Study On The Extraction And Membrane Concentration Technology Of Horse Blood SOD And Thrombin

This paper using horse blood as raw material, based on the single factor experiment, by responsesurface method and orthogonal analysis method to optimize the horse blood SOD ultrasonic extraction andpurification conditions of heating denaturation. By using RS ultrafiltration membranes with differentmolecular weight cut off maked comparative experiments on membrane flux, specific activity of SOD andthrombin after concentrate, the best membrane molecular weight cut off was chosen. On the basis of this,studied on effect of material liquid temperature, operating pressure, feed flow rate and concentrationmultiple on the membrane flux. At the same time, based on the single factor experiment, by orthogonalanalysis method to optimize membrane washing condition and determine optimal washing condition. Inaddition, on the basis of single factor experiment, thrombin activity as response value, prothrombinactivation conditions were optimized by response surface method and determine the optimal activationprocess.Based on the single factor experiment, by response surface method and orthogonal analysis method tooptimize the horse blood SOD ultrasonic extraction and purification conditions of heating denaturation.Determine the optimal horse blood SOD extraction conditions were ultrasonic time15min, ultrasonicpower220W, ultrasonic temperature31℃, under this condition the SOD specific activity was77.5U/mg.The best heating denaturation of the purified conditions: the thermal time was20min, thermal temperaturewas50℃, the dosage of Cu2+was2.5%, under this conditions the SOD specific activity was668.5U/mg.Using three RC ultrafiltration membrane with different molecular weight cut off, membrane flux andspecific activity of horse blood SOD crude enzyme liquid were compared. Determine the RC ultrafiltrationmembrane with MWCO10kDa was best for horse blood SOD crude enzyme liquid concentrate idea film.By studying on effect of several process parameters on the concentration process of horse blood SODcrude enzyme. Determine the optimal conditions of concentration: the material liquid temperature20℃,operating pressure0.20MPa, feed flow rate53.50m/s, concentration multiple of2times. The single factorand the orthogonal experiment results showed that, in the membrane concentration process whichpollutants as SOD crude enzyme solution, the membrane washing optimal conditions: cleaning agent was2%citric acid and0.3%NaOH, cleaning time was40min, the cleaning temperature was35℃, under theseconditions, the recovery rate reached94.12%.By comparative experiment, determine the applicable to concentration of prothrombin extract andcrude enzyme thrombin solution membrane were RC ultrafiltration membrane with MWCO50kDa andMWCO30kDa.By studying on effect of several process parameters on the concentration process, determine optimal conditions of the horse blood prothrombin extraction liquid membrane concentration: material liquidtemperature5℃, operating pressure0.15MPa, feed flow rate35.67m/s, concentration multiple of3times;The optimum membrane concentration conditions of thrombin crude enzyme liquid: liquid temperature6℃,operating pressure0.20MPa, feed flow rate71.34m/s, concentration multiple2times; Ultrafiltrationmembrane cleaning conditions: first, the use of distilled water cleaning for50min at55℃, secondly, usingthe2%citric acid and0.3%NaOH as cleaning agent cleaning for40min at35℃, then under the condition of30℃cleaning for25min with2%HCl, finally, using distilled water flushing membrane filtrationequipment30min.On the basis of single factor experiment, the conditions of prothrombin activation optimized byresponse surface method. Determine the optimal activation condition: the activation time was2.8h,activation temperature was32℃, the concentration of Ca2+was0.11mol/L, under the condition of thisthrombin activity reached51.16U/mg.