# Antioxidant and Antimicrobial Activity of Porcine Liver Hydrolyates Using Flavourzyme
## 3 Results and Discussion
### Antimicrobial Activity of Porcine Liver Hydrolysates  
The antimicrobial activity of porcine liver hydrolysates was evaluated by Gram-positive (_B. thermosphacta, L. monocytogenes, and S. aureus_) and Gram-negative (_P. aeruginosa, E. coli_, and _S. enterica_) bacteria. The zone of inhibition (mm) was measured as percentage of inhibition for porcine liver hydrolysates generated by Flavourzyme at different hydrolysis times (4, 6, 8, and 10 h) and filtration through three pore sizes (5, 10, and 30 kDa). The outcomes revealed that most hydrolysates caused growth inhibition of _Brochothorax thermosphacta_ but at different level (Figure 3).  
The highest antimicrobial activity was reached by a hydrolysis time of 10 h using filters with a pore size of 30-kDa. The effect of hydrolysis time showed that higher hydrolysis time led to higher inhibition. For instance, in the case of 30 kDa membrane, the inhibition percentage was 83.3% at 4 h and 116.7% at 10 h. Similarly, the same trend was obtained with 10 kDa membrane, where the  
Figure 3: Antimicrobial activity of porcine liver hydrolysates generated by Flavourzyme at different hydrolysis times (4, 6, 8, and 10 h) and filtration through three pore sizes (5 kDa, 10 kDa, and 30 kDa) against _Brochothorax thermosphacta_.  
Figure 2: Heat map analysis of amino acid profiles for each porcine liver hydrolysates. Six replicates were generated at different hydrolysis times (4, 6, 8, and 10 h) by Flavourzyme.  
inhibition percentage was 66.7% at 4 h, and 100% at 10 h. These findings are in accordance with the data reported by [PERSON] et al. [43], which showed the same behavior in the three microorganisms studied (_L. monocytogenes, E. coli, S. aureus_). On the other hand, the effect of the membrane seemed to indicate that the higher pore size achieved a greater level of inhibition except for hydrolysis time of 8 h. For the study of fresh meat, the _Brochothrix_ strains are one of the most predominant spoilage organisms [44]. Hence, the incorporation of these peptides into foods could be interesting from the antimicrobial safety point of view. Regarding the inhibition of _Listeria monocytogenes_, none of the extracts could inhibit it except for 30 and 10 kDa hydrolysates fractions obtained by Flavourzyme for 10 h (Figure 4). Both displayed antimicrobial capacity against this strain, reaching the same activity as the nisin standard in the case of 10 kDa and the double in 30 kDa fraction (the percentage inhibition of diameter growth was 100% and 200%, respectively).  
On the contrary, none of the porcine liver hydrolysates inhibited the growth of Gram-negative bacteria due to their greater resistance to antimicrobials. This higher resistance is conferred by the existence of an outer membrane in the cell wall structure of the bacteria [45]. This outer cell envelope is a highly hydrophobic asymmetric bilayer, which consists of lipopolysaccharides and phospholipids providing negative charge. This electrostatic charge may provide impermeability against antimicrobial agents, acting as a protecting selective barrier against hydrophobic molecules. Moreover, this bilayer contains porins, which are pore-forming proteins as transport channels [46], allowing the passive penetration of small hydrophilic molecules (\(<\)600 Da). Therefore, the permeability of hydrophilic compounds is compromised by size-exclusion [47]. The interactions of antimicrobial peptides with inner and outer membranes could also depend on the structural characteristics of the peptides, such as the total density of cationic charges, length of the peptide sequence, amount of hydrogen bonds donors, and 3-D conformation [48]. Overall, the antimicrobial activity of hydrolysates extracts is influenced by the cationic and hydrophobic character of the peptides, which can interact with the lipopolysaccharidesaccharides of the bilayer, causing the cell disruption [43].