Studies On Extraction And Enzymatic Preparation Of The Immunoactive Peptides From Bovine Placenta

Placenta has plentiful functional active materials and is the important resources for dairy corporation. The immunoactive peptides from different resources have been investigated widely. However, the study on immunoactive peptide from bovine placenta is little. To purify the immunoactive peptide from bovine placenta, discuss on the physiochemical characterization and the immune function in vivo/vitro provide the theoretical base for further study on development and utilization of bovine placenta and its immunoactive peptide.This paper firstly investigates the effect of different extraction buffer, extraction mode and extraction condition on water-soluble protein extraction rate of bovine placenta are studied. The best extraction parameter of phosphate buffer at one step extraction are temperature 20℃, time 2h, pH 7.2, material/solution rate 1:2 (w/v). At 0.10MPa working pressure and 60min working time of ultrafiltration and 0.6MPa and 90min of nanofiltration, the extracted protein solution is purified. The ammount of the sample by nanofiltration is 299.4mg/100g ret material (calculated according to protein). The sample by nanofiltration has significant immunomodulatory activity by determining spleen cell proliferation in vitro.DEAE Sepharose CL-6B, Sephadex G-25 and Sephasil C18 chromatography are used to purify the peptide with immunomodulatory activity in sample by nanofiltration with spleen cell proliferation for detecting immunomodulatory activity. The fraction 4 by RP-HPLC has significant immunomodulatory activity, shows single peak and is purity as detected by reverse phase chromatogrhphy and capillary electrophoresis.The structure, physical and chemical characterization and stability of the fraction 4 by RP-HPLC with immunomodulatory activity are studied. MALDI-TOF-MS, capillary isoelectric focusing and protein sequencer show that its relative molecular mass is 2134, pI is 3.82 and partial amino acid sequence is Tyr-X-Phe-Leu-Gly-Leu-Pro-Gly-X-Thr. It is peptide without carbohydrate as detected by phenol-sulphate reaction and ultraviolet chromatography. It can keep 6 months at -20℃; it has significant immunomodulatory activity at≤40℃and pH6-10. It is unstable to pepsin and trypsin hydrolyzation. It has the absorbance characterization ofα-helical,β-sheet,β-turn and nonregular coil and composition changs with the improved treating temperature as detected by the infrared spectrogram and circular dichroism.According to the immune function index of health food function, the relationship between amount and immunomodulatory function is studied at different dosage. Results show that at 500mg/kg,50mg/kg and 5mg/kg, for the normal mice, CH50, phagolytic activity of phagocyte and lymphocyte proliferation of the mice taking the water-soluble immunoactive peptide improve significantly (P<0.01) as compared with the mice not taking; and for the low immune function mice, spleen index, thymus index, cell immunity, body liquid immunity and monocyte-macrophage immunity of the mice taking the water-soluble immunoactive peptide improved as compared with model mice, this relaxes the low immune symptom.For the waste material of bovine placenta with plentiful protein after extracting the water-soluble immunoactive peptide, the immunoactive peptide prepared through protease hydrolyzing material and the hydrolysate used in medium are studied. At the optimal hydrolyzing condition of temperature 46.4℃, pH8.17,enzyme ratio 1:1, material/solution 1:3, total enzyme 4000u/g, the waste of bovine placenta is hydrolyzed by the mixed protease (neutral protease and trypsin) and got the hydrolysate with immunomodulatory activity. The hydrolysate is purified by RP-HPLC and fraction 7 has the highest immunomodulatory activity. Its amino acid sequence is Gly-Gly-Ser-Thr or Gly-Gly-Thr-Ser. Another, the decolored hydrolysate by active carbon is used to prepare peptone. The growth curves of Saccharomyces cerevisiae on the medium with this peptone and the Fluid Saboraud Medium are similar. The delay phase, index phase and stable phase are almost at the same time, and the absorbance is close. The growth of Escherichia coli on the medium with this peptone and Fluid Broth Medium is relatively close. The peptone made by bovine placenta hydrolysate has well application prospect.