**3. Results**

### *3.1. Characterization of Phenolic Extracts*

The main antioxidant parameters evaluated on the two phenolic extracts (PE A and PEB) were reported in Table 1. Significant differences were noted between the extracts, particularly for TPC and ABTS assays with higher results in PE A (TPC: 7895 mg GAE L−<sup>1</sup> PE; ABTS: 28,604 μmol TE mL−<sup>1</sup> PE) respect to PEB (TPC: 7258 mg GAE L−<sup>1</sup> PE; ABTS: 25,716 μmol TE mL−<sup>1</sup> PE).

**Table 1.** Antioxidant parameters and individual Phenolic Compounds of phenolic extracts.


Note: The data are presented as means ± SD. Student's t test performed between the two phenolic extracts (PEA and PEB): \* significant difference at *p* < 0.05; \*\* significant difference at *p* < 0.01. ns not significant. μmol TE mL−<sup>1</sup> PE for ABTS and DPPH and mg 100 mL−<sup>1</sup> PE for TPC and single phenolics.

Considering the partition coefficient of wastewaters phenols mixture, the extraction with ethyl acetate by different steps allows retaining most phenolic compounds soluble in the organic phase, as reported by Soberón et al. [22]. This explains the results observed in PE A. On the other hand, PEB was obtained by water extraction, so it was characterized by phenols insoluble in the organic phase. The only problem linked to the extract PE A could be represented by the typology of solvents used for the extraction, namely hexane and ethyl acetate, but the extractive procedure was carefully carried out with the aim to use the obtained antioxidant as functional ingredients in food matrixes. For this reason, the solvent has been totally evaporated at the end of the extraction and the solutes had to be recovered

with water. To verify if traces of solvent (hexane and Ethyl acetate) persisted in the phenolic extract before the food application, we analyzed the headspace of the hydrophilic phase (through a GC-MS) and have proved absent.

Similarly, differences in antioxidant activity of the phenolic extracts may be ascribed to the polarity of extracting solvents and thus to the chemical characteristics of extracted compounds [23]. The antioxidant activity measured by ABTS assay showed a higher value for both extracts compared to DPPH assay. Likewise, Bibi Sadeer et al. [24] investigated that ABTS cationic radical showed high solubility in organic and aqueous media, thus it is capable to screen the activity of both lipophilic and hydrophilic compounds. In contrast, DPPH radical dissolves in an organic medium reacting only with lipophilic phenolics. The principal phenolics in the extracts were hydroxytyrosol (PEA: 759 and PEB: 837 mg 100 mL−1) and tyrosol (PEA: 152 and PEB: 148 mg 100 mL−1), in agreemen<sup>t</sup> with literature [5,25]. Di Mattia et al. [26] reported that tyrosol and hydroxytyrosol are effective in preventing primary and secondary oxidation in o/w emulsion ensuring the oxidative stability during storage.

### *3.2. Qualitative and Quantitative Characterization of Enriched Vegan Mayonnaise (EM)* 3.2.1. Physicochemical Aspects

The colour of enriched mayonnaise was evaluated after the phenolic enrichment, considering that it is the main parameters which affect the consumer's choice (Table 2).


**Table 2.** Colour parameters of mayonnaise during storage period (days).

Note: The data are presented as means ± SD. Means within a row with different letters are significantly different by Tukey's post hoc test. Abbreviation: ns, not significant. \*\* Significance at *p* < 0.01. Small letters show differences among the different samples and capital letters show differences for the single sample during the storage period.

Moreover, the monitoring of its colour was considered crucial to verify the formation of compounds following an oxidative deterioration. The replacement of ingredients compared with the traditional formulation of mayonnaise, leads to a physical and chemical variation, and can have an effect on colour of the final products [27], in particular in this study which involved the use of brown extracts. The addition of phenolic extracts (PEs) and the storage time promoted a significant variation of colour parameters (*p* < 0.05) of enriched mayonnaise. Lightness decreased after phenolic extracts (PEs) addition, more with PEB, whereas vegan mayonnaises denoted higher a\* and b\* parameters after the enrichment. Storage time leads an increase of yellowness parameter and a decrease of redness parameter showing a trend opposite to that proved by Altunkaya et al. [28]. In contrast, no variations were observed for redness between the first and the last day of storage for Control sample. Previous research has proved that colour parameters, in particular lightness, are related to

fat droplet sizes [29]. Probably, the modification of fat droplet size that occurred following the addition of phenolic compounds may produce the colour detected changes [30].

Food safety and quality are important to consumers. As it is well known, the pH, acidity values and moisture content play an important role in chemical and microbiological stability of fat foods. For this, in order to evaluate the potential application of PEs, all samples were subjects to chemical and microbiological analysis. The pH values of mayonnaise samples analysed at 1st time ranged from 2.92 to 5.01 (Control: 5.01 > EMPEA: 2.92 > EMPEB: 3.74), therefore, the addition of PE allows an acidification of the enriched samples. slightly lower pH value (Control: 4.97 > EMPEA: 2.97 > EMPEB: 3.85) were observed at the end of storage period (45 days) according to Rasmy et al. [31]. The two enriched samples showed a decrease of the TA during the time of storage (Figure 2), instead the control sample showed increase of acidity. The highest measured value was in the sample EMPEA (9.41 g Oleic acid 100 g<sup>−</sup><sup>1</sup> mayonnaise). The high acidity value is consistent with pH of PE (pH 2), that induced a decrease of emulsion pH and an increase of TA value.

**Figure 2.** Changes in Total Acidity in the samples during storage time. Different letters show differences for *p* < 0.05.

The highest moisture content value was determined in EMPEA sample (40.43%) at 1st time, this value decreased during the storage period, indeed at the 45th day was of about 35.95% (Figure 3). In addition, the Control sample showed a significant variation of the moisture content from 38.38 to 30.88%. EMPEB showed instead a slight, no significant variation over time (35.18 to 35.72%).

**Figure 3.** Changes in Moisture content in the samples during storage time. Different letters show differences for *p* < 0.05.
