2.3.2. Well-Diffusion Assay

After selecting the day that presented the highest antibacterial activity, fermented whey (100%) was tested on a second screen for its antibacterial activity against several food outburst-related bacterial species, using the well-diffusion assay described by Rizzello et al. [11], modified by incorporation of inoculum into Plate Count Agar (bioMerieux, Marcy l'Etoile, France), 10 mL per plate, overlaid with 10 mL of water-agar (2% *w*/*v*). Wells of 15 mm were cut into agar plates and 200 μL of 120 h fermented whey were placed in each well. Plates remained at 4 ◦C for 4 h to allow diffusion.

To test dose-dependence, we also compared non-fermented whey, fermented whey, and milk against the strain of *L*. *monocytogenes*, using diluted solutions of 100, 50, and 25% (*v*/*v*). After incubation at 37 ◦C ± 1 ◦C, 24 h ± 2 h, plates were measured with a caliper rule for inhibition zone diameters, with averages of three independent trials calculated with standard deviations. As a positive control we used an anionic detergent surfactant and susceptibility was recorded by average diameters > 4 mm [44].

#### *2.4. Impact of Fermented Whey as a MP Lettuce Sanitizer*

Loose leaf lettuce (*Lactuca sativa* var. *crispa*) samples were purchased at a local biological market, always from the same organic grower, on the same day of the experiments. Samples were treated in parallel with whey solution and a 110 ppm chlorine solution by dissolving Amokina® (Angelini, Portugal) in sterile water, according to the manufacturer instructions. Outer leaves of the lettuces with signs of damage were discarded; the inner ones were cut using a cylindrical metal cutter of 6 cm diameter in order to take representative portions of all parts of the plant tissue. Circles of lettuce leaves were than washed in distilled water. Subsequently, three different solutions were used to sanitize and wash the lettuce shreds: (1) Distilled water acting as the reference; (2) chlorinated solution (110 ppm of Amokina®), as previously described; and (3) a 75% (*v*/*v*) fermented whey solution in distilled water [41]. The three different treatments were carried out in plastic bags filled with about 200 g of lettuce shreds, from the three different zones of the lettuces (outer, inner, and middle leaves) immersed in 1 L of the sanitizing or washing solutions. Bags were sealed and soaked for 5 min at 4 ◦C using an incubator with orbital shaking (Panasonic MIR 154, Gunma, Japan). After that, lettuce shreds were rinsed with sterile distilled water to remove sanitizers, and finally, the excess surface water was removed by a handheld salad spinner (IKEA Tokig, Lisbon, Portugal) for about 30 s. Processed lettuce were pooled and packed at 100 g of shredded lettuce per bag in heat-sealing bags (300 × 230 mm) of 30 μm oriented polypropylene (Amcor Flexibles Neocel, Portugal), graciously granted by Campotec SA. A bag prepared with 100 g of lettuce just soaked in distilled water, whose excess was also removed by the salad spinner, was marked as day 0 and served as the reference. To evaluate O2 and CO2 changes, three independent bags were separated. Samples were stored at 4 ◦C for subsequent evaluation of O2 and CO2 changes, pH, texture, color, sensory quality, and microbial growth, on processing day (day 0) and after 1, 3, 5, 7, 9, and 10 days of storage. The experiments were performed in three independent trials.
