## 2 Materials and Methods
### Determination of Antifungal Activity
#### 2.5.2 Challenge Test  
Microbiological challenge testing is a useful tool for determining the ability of a food to support the growth of spoilage organisms or pathogens. During this test, a deliberate inoculation of the product with the test microorganism is performed and the product is then stored and tested for this microorganism during its shelf life [31].  
A batch of mayonnaise (MY B_0), 250 g, was prepared under laboratory conditions by mixing the following ingredients: 2 egg yolks, 1 tablespoon mustard (15 g), 1 tablespoon white vinegar (15 g), 5 g salt, and 240 mL EVOO flavored with 40 g L\({}^{-1}\) dry majoram (similar to sample B described in Section 2.1). The final pH was 4.0. A second batch of mayonnaise (MY C) was prepared accordingly as a control by replacing the flavored with unflavored EVOO. Each batch was transferred into a stomacher bag and inoculated with 20 mL of inoculum of the spoilage yeast _Zygosaccharomyces bailii_ (104 cfu/mL), followed by mixing in a stomacher for 1 min for an even distribution of yeast cells. Each batch was apportioned into sterile jars, each containing 15 g of mayonnaise, which were then placed in an incubator at 25 \({}^{\circ}\)C for a total period of 20 days. The yeast counts were performed at 0, 5, 10, 15 and 20 days, in triplicate, under the following procedure: ten grams of mayonnaise was mixed under aseptic conditions in a stomacher bag with 90 mL of PW. This was followed by serial decimal dilutions and then spread-plated on YPD plates that were incubated at 25 \({}^{\circ}\)C for 48 h. The colony forming units (CFU) were counted and were expressed as the log CFU/g. Each sampling day, the pH was measured.  
Identification and Semi-Quantification of Volatile Compounds (VCs) by Solid Phase-Microextraction Gas Chromatography (SPME-GC-MS)  
The analysis of the VCs was performed according to the method of [PERSON] et al. (2021) [32]. Five grams of olive oil was put into a 15 mL vial along with 1 \(\upmu\)L of internal standard (\(\beta\)-ionone, Alfa Aesar, Ward Hill, MA, USA). The olive oil sample was heated at 50 \({}^{\circ}\)C in a water bath and stirred at 700 rpm for 30 min.  
A divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco, Bellefonte, PA, USA) was inserted and exposed to the headspace of the olive oil sample for 15 min. After the completion of sampling, the fiber was inserted into the injector of the gas chromatograph.  
Analysis of the VCs was performed using a gas chromatograph Thermo GC-TRACE ultra, coupled to a Thermo Mass Spectrometer DSQ II (Thermo Scientific Inc., Waltham, MA, USA) with a Restek Rtx-5 MS, 30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m (Restek, Bellefonte, PA, USA) column. The helium carrier gas flow rate was 1.0 mL/min. The GC inlet was operated for 3 min in splitless mode at a temperature of 260 \({}^{\circ}\)C. The column temperature was held for 6 min at 40 \({}^{\circ}\)C, then increased to 120 \({}^{\circ}\)C with a rate of 5 \({}^{\circ}\)C/min, the temperature increased to 160 \({}^{\circ}\)C with a rate of 3 \({}^{\circ}\)C min, heated to 250 \({}^{\circ}\)C with a rate 15 \({}^{\circ}\)C/min and held at 250 \({}^{\circ}\)C for 1 min. The temperatures of the MS source, quadrupole and transfer line were 240 \({}^{\circ}\)C, 150 \({}^{\circ}\)C and 290 \({}^{\circ}\)C, respectively. The MS operated at a mass range of 35-650 \(m/z\) with an acquisition mode of electron impact 70 eV.  
The VCs were identified by the comparison of the spectral data and the retention index (RI) of the eluting compounds to those of the [PERSON] [33] and [PERSON] 275 mass spectra library. An n-alkane (C8-C20) standards solution (Supelco, Bellefonte, PA, USA) was used for the RI calculation. Semi-quantification of the VCs was performed by dividing the peak area of each compound by the peak area of the internal standard. This ratio was multiplied by the initial concentration of \(\beta\)-ionone and expressed as mg kg\({}^{-1}\).