2.5. In Vivo Bactericidal Activity towards Induced S. aureus Contamination of Cheese

The antibacterial activity of PPW and PMW was evaluated against S. aureus inoculated in milk in order to simulate cheese contamination.

Briefly, a single colony of S. aureus was inoculated into Tryptone Soy broth (Oxoid, Basingstoke, UK) and shaken overnight to prepare a working solution. The concentration of the bacterial suspension was adjusted to 10⁹ CFU/mL in a sterilized 0.9% sodium chloride (NaCl) solution. An adequate quantity of bacterial suspension was centrifuged, and the obtained pellet was washed twice with sterile distilled water and added to milk to obtain an initial concentration of 10⁵ CFU/mL.

The inoculated milk was used to produce the experimental cheese following the procedure described in the Section 2.4. Cheese samples, both supplemented or not with the pomegranate extracts (cheese-PPW and cheese-PMW), were sealed, placed at 4 °C, and analyzed on the same day of production as well as after 1, 2, 4, 8, and 12 d of refrigerated storage. At each sampling time, cheese samples, contaminated or not with S. aureus, were aseptically transferred into a Stomacher bag containing a proportional amount of sterile Ringers solution (Oxoid, Basingstoke, UK) and homogenized for 5 min. Serial decimal dilutions of the obtained suspension were prepared with the same diluent. The growth of S. aureus, in contaminated cheese, was evaluated by spread-plating 0.1 mL of appropriate dilutions on Mannitol Salt Agar (MSA Oxoid, Basingstoke, UK) and incubation at 32 °C for 24 h. Cheese samples not inoculated with S. aureus was analyzed for total mesophilic bacteria (TMB), Enterobacteriaceae, and Lactobacillus spp. count, using Plate Count Agar (PCA, CM325, Oxoid, Basingstoke, UK), Violet Red Bile Glucose Agar (VRBGA, CM0485, Oxoid, Basingstoke, UK), and MRS Agar, respectively, and incubated at 32 °C for 24–48 h. Each microbiological count was performed in triplicate and expressed as log₁₀ CFU/g of cheese.