Optimization Of Enzymolysis Process Of The Donkey Serum Protein And Preliminary Activity Research Of The Hydrolysate

As the traditional Chinese medicine, Ejiao has various biological functions. Donkey serum protein is a major part of Ejiao protein. This study is mainly focused on hydrolyzing donkey serum protein and optimized the production process conditions using ultrafiltration method to obtain biologically hydrolysates (HMP) under 2500 Da. The preliminary research was carried on the activity of antitumor and immune. It supplied the certain theoretical and experimental basis of further development and utilization of the donkey serum as well as researching functional biological products.Firstly, using the single factor experiment and response surface method to determine the optimum parameters of serum protein by trypsin hydrolysis:the adding amount is 4000 U of enzyme, pH 8, temperature 50℃, enzymolysis time 6 h.The hydrolysates derived from donkey serum protein were determined to be high level under the conditions. Furthermore, the predictive value of hydrolysis was 54.05%. However, it was a 54.31% to be determined according actual experiment. The difference was 0.481% . The hydrolysates was filtrated by 2500 Da ultrafiltration membrane and then analyzed the map of the molecular weight distribution by ESI-MS spectrometry, the molecular weight of HMP were mainly distributed in 200-500Da.The effect of HMP on HepG2, HL-60 and L02 were repectively assessed in vitro by MTT assay. These results showed that the range of the effective concentration on HepG2 were from 0.0001 to 1 mg/mL with dependent of time and dosage. The best inhibition rate reached to 31.57% at 1 mg/mL. Similarly, the range of the effective concentration on inhibiting HL-60 cells were from 0.0001 tol mg/mL with dependent of time and dosage. The IC50 of HMP on the HL-60 cells after 24 h and 72h were 1.442 mg/mL and 0.731 mg/mL, respectively. Further study detected to evaluate its effect on the normal liver cells L02 and found HMP to have no significant effect on normal liver cells.By immune activity in vitro test, HMP with concentration of 0.1-1 mg/mL could promote significantly the proliferation of mouse spleen lymphocytes when it worked together with ConA except of itself.In the research, the donkey serum hydrolysates is obtained under 2500Da by enzymolysis and ultrafiltration, and the preliminary results proved the HMP has antitumor activity and immune activity in vitro.