# Enhanced Enzymatic Degradability of Livestock Blood Pretreated with Ultrasonic Technique
## 3 Results and Discussion
### Protein Solubilization by Ultrasonic Pretreatment
The morphological changes in RBCs before and after the grinding and ultrasonic treatments were observed using a phase-contrast microscope in three phases (Figure 3): (a) at the baseline (no treatment), RBCs exhibited a distinct spherical shape; (b) after grinding, hemoglobin within RBCs hemolyzed into the plasma due to RBC damage; and (c) after grinding and ultrasonic treatments, RBCs were completely destroyed and the boundaries between plasma and blood cells were indistinguishable, resulting from the complete destruction of RBCs due to two-stage solubilization of grinding followed by ultrasonic treatment. In summary, enzymes can easily access proteins without hindrance from blood cells, facilitating the cleavage of peptide bonds. This phenomenon is Likely to enhance enzymatic hydrolysis reactions. Figure 3: Morphological changes in red blood cell (magnification \(\times\)40); (a) before treatment, (b) only grinding (grinding speed: 10,000 rpm, grinding duration: 10 min), (c) after treatment (capacity: 1 L/batch, grinding speed: 10,000 rpm; grinding duration: 10 min; ultrasonic frequency: 20 kHz; ultrasonic density: 0.5 W/mL; ultrasonic duration: 30 min; agitation: 200 rpm). \begin{table}
\begin{tabular}{l l r r} \hline \hline  & **Properties** & **Grinding Treated** & **Ultrasonic Treated** \\ \hline Hemoglobin in whole blood (\%) & & & 15.25 \\ Hemoglobin in plasma (\%) & & 5.59 & 14.63 \\ Solubilization rate (\%) & & 36.32 & 95.91 \\ pH & & 7.48 & 7.14 \\ Salinity (\%) & & 0.96 & 0.92 \\ Particle size (um) & & 131.16 & 44.32 \\  & Moisture content (\%) & 81.69 & 81.07 \\
3-component & Organic content (\%) & 17.63 & 18.21 \\  & Inorganic content (\%) & 0.69 & 0.72 \\  & Carbon (\%) & 45.00 & 46.03 \\  & Hydrogen (\%) & 6.21 & 6.31 \\ Chemical component & Oxygen (\%) & 17.60 & 16.73 \\  & Nitrogen (\%) & 12.65 & 13.11 \\  & Sulfur (\%) & 0.14 & 0.15 \\  & Chlorine (\%) & 1.07 & 1.09 \\  & General Bacteria & 1.5 \(\times\) 10\({}^{5}\) & 1.3 \(\times\) 10\({}^{2}\) \\ Bacteria & Total Coliforms & 2.0 \(\times\) 10\({}^{3}\) & N.D. \\  & (CFU/mL) & & N.D. \\  & Salmonella & & N.D. \\  & (CFU/mL) & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Changes in the properties of Livestock blood after ultrasonic treatment. Pigs may carry various pathogens in their blood or may be exposed to them when they are slaughtered in a contaminated environment. Considering that pathogens in the blood can act as inhibitors of enzymatic hydrolysis [34], we analyzed these pathogens in the blood samples (Table 3). The analysis revealed a general bacterial count of 1.5 \(\times\) 10\({}^{5}\) CFU/mL in the untreated blood samples and 1.3 \(\times\) 10\({}^{2}\) CFU/mL in the post-treated blood samples, indicating a 99.93% reduction in bacterial count. Total coliforms were detected at 2.0 \(\times\) 10\({}^{3}\) CFU/mL in the untreated samples; however, they were not detected in the post-treated samples, indicating a 100% elimination rate. _Salmonella_ was not detected in both the untreated and post-treated samples. Microbiological contamination of Livestock blood may Lead to the production of foul odor and disease during the utilization of Livestock blood as a resource. It can also Lead to a decrease in amino acid conversion rates when using proteins from Livestock blood in enzymatic degradation processes. Furthermore, if pathogens survive enzymatic hydrolysis, they can have various negative effects, including gas generation. The eradication of pathogens is most significantly influenced by the cavitation effect of ultrasound, as confirmed in a previous study [35]. Additionally, ultrasound treatment results in a slight increase in temperature, which can contribute to the inactivation and eradication of microorganisms. Thus, ultrasonic treatment is advantageous for the solubilization of proteins within RBCs and eradication of pathogens in Livestock blood and is an efficient process for the utilization of Livestock blood as a resource.