The sausages samples were packed with a weigh of 200 ± 5 g, and showed a pH = (6.29 ± 0.11) and water activity Aw = (0.941 ± 0.008). The mortadella-type sausages were made in a pilot plant in the Products of Animal Origin Laboratory at the Federal University of Lavras (Brazil).

Lean beef, salt, phosphate and NaNO2 were placed in a cutter (Sire, Filizola S.A., Brazil) and mixed for approximately 1 min. Fifty percent 3-deazaneplanocin A chemical structure of the ice and spices were then added and mixed at a high speed. After complete homogenization, the speed of the cutter was reduced. Ground pork backfat was then added and mixed until the temperature of the mixture reached 10 °C. The remaining 50% of the ice, cassava starch, ascorbic acid and EO were added Ruxolitinib in vivo and mixed until the temperature of the mixture reached 13 °C. The total emulsification time was approximately 10 min, and the processing room temperature was approximately 20 °C. The batters were stuffed into nylon bags (Unipac Darlon, Brazil, 50 μm thickness) and were cooked by immersion in water according to the following program: 55 °C for 30 min, 65 °C for 30 min, 75 °C for 30 min, and 85 °C until the temperature of the mass reached 73 °C (measured

by a thermopar inserted into the center of the packed sausage batter). The cooked sausage was cooled in a water bath for 10 min and stored in a controlled chamber (Thermostat cabinets LS Logen Scientific) at 25 °C before analysis at 1, 10, 20 and 30 days. The EO concentrations used in manufacturing of the sausages were based GPX6 on the following factors: in vitro antimicrobial activity results; possible reductions in activity when applied to the food model (reported in literature); and combined effect of different nitrite levels used in the product manufacturing. The mortadella samples were inoculated with a microorganism culture (C. perfringens) to obtain an initial level of 107 CFU/g viable cells. Silicone was deposited on different points on the surface

of the product package. After drying, the grown culture of the target microorganism was injected with a sterile needle and syringe in a laminar flow biosafety cabinet. For the enumeration of C. perfringens, 10 g of the mortadella samples were weighed, transferred into sterile stomaching bags, combined with 90 ml of sterile peptone water (0.1% w/v) and homogenized in a Stomacher (Metroterm, Brazil) with 490 strokes/min for 2 min at room temperature. Stomached slurries were decimal serially diluted in peptone water (0.1% w/v), and aliquots (100 μl) of the sample dilutions were spread on Tryptose Sulphite Cycloserine differential selective agar (TSC, HiMedia, Mumbai, India) supplemented with 200 mg of d-cycloserine (inhibition of microbial anaerobic companion) and egg yolk emulsion (12.5 ml of yolk and 12.5 ml of 0.85% saline solution) to verify the phospholipase activity of α-toxin (lecithinase).