# Enhanced Enzymatic Degradability of Livestock Blood Pretreated with Ultrasonic Technique
## 2 Materials and Methods
### Analysis of Physicochemical Properties of Samples  
The characteristics of the Livestock blood used in the experiment were analyzed using standard methods to determine their moisture, organic matter, and inorganic matter content [29]. Sulfo-phospho-vanillin analysis was performed to quantify the total Lipid content of the blood samples. The concentrations of various proteins in the blood, including total proteins, albumin, fibrinogen, platelets, and hemoglobin count, were estimated using an automatic hematology analyzer (Coulter Ac*T; Beckman Coulter, Carlsbad, CA, USA).  
To determine the solubilization rate of the blood proteins after undergoing ultrasonic treatment, we performed hemoglobin quantification analysis using the cyanmethemoglobin method in the following order: hemoglobin within the blood proteins was reacted with potassium ferricyanide (K4[Fe(CN)6]-3H2O) to generate methemoglobin; the generated methemoglobin was reacted with potassium cyanide to form cyanmethemoglobin; and the concentration of the final product was determined using a UV-Vis Spectrophotometer (Cary 4000; Varian Medical Systems, Belrose, New South Wales, Australia) at a wavelength of 540 nm, utilizing hemoglobin standard reagents. The protein solubilization rate was calculated using the measured hemoglobin concentration according to the following formula [30]:  
\[\text{SR}(\%)=\frac{H_{t}-H_{u}}{H_{w}-H_{u}}\times 100(\%) \tag{1}\]  
where SR is the solubilization rate; \(H_{t}\) is hemoglobin in treated plasma; \(H_{u}\) is hemoglobin in untreated plasma; and \(H_{w}\) is hemoglobin in whole blood.  
To evaluate the morphological changes in the blood samples subjected to ultrasonic treatment, the external appearances of the blood samples before and after treatment were compared using a phase contrast microscope (TS100F; NIKON, Tokyo, Japan). Changes in particle size distribution before and after treatment were assessed using a particle size analyzer (109 OLD; CILAS, Orleans, France). Additionally, to determine whether any potential pathogens present in the pulmonary blood were eliminated, the dry rehydtable film method (3 Mtm Petrifilm, NEOGEN, Lansing, MI, USA) was applied to analyze the general bacteria and pathogenic _Escherichia coli_, while _Salmonella_ sp. was analyzed using the standard plate culture method [31].  
To assess the extent of protein degradation resulting from enzymatic hydrolysis, the concentrations of proteins and amino acids in the blood samples were analyzed.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline  & & & & **Activity** \\ \cline{3-5}
**Enzyme** & **Characteristic** & **Source** & **pH Range** & **Temperature Range (\({}^{\circ}\)C)** \\ \hline Savinase\({}^{\circledR}\) & Endopeptidase & - & 8–12 (10) & 20–60 (55) \\ Alcalase\({}^{\circledR}\) & Endopeptidase & _Bacillus licheniformis_ & 6.5–8.5 & 45–65 (60) \\ Flavourzyme\({}^{\circledR}\) & Exopeptidase & _A Aspergillus oryzae_ & 5–7 & 50 \\ \hline \hline \end{tabular}  
Activity: indicates the range of conditions under which the enzyme shown by the manufacturer is active. The optimal conditions are given in parentheses.  
\end{table}
Table 2: Characteristic of commercial enzymes used.