysis, and recovered the target products using polymeric resins. In our work, the retentate from DD was subjected to acid hydrolysis to release a number of valuable compounds, including gallic, vanillic, and p-coumaric acids; aldehydes such as vanillin; and flavonoids such as catechin and (-) epicatechin.

Figure 4 shows the results achieved for the three streams involved in the membrane processing. The methods employed for this purpose included the total phenolic content (TPC, expressed as gallic acid equivalents/L or GAE/L), antioxidant activities determined using the methods TEAC (measured as g Trolox/L), FRAP (measured as g FeSO4·7H2O/L), and DPPH (measured as g GAE/L needed for EC50) [35–38].

The composition of phenolics (TPC) in AL210 was 1.63 g GAE/L, which is in the range reported for AL of other biomasses, i.e., eucalypt (1.64–1.98 g GAE/L) [47], vine shoots (1.33 g/L) [48], or peanut shells (1.58 g/L) [24]. The TPC of retentate decreased by 53.74% relative to the AL210 stream, and followed a behavior in agreement with reported literature concerning OS purification by membrane processes [23,24,27]. The TPC in the retentate accounted for 0.87 g/L, which implies a contribution of 5.00% relative to the NVC (17.44 g/L).

Despite the decreased phenolic content of retentate, the presence of phenolics is interesting due to their antioxidant activity, as these provide additional value to the target products as functional food ingredients [23,24,27]. According to the data in Figure 4, AL210 showed acceptable FRAP, TEAC, and DPPH activities. Interestingly, the DPPH radical scavenging capacity was not affected by DD, leading to the same EC<sup>50</sup> value in feed and retentate. On the contrary, the ABTS radical scavenging activity determined by the TEAC assay dropped by 58.19% in retentate relative to the value measured for AL210. However, this antioxidant activity is still an interesting contribution to the functional properties of the retentate.

*Agronomy* **2020**, *10*, 760

The assays based on radical scavenging reactions frequently show a dose-dependent response [23,49,50]. The data in Figure 5 show that the DPPH assay presented this type of behavior in the AL210 and retentate streams.

TEAC assay FRAP assay DPPH assay TPC

**Figure 4.** Total phenolic content (TPC) and antioxidant activity determined by TEAC, DPPH, and FRAP assays of AL210, retentate, and permeate. TPC, expressed as g GAE/L; TEAC, as g Trolox/L; FRAP, as g FeSO<sup>4</sup> ·7H2O/L; DPPH (EC<sup>50</sup> ), as g GAE/L.

**Figure 5.** Effects of the concentration on the DPPH radical scavenging activity, expressed as percentage of inhibition, PI (%), for autohydrolysis liquors at 210 ◦C (AL210) and retentate.

In general terms, the data obtained in this work for antioxidant activities follow general trends similar to the ones reported in studies dealing with other lignocellulosic feedstocks (such as rice husks, pine and eucalypt woods, peanut shells or almond shells) [23,24,26].
