Enzymatic Preparation And Anticoagulant Activity Of Rapeseed Peptides

Rapeseed is the second largest oil crops in the world. Rapeseed protein has beenrecognized as a complete protein, and its essential amino acids are well-balanced. So, it is ahigh quality protein for human consumption. In this dissertation, rapeseed protein wasextracted from cold-pressed rapeseed meal, further enzymatic to produce rapeseed peptides.And the anticoagulant activity of rapeseed peptides was explored. Rapeseed proteinhydrolyzate was desalted and decolorized by macroporous resin adsorption and activatedcarbon, and the amino acids composition, molecular weight distribution and anticoagulantactivity of different rapeseed peptides were determined. Then high anticoagulant activity ofrapeseed peptides was prepared by SephadexG-15filtration chromatography,semi-preparative RP-HPLC and SEC-HPLC.Though orthogonal experiment, the best process conditions alkaline extraction rapeseedprotein was determined: liquid ratio (g/mL)1:15, pH12, temperature55℃, extraction time80min. Under this condition, the extraction rate of protein was88.5%. Acid precipitation ofrapeseed protein was at pH4.5, and the protein content was85.1%after freeze-dried undervacuum.Alcalase2.4L was the best hydrolase compared with other protease enzymes (Alkalineprotease2709, Protex6L, Protex7L and Neturase1.5MG). Though single factor test, theoptimal hydrolysis conditions of Alcalase2.4L were: substrate concentration3.0g/100mL,enzyme dosage7000U/(g protein), reaction temperature60℃, pH8.5, hydrolysis time2h.Under these conditions, the liquid enzyme thrombin inhibition rate reached98.8%, the degreeof hydrolysis of rapeseed hydrolyzate was18.1%.pH had a great effect on the color of rapeseed proteolysis solution in this experiment.Under acidic conditions (pH2.5), the discoloration effect of powdered activated carbon wasbetter than granular activated carbon on rapeseed protein hydrolyzate. However, the loss rateof peptide was higher than granular activated carbon. The amount of powdered activatedcarbon added to hydrolyzate also had the effect of discoloration. With the increase in its amount added, the bleaching effect was getting better and better, while peptide loss rate wasalso increasing rapidly. In order to get good discoloration and low peptide loss rate, thepowdered activated carbon of3%added into hydrolyzate was determined.After desalted by DA201-C macroporous adsorption resin and decolorized by powderedactivated carbon, discoloration peptide (protein content of68.9%) and desalted peptide(protein content of90.3%) were obtained. The hydrophobic amino acids of discolorationpeptide were decreased, while part hydrophobic amino acids were improved in desaltedpeptide. However, the proportions of component molecular mass below1000were declinedin both of them.The thrombin inhibition rate of rapeseed peptides was measured by microplate method.The half inhibition rate IC50of rapeseed crude peptide, desalted peptide treaded bymacroporous resin and discoloration peptide treaded by powdered activated carbono were16.5mg/mL,74.4mg/mL and52.5mg/mL.Rapeseed peptide was separated by SephadexG-15, and rapeseed peptide separationworked well when sample mass was100mg and flow rate was150mL/h. Meanwhile, fourmajor components were collected, and the thrombin inhibition rate of G1was96.8%at1mg/mL. G1was separated by Semi-preparative RP-HPLC, and the optimal condition forseparation was:0～5min l00～80%A；5～20min80～60%A；20～25min60～0%A；25～27min0%A；27～29min0～100%A；29～40min100%A (A:5%acetonitrile containing0.1%TFA; B:80%acetonitrile containing0.1%TFA), flow rate1.5mL/min. Under theseconditions, eight major components were obtained, and G1-R8had a strong anticoagulantactivity since it could completely inhibit the activity of thrombin at0.41mg/mL. FurtherG1-R8was separated by SEC-HPLC, and the result showed G1-R8wasn’t a singlecomponent.