Fiorentino, Florence, Italy). They consisted of grape seed and chestnut extracts, tocopherol and 66 hydroxytyrosol (extracted by defatted olive pomace). The manufacturer provided the phenolic 67 profile (Table 1), total phenolic content and antiradical scavenging activity (EC50) (Table 2) of each 68 extract. The grape seed and chestnut extracts were combined with the same amount of 69 hydroxytyrosol and tocopherol to form two different mixtures; grape seed (GSE) and chestnut 70 (CHE) mixtures. 71 2.2. Sausages manufacturing 72 4In an industrial plant (Azienda Agricola Savigni, Pistoia, Italy), 24kg of pork lean and 6kg of 73 subcutaneous backfat from Cinta Senese pig breed were minced and equally divided in three 74 batches. Salt (23g/kg), sucrose (35g/kg) and black pepper (0.2g/kg) were added to each batch 75 following the recipe traditionally used by the manufacturer. Thirty ppm of sodium nitrite (E250) 76 were added to the first batch to constitute the control (NIT). In second batch, 10g/kg of GSE 77 mixture were used to replace sodium nitrite, while 10g/kg of CHE were added to the third batch.
78Sausages were weighed, dried at 28°C and RH 85% for 4 days and then ripened 21 days (T 13°C, 79 RH 70%). Once ripened, six samples of each batch were collected, pH, color, and processing loss 80 were immediately measured. Samples were vacuum packed and stored at -80°C for physical, 81 chemical and aromatic analysis. Another 3 samples of each batch were stored at 4°C to be 82 employed for sensory analysis the following day. This design was replicated to have two totally 83 independent batches for each treatment. 84 2.3. Physical, chemical and microbiological parameters 85 At the end of ripening, physical parameters were assessed on 12 samples of each batch (6 for each 86 replication). Sausage pH was measured at room temperature (20 °C) using a pH meter Crison 87 GLP21 (Barcelona, Spain), the instrument was introduced in a sausage portion. Color (L*, a* and 88 b*) was determined by a Minolta Chromameter CR-200 (Tokyo, Japan) immediately after slicing. 89 aw was measured following the method ISO 21807:2004. Two 10 mm-thick and 10 mm-width slices 90 of each sample, were cut and immediately analyzed at room temperature (22 °C), using a Zwick 91 Roell Z2.5 apparatus (Ulm, Germany) with a loading cell of 1 kN at the crosshead speed of 1 92 mm/sec. Texture profile analysis (TPA) was performed assessing the following parameters: 93 hardness, cohesiveness, gumminess, springiness and chewiness. Moisture was determined by 94 lyophilizing to constant weight 40g of sample, according to AOAC methods (1990). Weight loss 95was measured as the difference between weight at time zero and end of ripening (after 24 days).
96Total protein, fat and ash contents were determined following AOAC (1990) methods. Lipid 97 5 oxidation was determined according Vyncke (1970), using a PerkinElmer Lambda EZ150 98 spectrophotometer (Waltham, MA, USA). Results were expressed as mg of malondialdehyde 99 (MDA)/kg of samples. Fatty acids were determined ...