Preliminary Research On Peptide Hydrolyzed From Pine Pollen By Enzyme And Its Antioxidant Activity

A pine pollen contains a wide range of nutrition.There are multiple researches have been done on fatty acids and carbohydrates in Pine pollen, but the researches on proteins are very few reported. Proteins are the most important material of life,and the peptide are product of protein decomposition.Active peptides have so many functions,for example,antioxidant, deduce blood pressure,anti-fatigue,suppress tumor and promote the human body to absorb useful protein and many beneficical microelements. That is why the active peptides reproduction is more popular into the field of biological engineering. This subject take the yunnanensis pollen crude protein which is made by laboratory as the raw material.Using the alkaline protease to hydrolyze the pollen crude protein,and want to get the optimization of hydrlysis condition.We take the peptide hydrolysis to ultrafiltration purification,and then take the preliminary antioxidant study in the chemical system in vitro and the cell system.The aim of the study was to explore the effects of hydrolysates of yunnanensis pollen crude protein.This result of study as follows:1. The hydrolyzation condition of yunnanensis pollen crude protein by alkaline protease with the criterion of scavenging activity and degree of hydrolysis.Hydrolysed yunnanensis pollen crude protein was studied by single factor and orthogonal experiment design. The optimum condition of pollen crude protein concentrate has been found as follows: pH9.0, hydrolysis temperature 50℃, substrate concentration 3%, enzyme concentration 4000U/g, hydrolysis time 2 hours and the scaveging rate of hydroxyl radical is 67.6%.2. The research on antioxidantion of peptides hydrolyzed from Pinus yunnanensis pollen (PHPP) by enzyme in vitro. With the concentration of PHPP at 7.5mg/mL , the eliminating rate of superoxide anion radical reached to 64.45%; when PHPP was 30mg/mL, the hydroxyl radicals on the removal rate was 72.99%; when PHPP was 3mg/mL, the clearance rate to DPPH free radical was 42.48%.3. Using centrifugal ultrafiltration to make PHPP purification and concentration, then we can get the purified peptides hydrolyzed by enzyme, it referred to PPHPP. We take a depth antioxidantion research with the PPHPP.With the concentration of PHPP at 7.5mg/mL, the eliminating rate of superoxide anion radical reached to 92.48%; when PHPP was 30mg/mL, the hydroxyl radicals on the removal rate was 80.80%; when PHPP was 3mg/mL, the clearance rate to DPPH free radical was 70.83%.Compared with PHPP, the eliminating rate of PPHPP on the radical has increase to a certain extent.This result explain that the method of centrifugal ultrafiltration can play an important role in the protein purification, removal of impurities and increased activity.4. The method of MTT is been used to detect the protection of myocardial cells from peroxide injured by PPHPP. The result show that, compared with injury group, OD value of the group which adds PPHPP has icreased more or less, and sample group which final concentration was 50, 100, 500μg/mL has more visible protection, the OD value has significant difference (P<0.01) compared with injury group.5. The detection of SOD and MDA showed that, SOD vitality in the injury group has notable lower than control (P<0.01), MDA value has increase more (P<0.01). But in the PPHPP sample group,SOD vitality is more than injury group (P<0.01), the peroxide products MDA is much less(P<0.05).The result illustrate that the PPHPP reduce the stress response to H2O2, and the PPHPP paly a role in the protection of the myocardial cells.