*2.8. Statistical Analysis*

The inhibitory concentration 50 (IC50) was calculated by non-linear regression with the use of Prism Graph- Pad Prism, version 4.0 for Windows (GraphPad Software). One-way analysis of variance test (ANOVA) followed by a multicomparison Dunnett's test were applied. All toxicity essays were performed in triplicate. Statistical analyses were made with Graph Pad. The data were evaluated by one-way analysis of variance followed by the Mann–Whitney U test. A value of *p* < 0.005 was considered significant.

### **3. Result and Discussion**

### *3.1. Oil Mill Wastewater Treatment*

Oil mill wastewater (OMW) displays a variable composition that is influenced by different issues including agronomic parameters, such as cultivar and maturation of the olive fruit, region of origin and climatic conditions [32]. In particular, OMW is an acidic and dark suspension mainly composed by water (83–94% *w*/*w*) and also containing inorganic (0.4–2.5% *w*/*w*) and organic substances (4–18 % *w*/*w*), including mucilage, lignin, tannins and pectins, as well as cation species such as magnesium, sodium, calcium and potassium. In addition, OMW contains phenolic compounds, the most popular high added-value ingredients, varying from 0.5 to 24 g/OMW), that represent about 98% ( *w*/*w*) of the phenols typically present in olive fruit [33]. A high concentration of polyphenols generally involves a condensation step by ultrafiltration, thermal concentration or freeze-drying processes [34]. In this regard, OMWs from Roggianella *cv* were subjected to a freeze-drying process after preliminary filtration and centrifugation providing a vaporous solid (LOMW) that was deeply characterized by chromatographic and spectroscopic techniques, as well as in terms of antioxidant performance.

### *3.2. HPLC-MS/MS and 1H-NMR Analyses of Lyophilized Oil Mill Wastewater*

Separation of the main polyphenols in LOMW was carried out by HPLC-MS/MS analysis and their identification was based on mass measurements of deprotonated [M–H] − ions and MS/MS fragmentation patterns (Table 1). The compound at RT = 1.475 min (at concentration of 0.71 μg mL−1) was assigned to a verbascoside residue lacking the rhamnose moiety due to its [M–H]− at *m*/*z* 477 together with the fragments at *m*/*z* 459 (water loss) and 161 (ascribable to the dehydrated ion of the caffeic acid unit), as already hypothesized in literature [31]. The compound at RT = 1.515 with a concentration of 0.19 μg mL−1, exhibiting [M–H]− at *m*/*z* 169 and a fragment at *m*/*z* 151 probably produced by the loss [M–H–H2O]<sup>−</sup>, was tentatively identified as 3,4-dihydroxyphenylglycol [15].

Six phenolic acids and derivatives were also revealed, namely quinic acid (4.1 μg mL−1),HyEDA (0.27 μg mL−1) and decarboxymethyl-elenolic acid derivative (1.55 μg mL−1), hydroxylated product of dialdhydic form of decarboxymethyl elenolic acid (3.3 μg mL−1), caffeic acid (1.9 μg mL−1) and *p*-coumaric acid (2.1 μg mL−1) on the basis of their [M–H]− ions as well as the presence of [M–H2O]− and [M–CO–2H2O]− in their fragmentation patterns [15,17]. The compounds at RT = 2.245 min and 2.470 min (having concentrations of 4.3 and 2.9 μg mL−1, respectively) were two 3-hydroxytyrosol glucoside isomers because they showed the same [M–H]− at *m*/*z* 315 and similar MS/MS spectra characterized by two main fragment ions at *m*/*z* 153, which were formed through the loss of a glucose moiety and at *m*/*z* 123 corresponding to the subsequent loss of the CH2OH group. However, since different hydroxytyrosol glucoside isomers (i.e., hydroxytyrosol-1-O-glucoside, hydroxytyrosol-3-O-glucoside and hydroxytyrosol-4-O-glucoside) have been identified in *Olea europaea* [32], they were not furtherly distinguishable by MS analysis. Finally, oleuropein aglycone derivative (5.8 μg mL−1) and 3-hydroxytyrosol (0.09 μg mL−1) were recognized by matching chromatographic and MS characteristics to those of previous literature reports in the former case [15] and of a pure standard in the latter one.

As reported in Figure 1, the 1H-NMR of LOMW revealed the main features typical of minor components in olive oil reported in Table 2, responsible for its interesting biological activities.


**Table 1.** Identified polyphenol compound in LOMW (in μg mL−1). Data represent mean ± RSD (*n* = 3).

RT = retention time; LOMW = Lyophilized olive mill wastewater.

