# Antioxidant and Antimicrobial Activity of Porcine Liver Hydrolyates Using Flavourzyme
## 3 Results and Discussion
### Antioxidant Activity of Extracts from Flavourzyme Hydrolysates  
ABTS and DPPH scavenging activity was determined for liver hydrolysates generated at different hydrolysis times (4, 6, 8, and 10 h) and filtered with the three pore sizes (5, 10, and 30 kDa) (Table 2). All extracts were significantly affected (\(p<\) 0.001) by the membrane pore size employed for collecting the fractions. The ABTS activity increased with pore size, reaching the highest values in the 30 kDa fraction (433.77, 497.26, 474.40, and 426.15 mg ascorbic acid/100 g at 4, 6, 8, and 10 h, respectively). Our results were lower than those reported in poultry viscera with values of 794.6 mM TEAC/mg and 675.5 mM TEAC/mg for fractions below 10 and 3 kDa, respectively [49]. Concerning to the hydrolysis time, porcine liver hydrolysates subjected to ultrafiltration through 5 kDa and 30 kDa showed significant differences. The highest ABTS value was achieved at 6 h with a filtration of 5 kDa and 30 kDa (388.91 and 497.26 mg ascorbic acid/100 g, respectively), hence there is no need to extend the hydrolysis to maximize the ABTS activity. Figure 4: Antimicrobial activity of porcine liver hydrolysates using Flavourzyme during 10 h for 30-kDa (**a**) and 10-kDa (**b**) against _Listeria monocytogenes_. Another antioxidant assay is the DPPH which is based on the ability of compounds as hydrogen donors or free radical scavengers [50; 51]. As ABTS assay, the antioxidant activity was significantly higher in hydrolyses filtered through a pore size of 30 kDa (970.06, 484.00 and 441.67 \(\upmu\)g Trolox/g for 4, 6, and 8 h, respectively) except for a hydrolysis time of 10 h. Indeed, the antioxidant activity was not significantly different (\(p>0.05\)) at 10 h of hydrolysis (400.82, 397.85, and 403.05 \(\upmu\)g Trolox/g for 5, 10, and 30 kDa, respectively). These outcomes followed the same trend as ABTS assay, as expected. Regarding hydrolysis time, the maximum of antioxidant activity was observed at 6 h for 5 kDa and 4 h for 10 and 30 kDa. Considering ABTS and DPPH assays, it does not seem necessary to reach 8 h to achieve the highest antioxidant capacity. Similar results were observed by [PERSON] et al. [35] in peptide hydrolyses from pig red blood cell fractions using papain and bromelin. Although a slight increase in DPPH values was observed after two hours of hydrolysis, little effect of hydrolysis time was observed over time. In addition, small decreases were also observed at 4 h and 24 h in the case of bromelin and papain, respectively. The effect was observed in porcine plasma and hemoglobin protein hydrolyses, which lower molecular weight fraction of \(<\)3 kDa presented higher values of antioxidant capacity than \(>\)10 kDa fraction [52; 53]. In this sense, [PERSON] et al. [54] reported that hydrolyzed extracts of duck breasts filtered through (\(>\)30 kDa) showed 63% lesser antioxidant activity than corresponding ones (\(<\)10 kDa). These authors have separately evaluated the antioxidant capacity with respect to peptide size. In our work, the smallest (\(<\)5 kDa) peptides of porcine liver hydrolyses are included within the three types of hydrolyses. Additionally, the other two hydrolyses had higher amount of intermediate  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Hydrolysis Time** & \multicolumn{2}{c}{**Pore Size of Membrane**} & **Sig. ** \\ \hline  & **5 kDa** & **10 kDa** & **30 kDa** & \\ \hline  & \multicolumn{3}{c}{**ABTS (mg Ascorbic Acid/100 g)**} \\
4 h & 337.27 \(\pm\) 6.39 \({}^{\text{a\alpha}}\) & 399.07 \(\pm\) 3.69 \({}^{\text{\text{\text@underline{\text{\text{\text  
peptides. For this reason, it seems logically that the fraction with more amount of peptides including small peptides display a higher antioxidant capacity. Indeed, the antioxidant capacity of pork liver hydrolysates have been evaluated in fractions (<1 kDa, 1-5 kDa, 5-10 kDa, and >10 kDa) resulting lower than the whole fraction [55].