*2.2. Beef Burger Formulation*

Beef burgers were produced in an EU-approved meat processing plant located in Umbria, Central Italy. The meat for the preparation of beef burger was obtained from cuts (beef rump and shoulder muscle) of 18-month-old female Chianina cattle reared

and slaughtered in Italy in accordance with European Union Regulation (Regulation (EC) No. 853/2004 s.m.i.) [29]. After 12 days of carcass aging, 40 kg of meat cuts was ground twice in a professional trimmer equipped with a 4 mm-hole plate. The grinded meat was divided into four different formulations, each 5 kg in weight, and aseptically handmixed with the following ingredients for 2 min (basic recipe): 10 g/kg NaCl, 60 g/kg grated parmesan, 80 g/kg breadcrumbs and 2 eggs/kg. The four different formulations were elaborated as follows: C, basic recipe with no addition; A, basic recipe plus 10 g/kg commercial antioxidant mix (CM) (CondiHamb, MEC Import, Perugia, Italy); AP, basic recipe plus 5 g/kg CM plus 350 mg/kg PE crude extract; P, basic recipe plus 700 mg/kg PE crude extract. The chemical composition of the burger was characterized by an average of 22% protein and 8 % fat. Each batch was further mixed for 2 min, and the burgers were then molded (about 100 g each) and placed in a display refrigerator at 4 ± 2 ◦C for 7 days, under alternating exposure to fluorescent light (12 h light/12 h darkness) to simulate retail storage conditions.

### *2.3. Antioxidant Capacity of PE and Mix Extracts and Beef Burger*

The antioxidant capacity was evaluated for the synthetic and natural additives and burgers over the course of their shelf-life using the oxygen radical absorbance capacity method (ORACFL). One gram each of CM, PE and burger samples was separately mixed with a buffer, 75 mM, pH 7.2, containing 13.19 g of K2HPO4 and 10.26 g of KH2PO4 in 900 mL of deionized water, homogenized with an Ultra-Turrax homogenizer (Ultra Turrax T25 Basic, IKA Labortechnik Janke & Kunkel GmbH, Stavfen, Germany) for 1 min, and then vortexed for 2 min. The homogenates were centrifuged at 6000 rpm at 4 ◦C for 20 min, and the supernatant was used for the determination of the antioxidant capacity using the oxygen radical absorbance capacity method (ORACFL) based on the fluorescence decay rate of a probe in the presence of a radical oxygen species (ROO) and compared with that of a reference standard, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich, Steinheim, Germany). The ORACFL assays were carried out on a FLUO-star OPTIMA microplate fluorescence reader (BMGLABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The results are expressed as μg of Trolox equivalents (TE) per 100 g sample.

### *2.4. Lipid Oxidation of Beef Burger*

Lipid oxidation over the course of shelf-life was measured by thiobarbituric reactive substance (TBAR) determination, performed according to Tarladgis et al. [30] and measured by an Ultrospec 2100 pro UV–visible spectrometer (Amersham Pharmacia Biotech, Amersham, UK) at 532 nm. Quantification was performed using a standard calibration curve and with a concentration range of 1E6 to 1E5 M (y = 2E + 07x + 0.0046, R<sup>2</sup> = 0.9999), corresponding to a range of 0.4–4 mg of malonaldehyde (MDA)/kg meat. The MDA recovery was determined by spiking the samples with a known volume of 0.2 mM TMP. The TBAR concentration was expressed as mg MDA/kg meat.

### *2.5. Antimicrobial Activity of Commercial Mix and PE Extract*

The evaluation of the antimicrobial activity of CM and PE extract was performed through the agar well diffusion technique [31,32] on some micro-organisms relevant for the food industry, such as *Staphylococcus aureus* (WDCM 00034), *Escherichia coli* (WDCM 00013) and *Pseudomonas fluorescens* (WDCM 00115). The selected reference strains were revitalized in Brain Heart Infusion (BHI) broth and incubated at 37 ◦C for 24 h with the exception of *P. fluorescens*, which was incubated at 25 ◦C for 24–48 h. An initial suspension of 0.5 McFarland in 0.9% sterile saline solution was prepared for each micro-organism, and 100 μL was then distributed on Mueller–Hinton Agar (MHA, Thermo Fisher Scientific, Milan, Italy) plates with a swab, making four 90◦ rotations. At the time of use, the extracts were suspended with sterile demineralized water to obtain a concentration of 750 mg/mL, and then two dilutions were performed at 375 and 187 mg/mL. In each inoculated MHA plate, 7 mm-diameter holes were produced with a sterilized cork borer and then filled with 50 μL of extract suspension at different concentrations [31,32]. The plates were incubated according to the most suitable growth conditions, as reported above. For each bacterial strain, CM and PE extract were tested, and a negative control was set up with sterile demineralized water. At the end of the incubation period, the diameter of the inhibition halo was measured by a gauge and expressed in mm.

### *2.6. Microbial Analysis of Beef Burger*

After 0, 2, 5 and 7 days of storage (T0, T1, T2 and T3, respectively), 10 g of each sample was aseptically removed and placed in a sterile stomacher bag with 90 mL of Buffered Peptone Water (Oxoid Ltd., Basingstoke, UK). After homogenization (Stomacher 400 circulator, Seward Ltd., Norfolk, UK), decimal serial dilutions were performed, and the below-reported microbiological determination was carried out in duplicate. Total viable count (TVC) was performed on Plate Count Agar (PCA, Oxoid Ltd.) incubated at 30 ◦C for 72 h according to ISO 4833-1 [33]. *Enterobacteriaceae* were enumerated according to a validated alternative method of ISO 21528-2 [34] (AFNOR AES 10/07-01/08) on Rebecca ™ EB (bioMérieux, Mercy Etoile, France) incubated at 37 ◦C for 24 h. The lactic acid bacteria (LAB) count was performed on de Man, Rogosa and Sharpe agar (MRS, Oxoid Ltd.) incubated at 30 ◦C for 72 h, while the *Pseudomonas* spp. count was performed on Pseudomonas Agar Base with CFC selective agar supplement (Oxoid Ltd.) and incubated for 48 h at 25 ◦C. Coagulase-positive staphylococci were enumerated on Baird Parker Agar with the addition of RPF supplement (Biolife, Milano, Italy) and incubated at 37 ◦C for 48 h. Results were recorded as colony-forming units (CFUs) and converted into log10 values to obtain Log CFU/g of meat prior to statistical analysis according to Gill and Jones [35]. In order to assess the microbiological safety over the course of beef burgers' shelf-life, the *Salmonella* spp. detection was performed according to ISO 6579–1: 2017 [36] at T0, T1, T2 and T3, in compliance with Regulation (EC) No. 2073/2005 [37].

Corresponding with the end of the manufacturing process (T0), the enumeration of *E. coli* was performed according to ISO 16649 [38] on all experimental groups as a process hygiene criterion of Regulation (EC) No. 2073/2005 [37].
