Extraction Of Superoxide Dismutase From Rabbit Blood And Its Application In Yogurt

Superoxide Dismutase is one of metalloproteinase that widely exist in animals, plants and microorganisms, it could catalyze super oxygen anion radical (O 2·), disproportionation reaction occurs, remove free radicals within the biology, in food, cosmetics, medical and other industries widely used. Our country has very abundant rabbit blood resources, but the amount of rabbit blood are underused. The SOD is rich in rabbit blood. This research intend to use the fresh rabbit blood as raw materials to extract SOD, and to prepare SOD-yogurt with SOD for functional factor and milk as the carrier, it provide an effective way for SOD oral. In the process, the main researches were that the ultrasonic dissolving blood preparation and the optimum process conditions of removing impurity protein by thermal denaturation method; SOD purified and part of the nature of the research; the stability in the artificial gastrointestinal digestive juices and the changes in SOD activity of SOD-yogurt during the storage time. The main results were as follows:1. The dissolving blood was prepared combining ultrasonic extraction with swelling method, the effect of the volume ratio of red blood cells with water, ultrasonic treatment time and ultrasonic power on SOD activity were investigated, optimum technology parameter was obtained through a single factor and orthogonal experiment as follows:the volume ratio of the red blood cells and water 5:1, ultrasonic time 10 min, ultrasonic power 200W, under this condition, the dissolving blood has the highest SOD specific activity.2. The thermal denaturation method is used to purify proteins, the effect of thermal denaturation time, thermal denaturation temperature and the volume fraction of CUCl2 were investigated, by using the response surface analysis to optimize the process conditions, the results show that:the volume fraction of CuCl2 has a highest influence on SOD specific activity to extracting SOD by thermal denaturation method, followed by thermal denaturation temperature and time. Regression model established by the response surface method was significant (P<0.01), can be used to predict the actual value. The optimum technology parameter of the thermal denaturation method to2. purify proteins was optimized, that is, the thermal denaturation time 10.60 min, thermal denaturation temperature 10.60 ℃, CuCl2 volume fraction 1.09%, in this condition, SOD specific activity was 2165.39 U/mg. The validation experiment obtained SOD specific activity was (2204.50±2.63) U/mg, with the relative error of the predictive value was 1.77%, show that the response surface method can be used to optimize the optimum technology parameter of removing impurity protein by thermal denaturation method.3. Using acetone precipitation and column chromatography to purify SOD, the results show that:when the cold acetone is 1.0 times the volume of crude enzyme and the standing time was 30 min, the SOD specific activity was the hightest. By Sephadex G-75 gel filtration and Macro-Prep DEAE support anion exchange chromatography, collected enzyme fluid to concentrated by ultrafiltration, measured the total enzyme activity was 20474 U, the specific activity of the enzyme was 15749.23 U/mg, recovery rate was 19.08%, and its purification time was 487.47.4. The purified SOD for purity and species identification, and investigate some of its properties, the results showed that:the purified SOD has reached the electrophoresis purity, measured the subunits molecular weight of rabbit blood SOD was about 16.0 KDa, the ultraviolet absorption peak of the enzyme was found at 270 nm, and the enzyme was inhibited by H2O2. The enzyme was found to have good stability at the pH range from 5.0 to 9.0 and the temperature under 50 ℃, showed that the purified rabbit blood SOD is Cu, Zn-SOD.5. The stability of SOD-yogurt in simulate gastrointestinal environment was investigated. The results showed that the SOD residual activity of SOD and SOD in SOD-yogurt were 0.1% and 8.7% after 3 h of incubation in artificial gastric fluid with pH 2, however, in artificial gastric fluid pH value of 3 and 4 can keep high activity, were 50.4%,60.7% and 65.4%,84.4% respectively. After 4 h incubation in artificial intestinal fluid, the residual activity was more than 44.8%. The SOD residual activity was higher than 87.4% after 4 h of incubation in bile salt with different concentration. After continuous digestion in the artificial gastric fluid and artificial intestinal fluid, the SOD residual activity of SOD and SOD in SOD-yogurt were 15.4% and 72.4%.4. Visibly, SOD has excellent tolerance in artificial gastric fluid with pH 3, artificial intestinal fluid, and bile salt environment, specially, the SOD in SOD-yogurt has higher stability to the simulated gastrointestinal digestive fluid.6. SOD-yogurt was storage at 4 ℃, the results showed that the SOD residual activity of SOD-yogurt was 84.5% after 6 days of storage; storage for 14 days,the SOD residual activity of SOD-yogurt was 64%. The changes of pH and acidity was not obvious, and the difference between groups was not significant. The TBA value was on the uptrend and reduced after the storage of 12 days, the TBA value of experimental groups are smaller than the control groups(the yogurt without SOD). The whey separation of each samples was increased with the extension of storage time, and the experimental group were lower than the control group. Texture properties of each group did not change significantly, the firmness, consistency, cohesiveness and index of viscosity of control group and experimental group were not significant, this result showed that SOD added to yogurt has a little effects on its texture properties.