**Figure 1.** 1H-NMR of 16.7 mg of LOMW sample in 0.6 μL of D2O.


**Table 2.** Characterization of LOMW by 1H-NMR spectroscopy.

The corrected assignment was obtained comparing the NMR spectra with those available in literature and using the Human Metabolome Database (HMDB).

Many signals belonging to sugar residues derived from glycosides and the OH group of Verbascoside, together with Ph-CH2 and CH2-O of Hydroxythyrosol and the CH3 and the enantiotopic CH2-OH of Oleuropein are detected. Furthermore, at 0.89, 1.3, 1.64, 2.02, 2.35 ppm, the signals of oleic acid are visible.

### *3.3. Antioxidant Properties of Lyophilized Oil Mill Wastewater*

LOMW was characterized by evaluation of APG, FC, PAC and AC in order to provide a straight measure of the antioxidant potential of this by-product and the results are reported in Table 3.



LOMW = Lyophilized olive mill wastewater; APG = Available phenolic groups; PAC = Phenolic acids content; FC = Flavonoid content; AC = Anthocyanin content; TAC = Total antioxidant activity; DPPH = 2,2-diphenyl-1- picrylhydrazyl radical; ABTS = 2,2-azino-bis(3-ethylbenzothiazolin-6-sulphonic radical; CT = catechin.

By-products from the olive oil extraction process are found to be particularly rich in phenolic compounds [35,36]. The available phenolic groups of LOMW was 75.0 mg CT per gram. This value appears in the same magnitude of other studies reported in literature, showing high concentration of phenolic compounds present in the OMWs [37,38]. Typically, during the extraction process, the highest (98%) amount of polyphenols in olive fruit can be found in the OMWs (0.5–24 g L−<sup>1</sup> of OMW), while only 2% of them is in the oil phase [30]. A correlation of this parameter with literature data appears quite difficult because total polyphenol concentration is strictly related to many factors, such as type and region of origin, maturity of olives, method of extraction, climatic conditions and cultivation and processing techniques [39].

The analysis of FC in LOMW was carried out by using AlCl3 reagen<sup>t</sup> and the results were expressed as milligrams of CT per gram of sample (Table 2). In plants, the number of glycoside fractions of these compounds can vary from one to three. In particular, flavonoids are found glycosylated with carbohydrates such as glucose or rhamnose, but they can also be found linked to glucose units such as galactose, arabinose or other sugars [40]. The results showed that phenolic compounds with a flavonoid structure in LOMW are equal to 34.0 mg CT per gram, corresponding to 45.3% of the APG.

The evaluation of PAC in LOMW was carried out using the Arnov's method by expressing the results as milligrams of CT per gram of LOMW. Such compounds are hardly found in the free form, due to their ability to link quinic and tartaric acids forming esters and/or glycosylated derivatives [41]. LOMW sample provided high amounts of PAC (50.8 mg CT per gram), showing values that were equal to 67.7% of the total polyphenols. As can be seen from the data obtained by LC-MS analysis, the phenolic acid content was mainly related to the amounts of quinic, coumaric and caffeic acids.

Finally, a colorimetric assay was employed to quantified anthocyanin compounds, a class natural pigments responsible for the coloring of most fruits and vegetables [42]. Recorded results displayed that AC in LOMW was equal to 0.15 mg CT g<sup>−</sup>1, almost two orders of magnitude lower than FC and PAC.

Antioxidant properties of the food matrix were deeply investigated by specific tests, including total antioxidant capacity and scavenging activity of the LOMW against DPPH and ABTS radicals. TAC of LOMW was determined using (NH4)2MoO4 reagen<sup>t</sup> and by expressing the results as mg CT per gram of matrix. According to Prieto et al. (1999) [43], this reagen<sup>t</sup> can be systematically applied for the evaluation, both in aqueous and organic environments, of the antioxidant activity of matrices with a complex composition.

The recorded results (Table 3) depicted as a high APG value was not always related to a significant antioxidant capacity according to literature data [44], highlighting as the class of phenolics deeply influenced the total antioxidant capacity of the food matrix.

In order to determine scavenger activity both in aqueous and in organic environments, LOMW was tested against DPPH and ABTS radicals. The ability of LOMW to inhibit these reactive species, was expressed in terms of IC50 (mg mL−1), as reported in Table 3. In particular, the ability to inhibit the ABTS radical is a highly used parameter for the determination of antioxidant activity in food and biological samples [45]. Based on the inhibition kinetics of the ABTS radical, recorded LOMW IC50 value was 0.019 mg mL−1, almost five times less compared to the activity against DPPH, suggesting that LOMW is a matrix rich in highly hydrophilic moieties.

