# Antioxidant and Antimicrobial Activity of Porcine Liver Hydrolyates Using Flavourzyme
## 3 Results and Discussion  
### Molecular Weight Profile and Amino Acid Profile of Pork Liver Hydrolysates  
The hydrolysis of the porcine liver was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein profile of the control and the four Flavourzyme treatments at different times (4, 6, 8, and 10 h) are displayed in Figure 1. Porcine liver protein used as control was separated into seven bands at 100, 76, 64, 54, 49, 41, and 23 kDa. After the treatment with Flavourzyme, most of the protein bands disappeared, and only, four weak bands (68, 60, 54, 41 kDa) remained to confirm the enzymatic reaction of pork liver. However, there were no significant differences among the hydrolysis times. Several authors hypothesized that Flavourzyme contains a mixture of endo- and exo-peptidases with more capacity to hydrolyze peptide bonds than other enzymes which contain only one type of peptidase [34], although red blood fractions from different species required 24 h for total digestion with Flavourzyme [35]. In another case, proteolysis of Cantonese bacon by Flavourzyme was carried out in 28 h [36]. This suggests us that complete degradation of the pork liver need longer time of enzymatic reaction. The peptides with higher bioactivity are small peptides of approximately 4-16 amino acids (400-2000 Da) [37]. Consequently, the bioactive peptides are not showed in the SDS-PAGE gel, they are smaller degradation products. In these sense, the bioactivity depends on the amino acid sequence and length [38]. The amino acids may also have bioactivity, consequently the free amino acid content is of great importance. The free amino acid content was analyzed in each porcine liver hydrolysate at different hydrolysis time of Flavourzyme (Table 1). In general, hydrolyses revealed that they are rich in glutamic acid, lysine, and leucine. Both lysine and leucine are essential amino acids, hence they cannot be synthesized by the body so they must be supplied from the diet [39]. Total free amino acids significantly increased with the hydrolysis time as expected. The presence of free amino acids within the hydrolyses could increment its antioxidant activity [40]. This suggests that the porcine liver hydrolyses obtained from Flavourzyme at 10 h could present the highest antioxidant capacity. Figure 1: SDS-PAGE of proteins from control and pork liver hydrolyses obtained from Flavourzyme at different times of hydrolysis (4, 6, 8, and 10 h). The effect of hydrolysis time significantly affected almost all the analyzed amino acids. The hydrolysates subjected to the longer reaction time of enzyme, generated more amount of free amino acids, excluding, arginine, threonine, tyrosine, and methionine. This fact implies that a longer reaction time of enzyme produced a greater protein degradation releasing peptides and amino acids. It has been reported that hydrophobic amino acids such as aliphatic ones (proline, valine, methionine, isoleucine, and leucine) increase the solubility in the lipid tissue, while aromatic amino acids (phenylalanine and tyrosine) facilitate the scavenging of free radicals [41]. Particularly interesting is the increase of these type of amino acids to enhance the antioxidant activity of the hydrolysates. To evaluate the amino acid profiles, a heat map cluster analysis was performed as shown in Figure 2. To build the heat map analysis, we use the six replicates of the amino acid profile of the porcine liver hydrolysates generated at different reaction times. The samples were mainly separated into four clusters demonstrating the strong changes in the amino acid profile. The first cluster only contains samples which were hydrolyzed during 8 h, meanwhile, the second cluster only links samples hydrolyzed during 10 h. Nevertheless, the third and fourth clusters display that the amino acid pattern in reaction times of 6 and 4 h did not show differences. It is possible to think that reaction times between 4 and 6 h produce the same pattern of amino acids.