Study On Trypsin Inhibitory Activity Of Hydrolysates Of Oyster(Crassostrea Gigas)

Enzymatic hydrolysis conditions of Crassostrea gigas and trypsin inhibitory activity of peptides from oyster hydrolysates were studied in this paper. Oyster was hydrolysed with six kinds of commercial proteinases including pepsin, trypsin, bromelin, papain, 3942 neutral proteinase and Alcalase 2.4L. Three enzymes of better hydrolysis effect were chosen as the target enzymes, taking the α-amino nitrogen in the hydrolysates as the index. The hydrolysis conditions of the three enzymes were investigated by single factor experiment and orthogonal test. The results show that the optimal hydrolysis conditions of these three enzymes were:50℃,pH 8.5, substrate concentration ( W/V ) 1:3,enzyme dosage 1200U/g,time 4h for Alcalase; 45℃,pH 8.1, substrate concentration 1:3, enzyme dosage 1200U/g, time 4h for Trypsin;50℃, pH5.5, substrate concentration 1:3, enzyme dosage 1400U/g, time 6h for Bromelin, respectively. The compound enzymes hydrolysis was investigated on the basis of the single enzyme hydrolysis. The optimal compound conditions were:substrate concentration 1:3, the original pH was 8. 5, adding 600U/g Alcalase hydrolyzing for 3h at 50℃;then pH was adapted to 5. 5, adding 700U/g Bromelin for another 3h;the reaction was terminated by heating the solution to 100℃ for 5min. The effect of heating treatment of the substrate on hydrolysis effect before hydrolysis was also investigated.The assay method of trypsin inhibitory activity was established. The hydrolysates were separated into four fractions with different molecular weight ultrafiltration membranes whose cutoff is 5K ,3K and 1K, respectively. Then the inhibitory activity was measured for these four fractions. Size exclusion chromatography was applied to make fartherseparation. The inhibitory activity of every peak collected was determined simultaneously. Results reveal that peptides with high molecular weight inhibited trypsin activity strongly.The relationship between hydrolysis time and hydrolysates inhibitory activity was studied. The longer hydrolysis time was, the lower hydrolysates activity was. UV scan spectrum was conducted for different components of oyster. Further separation was performed for those with high inhibitory activity. Results show that the first peak had the highest activity. The hydrolysates of 2h compound enzymatic hydrolysis was separated using Sephadex G-25 and the IC50 of the first peak was 1. 894mg/ml. The extractives without enzymatic hydrolysis gained the first peak with the ICM0. 658mg/ml.Amino acid analysis and SDS-PAGE were performed for different fractions of oyster extractives in order to determine their molecular weight. Many bands were detected in oyster extractives without hydrolysis and four proteins with molecular weight of approximately 32. 6i^ 27. 8KD% 21-22KD and 15-16KD were the dominant ones. However only two obvious bands with the molecular weight of 31KD and 33KD were found in oyster hydrolysates.