### *3.4. Synthesis of the Tara Gum Conjugate*

An innovative strategy to synthesize versatile materials with antioxidant features involved the covalent linkage of the biomolecules of LOMW on the tara gum (TG) chain. Literature data largely proposed TG as starting materials in the synthesis of innovative polymeric carries of nutraceuticals able to be employed in the food industry [13,14,46], but to the best of our knowledge covalent grafting of bioactive molecules on TG chain was not investigated. This approach allowed the preparation of a high molecular weight antioxidant compounds showing improved chemical stability, as well as lower degradation rate compared to the low molecular weight antioxidants [47]. Conjugate polymers with antioxidant features were synthesized by employing an eco-compatible, radical initiated grafting procedure. Antioxidant conjugate was synthesized by anchoring the LOMW reactive species to TG chains, using a water soluble in a radical reaction initiated by a biocompatible redox pair (H2O2/ascorbic acid). This redox couple showed numerous advantages, such as the opportunity of inducing polymerization reaction at room temperature drastically reducing the risks of degradation of phenolic compounds and avoiding the generation of any type of toxic products [48]. A specific LOMW/TG ratio (*w*/*w*) was used in the polymerization mixture. In particular, a quantity of LOMW equivalent to 35 mg of catechin (calculated by APG value) for each gram of commercial TG was found to be optimal. After 24 h, to remove the unreacted molecules, the conjugate was subjected to a purification process by dialysis and the resulting solution was lyophilized which led to obtaining a vaporous solid (PLOMW) whose antioxidant properties were investigated. To verify the antioxidant performance of the conjugate, a control polymer (labelled BTG), was prepared in the same conditions, but in the absence of LOMW.

### *3.5. 1H-NMR Analysis of the Polymers*

The 1H-NMR spectra of BTG (Figure 2A) and PLOMW (Figure 2B) showed the main residues of galactomannan [49], scaffold of TG and some fragments of LOMW (close to 1.12–1.40 ppm and 8.32 ppm) that confirm the conjugation.

**Figure 2.** Panel (**A**): 1H-NMR spectrum of BTG; Panel (**B**): 1HNMR spectrum of PLOMW. In the panel (**B**), the signal at 5.029 ppm belongs to H1 of α-D-galactopyranose which in the panel (**A**) collapsed into the D2O signal.

### *3.6. Calorimetric Analysis of the Polymers*

The Differential Scanning Calorimetry analysis of PLOMW, BTG and the commercial tara gum (CTG) was performed subjecting the samples to an isotherm at 25 ◦C for 20 min and heat ramp from 25 to 650 ◦C at a temperature scanning speed of 10 ◦C min−<sup>1</sup> under nitrogen flow. The results obtained (Figure 3) allow to highlight the presence of two significant peaks in all the samples: an exothermic peak (100–150 ◦C) and an endothermic peak (around 300 ◦C).

**Figure 3.** Differential scanning calorimetry (DSC) of PLOMW, BTG and CTG.

Specifically, the graph shows that the two polymer samples show the same peaks as TG sample, but the values are slightly shifted with respect to the reference (CTG) and this phenomenon is most likely due to the different molecular structure.

The enthalpy was then calculated for each peak and the results were reported in Table 4.


**Table 4.** Enthalpy and temperature values of the polymers and commercial tare rubber peaks.

PLOMW = Polymer conjugate lyophilized olive mill wastewater and tara gum; BTG = Blank tara gum; CTG = commercial tara gum.

Measuring the enthalpy of fusion allows to calculate the degree of crystallinity of a substance. Therefore, a higher enthalpy corresponds to a greater interaction between the molecules. The results show that as regards the enthalpy values measured on the peak (1), these are significantly lower for the PLOMW and BTG polymers. This behavior denotes a lower level of interaction between the polymer molecules than the commercial tare rubber sample. On the other hand, the values recorded for the peak (2) show how the blank polymer has a higher enthalpy value ( −11.6 J g<sup>−</sup>1) and, therefore, has a greater degree of interaction between its molecules than CTG and PLOMW polymer. In particular, the latter, having the lowest enthalpy value ( −154.3 J g<sup>−</sup>1), is the one with the least interaction between its molecules.

### *3.7. Toxicity Evaluation of the Conjugate*
