Enzymatic Preparation And Purification Of Hypoglycemic Peptide From Hemp Seed Meal

Hemp seeds are normally used to extract the oil in most Chinese areas ofproduction.While hemp seed meal is rich of protein, it is abandoned or used as feed, resultingin a waste of resources. Therefore, extracting the highly hypoglycemic peptides from hempseeds is of importance for the utilization of waste and the prevention and cure of diabetes. Inthis paper, the protein extraction, enzymatic hydrolysis and bioactive peptides of defattedhempseed meal were studied. The main conclusions are as follows:First, the process of the protein extraction from defatted hempseed meal through alkaliextraction and acid precipitation method was studied. The pH value of4.5was determined bysingle factor experiments in acid precipitation process. By the L9(34) orthogonal test, themost optimal extraction conditions were as follows: the alkali-soluble pH value was8.5, theextraction time was60min, the solid-liquid ratio was1:20, and the temperature was40℃.Under this condition the protein extraction rate of hempseed meal was86.17%, with theprotein purity93.94%.Second, the enzymatic hydrolysis degree had been measured by formaldehyde titrationand pH-stat methods after the enzymolysis of six common commercial protease(Alcalase2.4LFG, Flavourzyme, Protamex, Neutrase, Typsin and Papain) on HMPI. The results showed thatthe two methods had no significant differences. What’s more, the hydrolysate of Alcalase AF2.4L had the maximum degree of hydrolysis. With the substrate concentration50g/L and pH8.5fixed, the optimum enzymatic hydrolysis conditions were achieved as follows: the amountof enzyme was10190U/g, the temperature was62.04℃, the reaction time was4.21h. Underthese conditions, the experimental average degree of hydrolysis was21.87%.Third, the ACE inhibitory activity, the inhibitory activity of the cholesterol micellessolubility, α-glucosidase inhibitory activity and antibacterial activity of the different HMPIhydrolysate of Alcalase AF2.4L were tested.It showed that the hydrolysate with the degree ofhydrolysis of20.14%had the highest ACE and α-glucosidase inhibitory activity. The IC50value was27.22mg/ml and63.26mg/mL respectively. When the concentration of eachhydrolyzate was30mg/mL, the inhibition rate of cholesterol micelles solubility was less than20%. And they had no antibacterial activity.Fourth, the hydrolysate with the degree of hydrolysis of20.14%was separated bymacroporous resin DA201-C. The total peptide yield of the distilled water,25%,50%,75%, 100%elution fractions was89.91%.75%ethanol elution fraction had the highest α-glucosidase inhibitory activity(IC50=6.77mg/mL). The molecular weight of most componentsof75%ethanol elution fraction was less than1500Da. Four components(G1, G2, G3, G4) werecollected from D3by Sephadex G-15gel separation. The component with the lowestmolecular weight had the highest α-glucosidase inhibitory activity(IC50=0.11mg/mL).Fifth, G4was separated into seven active ingredients by semi-preparative RP-HPLC. R7had highest activity(IC50=43μg/mL). R7mainly consisted of a piece of pentapeptide and apiece of pidipeptide with mass at568.4Da and287.2Da separately by MALDI-TOF/TOFMS/MS sequence analysis.Their amino acid sequences are Pro-Leu-Met-Leu-Pro andLeu-Arg respectively.