2.4.2. Microbiological Analysis

The viable populations of the principal groups of microorganisms were determined by plate inoculation and incubation at 32 ◦C up to 3 days before counting the colonies in the following selective media: total mesophilic bacteria in Plate Count Agar (Plate Count Agar, Conda-Pronadisa, Spain), lactic acid bacteria in MRS Agar (LAB) (Oxoid), yeasts and moulds in OGYA (Oxoid).

### 2.4.3. Oxidative Stability in Accelerated Storage Test

To investigate the effect of PE extracts in delaying or inhibiting of fat oxidation, mayonnaise samples with and without extract were subjected to high oxidative stress in OXITEST reactor. Oxitest analysis allows to detect the time necessary to reach an end point of oxidation that corresponds to a detectable rancidity or a rapid change in the oxidation rate. An oxidation Test Reactor (VELP Scientifica, Usmate Velate, MB, Italy) was used in order to evaluate the opposition to fat oxidation. This method is recognized by AOCS International Standard Procedure (Cd 12c–16) for the determination of oxidation stability of food, fats, and oils (AOAC, [21]). The analysis consists of monitoring the oxygen uptake of the reactive constituent of food samples to determine the oxidative stability under conditions of accelerated oxidation. Briefly, 5 g of oil sample were distributed homogenously in a hermetically sealed titanium chamber; oxygen was purged into the chamber up to a pressure of 6 bar. The reactor temperature was set at 90 ◦C. These reaction working conditions allow obtaining the sample Induction Period (IP) within a short time. The OXITEST allows to measure the modification of absolute pressure inside the two chambers and, through the OXISoftTM Software (Version 10002948 Usmate Velate, MB, Italy), automatically generates the IP expressed as hours by the graphical method.

### 2.4.4. Analysis of Antioxidant Compounds

The extraction of antioxidant compounds from Mayonnaise samples (EM) and the evaluation of antioxidant parameters were carried out following the method reported by Romeo et al. [5], opportunely modified. Two grams of EM were added with 2 mL of methanol: water (70:30) and 2 mL of hexane and mixed with a Vortex for 10 min. The hydroalcoholic phase was separated from the oil phase in a refrigerated centrifuge apparatus (NF 1200R, Nüve, Ankara, Turkey) at 5000 rpm, 4 ◦C for 10 min. Hydro-alcoholic extracts were recovered with a syringe, filtered through a 0.45 μm nylon filter, diameter 15 mm (Thermo Fischer Scientific, Waltham, MA, USA), and utilized for the phenolic compounds quantification and antioxidant activity.

For the total phenolic determination in EM, an aliquot of the diluted extract was mixed with 0.300 mL of Folin reagen<sup>t</sup> and 0.25 mL of deionized water and, after 4 min, with 2.4 mL of an aqueous solution of Na2CO3 (5%). The mixture was maintained in a 40 ◦C water bath for 20 min and TPC was determined at 750 nm. The results were expressed as mg of gallic acid equivalent kg−<sup>1</sup> of Mayonnaise. The total antioxidant capacity assays (DPPH and ABTS) and the determination of the main bioactive phenolic compounds in EM samples were analysed with the same methods reported in Section 2.3, with some modifications. For DPPH and ABTS assays, the radical scavenging activity was expressed as μmol Trolox 100 g<sup>−</sup><sup>1</sup> of EM; while the individual phenolic compounds were expressed as mg kg−<sup>1</sup> EM.
