### 1. Introduction

### 2. Materials and Methods

### 2.1. Materials

### 2.2. Experimental System

### 2.3. Method

### 2.4. Physico-chemical Analysis

^{2+}) of hemoglobin are oxidized to the ferric (Fe

^{3+}) state by potassium ferricyanide(K

_{4}[Fe(CN)

_{6}]·3H

_{2}O) to form methemoglobin. Methemoglobin subsequently reacts with the cyanide ions of potassium cyanide to form cyanmethemoglobin. The amount of cyanmethemoglobin can be measured spectrophoto-metrically at a wavelength of 540 nm on a UV-Vis spectrophotometer (Cary 4000; Varian Australia Pty., Ltd, Australia), and can be compared to known hemoglobin standards in order to determine the hemoglobin concentration of the blood sample.

### 2.5. Experimental Design and Data Analysis

_{f}+ n

_{a}+ n

_{0}[16, 17]. In this study, the dependent variables were ultrasonic density (UD), (X

_{1}) and ultrasonification time (UT), (X

_{2}). A total of 11 different combinations of 4 factorial points coded to the (±1, ±1), 6 axial points coded to the (0, ±1) and (±1, 0) and 3 center points coded to the (0, 0) were chosen in random order according to a 2

^{2}full factorial CCD configuration for two factors. UD (X

_{1}, 0.5–2.0 W/mL) and UT (X

_{2}, 10–60 min) are coded in Table 1, and we attempted to confirm optimization conditions through observing interaction between their variables.

##### (2)

$$\eta ={\beta}_{0}+\sum _{i=1}^{k}{\beta}_{i}{x}_{i}+\sum _{i=1}^{k}{\beta}_{ii}{x}_{i}^{2}+\sum \sum _{i<j}^{k}{\beta}_{ij}{x}_{i}{x}_{j}$$_{i}and x

_{j}are the input variables, β

_{0}is the intercept term, β

_{i}is the linear effects, β

_{ii}is the squared effect, and β

_{ij}is the interaction tern. The effect and regression coefficients of individual linear, quadratic and interaction terms were determined through the analysis of ANOVA.

### 3. Results and Discussion

### 3.1. Physico-chemical Characteristics of Slaughter blood

### 3.2. Changes in Slaughter Blood with Different Ultrasonic Frequency

### 3.3. Response Surface Modeling by CCD

_{1}) and UT (X

_{2}) : Solubilization rate = 101.304 − 19.4205 X

_{1}+ 0.0398 X

_{2}+ 7.9411 X

_{1}

^{2}+ 0.0001 X

_{2}

^{2}+ 0.0455 X

_{1}X

_{2}.

*p*values less than 0.05 indicate that model terms are significant, while

*p*values greater than 0.1 indicate that model terms are not significant. In this model, F value and

*p*values were indicated to be 79.99 and < 0.001. In addition, the linear value was 0.001 (< 0.05), with a very high validity, and the

*p*value of the square and interaction were 0.001 (< 0.05) and 0.027 (< 0.05) respectively, illustrating a significant effect. In the verification of lack of fit, if the value of

*p*is smaller than 0.05, it is assumed to be inappropriate for a prediction model [19]. Yet, in this study, the calculated value of

*p*was 0.059 which indicates that it is appropriate for use as a prediction model. As a result of variance analysis with complete second-order formula for the verification of the validity of the model, the coefficient of determination (R

^{2}) of the SR model formula was 98.77%, and the modified coefficient of determination was 97.53%, displaying the appropriateness of the experimental design [18, 20].

### 3.4. Optimization of Ultrasonification of Slaughter Blood

### 4. Conclusions

_{1}+ 0.0398 X

_{2}+ 7.9411 X

_{1}

^{2}+ 0.0001 X

_{2}

^{2}+ 0.0455 X

_{1}X

_{2}. The value of correlation coefficient (R

^{2}) in SR model was 98.77%, and the adjusted correlation coefficient (R

^{2}) was 97.53%. The result of regression and variance analysis,

*p*value of linear, square, and interaction effects were 0.001, 0.001, and 0.02, respectively. And the

*p*value of lack of fit was 0.059. Thus, the statistical analysis indicated that the proposed model was adequate, possessing no significant lack of fit and very satisfactory values of R

^{2}for all responses. The best combination of each significant factor was determined by RSM and optimum pretreatment conditions for 95% of SR were established when blood was treated with 0.5 W/mL of UD for 22 min. Under these conditions, 95.53% of SR was observed experimentally, similar to the theoretical prediction of 95%. As a result, it is expected that enzymatic degradation of SB is most effective when the optimum conditions determined by this research are applied.