# Antimicrobial and Antioxidant Properties of Bovine Livers and Hearts Hydrolysates
## 3 Results and Discussion  
### Amino Acids  
The quantity of free amino acids in the hydrolyses of bovine liver and hearts showed significant differences at different times of hydrolysis (Table 1). The composition of amino acid profiles in hydrolyses consists of both free amino acids and short-chain peptides [28]. The levels of free amino acids in the by-products of bovine meat by-products exhibited the lowest values following a 24-h incubation treatment time with papain and popsin enzymes. The amounts of hydrophobic amino acids (Ala, Val, Ile, Leu, Tyr, Phe, Pro, Met) tend to decrease with increasing hydrolysis time. [PERSON] et al. reported similar findings, indicating that an increase in hydrolysis time resulted in a decrease in the levels of free amino acids [13]. In our study, papain was the most effective in reducing free amino acids in both hydrolyses: heart and livers. According to [PERSON] et al., papain is the most effective enzyme at hydrolyzing meat proteins rich in glycine and hydroxproline, while other enzymes, such as trypsin and pancreatin, are less effective in dissolving meat, producing hydrolyses with less glycine and hydroxproline [29]. It has been observed that the hydrophobic amino acids Gly and Pro can enhance antioxidant activity due to increased lipid solubility and free radical reactions [30]. Furthermore, it has been observed that certain amino acids, including Lysine (Lys), Tyrosine (Tyr), and Histidine (His), possess inherent antioxidant capabilities when present in peptide structures. In addition, Cysteine (Cys) has proton-donating properties, while essential amino acidscan chelate metal ions [31]. Considering the outcomes that have been obtained, we may conclude that bioactive peptides can also be formed from free amino acids, connecting them to specific sequences. Some amino acids, particularly sulfur-containing amino acids like Cys, can act as antioxidants themselves, so the resulting peptide sequences with these amino acids in extremely short chains can have an excellent antioxidant effect. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{2}{c}{**Bovine Liver**} & \multicolumn{3}{c}{**Bovine Heart**} \\ \hline
**Amino Acids** & **Time (h)** & **Pepsin** & **Papain** & **Pepsin** & **Papain** \\ \hline \multirow{4}{*}{Ala} & 3 & 0.31 \(\pm\) 0.10 & 0.26 \(\pm\) 0.30 & 0.34 \(\pm\) 0.02 & 0.28  
Three methodologies, DPPH\(\bullet\) and ABTS\(\bullet+\) radical scavenging activity and iron-reducing antioxidant power (FRAP), were applied to evaluate the antioxidant activity of specific samples, as outlined in Table 2. The antioxidant activity values of bovine heart and liver hydrolyses were highest after 24 h (\(p<0.05\)). The highest values of antioxidant activity values were determined ABTS\(\bullet+\) (98.10 \(\pm\) 1.76%) and DPPH (92.56 \(\pm\) 0.56%) by hydrolyzing bovine liver with papain for 24 h (\(p<0.05\)). In addition, it was observed that bovine liver hydrolyses exhibited better antioxidant activity compared with heart hydrolyses. Other authors revealed that liver hydrolyses have a higher antioxidant capacity compared to other tissues, including the colon, pancreas, and appendix. In this regard, papain treatment resulted in higher antioxidant activity in bovine hearts and livers and was more effective than popsin enzyme [32]. Also, the hydrolysis time of 24 h increased the free radical scavenging activity of DPPH\(\bullet\) and ABTS\(\bullet+\). Immune system performance is generally enhanced by substances with antioxidant characteristics [33]. It is thus a very helpful method to evaluate the antioxidant potential of protein hydrolyses [34]. Among the bovine hydrolyzed samples, the highest FRAP value was measured in hydrolyzed bovine hearts (18.47 \(\pm\) 1.22) and similar-in bovine livers (17.88 \(\pm\) 0.98) after 24 h of hydrolysis with papain enzyme. FRAP values varied significantly (\(p<0.05\)) depending on the duration of treatment. Prolonged hydrolysis periods enhanced the FRAP activity of the peptides generated during enzymatic hydrolysis.