Proteolysis of Silkworm Pupa and Antioxidative Activity of the Hydrolysate
|Course||Of Food Science|
|Keywords||silkwormpupa proteolysis hydrolysate antioxidative activity peptides separation purification|
The protein from silkwormpupa (silkworm of Bombyxmori L.)contains more than 60% of the essencial amino acids and a variety of other nutrients for human nutrition, and it can be utilized as a functional ingredient to improve human health. This research was focused on the antioxidative activity of silkwormpupa protein hydrolysates including the optimization of its hydrolysis process, isolation, purification, and structural analysis of the hydrolysates with the highest antioxidative activity.Slkwormpupa protein with high purity was prepared by n-hexane extraction under optimized conditions. The defatted silkwormpupa protein was hydrolyzed with six different proteases (alcalase, protamex, neutrase, papain, chymotrypsin, and flavourzyme), and the angiotensinⅠ-converting enzyme (ACE) inhibitory activity and antioxidative activity of the hydrolysates were examined. Results show that the hydrolysate has a low activity of inhibiting ACE, but a strong radical scavenging activity in eliminating DPPH,·OH, and superoxide anion radicals. Meanwhile the research found that the hydrolysates using papain has the strongest activity.The effects of pH, temperature, enzyme and substrate concentration on degree of hydrolysis (DH)and the radical scavenging activity of the papain hydrolysates were studied. With responsive surface methodology, the preparation conditions of the hydrolysates with high antioxidative activity were optimized. Under a concentration of 30mg/mL hydrolysate, the hydrolysis conditions for optimal DPPH·scavenging activity were:pH=7.3, T=49.7℃, E/S=2.1 %, t=1.9 h, the activity is i=92.38 %;superoxide anion radical scavenging activity: pH=7.4, T=50.2℃, E/S=2.0 %, t=1.4 h,the activity is i=48.56 %;·OH radical:pH=7.5, T=49.9, E/S=2.1 %、t=2.0 h,with the activity of i=93.97 %.Papain hydrolysate was further analyzed using a series of techniques. Four factions(P1-P4) were shown in the SephadexG-25 chromatography profile, and the activities of scavenging DPPH·radical for the two sub-fractions P4F and P4L(from fraction 4) increased 5.28 and 6.49 folds, respectively, than that of the initial hydrolysate. Fraction P4L has the same activity level as VC in scavenging DPPH·radical, and the IC50 of fraction P4L is 0.1524 mg/mL.The fractions P3、P4F、P4L with strong DPPH scavenging activity were further analyzed using RP-HPLC and LC-MS. Two major fractions (P4F2,P4F3) were shown by RP-HPLC, and their molecular weights are 168 and 222, respectively. Molecular structure of fraction P4F3 was determined to be a dipeptid Phe-Gly, and the fraction with a Mw of 168 is present in both fractions of P4L and P4F2. The antioxidative activity in scavenging DPPH·radical was shown to be 49.80 % and 28.50 % for fraction P4F2(Mw168)and P4F3(Phe-Gly), respectively, under a concentration of 1mg/mL, and no synergistic effect was shown for these two fractions. Further work is suggested to improve the understanding of the antioxidative activity of the hydrolysate.