Scavenging effect of the hydrolysates from Hypophthalmichthys molitrix meat protein on hydroxyl radical

The purpose of this study was to investigate the scavenging activity (SA) of the fish protein hydrolysates (FPH), which were produced using five different hydrolytic enzymes from Hypophthalmichthys molitrix protein, on hydroxyl free radical, and to separate the active peptides produced by hydrolysis and measure the molecule weight distribution of the active peptides. The fish protein hydrolysates (FPH) are of increasing interest due to their potential applications as a source of bioactive peptides or as nutritional ingredients of food products or as nitrogenous substrates for the fermentation media. According to the scavenging activity (SA) of Hypophthalmichthys molitrix protein hydrolysates on hydroxyl free radical produced by Fenton reaction, papain and trypsin, whose hydrolysates had higher scavenging activity (SA=81.5% and 83.5%, respectively) on hydroxyl free radical, had been chosen to be the good hydrolytic enzymes from the five applied enzymes such as trypsin, papain, pepsin, subtilisin and flavourzyme. Furthermore, the optimal hydrolysis parameters of papain and trypsin hydrolysis reaction, including the temperature, time, pH, enzyme content, and the substance concentrations in enzymatic reaction system, were determined by the orthogonal design L_9 (3~4) , respectively. The influence of the process variables enzyme substrate ratio; effect of intermediate substrate and enzyme addition was studied with regards to the extent of proteolytic degradation and the scavenging activity on hydroxyl free radical, and to the molecular weight distribution of the active peptides. Although the degree of hydrolysis (DH) increased with the time and the enzyme addition, there were no direct relationship between degree of hydrolysis (DH) and scavenging activity (SA). Then the Hypophthalmichthys molitrix protein hydrolysates produced by using papain and trypsin with higher scavenging activity on hydroxyl free radical were fractionated with Sephadex G-25 resin, respectively. The absorbance of every fractionated component was measured at 280nm and its molecular weight distribution was calculated according to the absorbance and standards graphs drawn. The results showed that the optimal enzymatic hydrolysis conditions that could produce the protein hydrolysates with the highest scavenging activity on hydroxyl free radical for using papain were temperature 50℃, time duration 15 min, pH 6.5, enzyme content 1.50% (w/w) and enzyme/substances ratio 1:2, and that the highest scavenging activity (SA) was 88.2%; And the optimal enzymatic hydrolysis conditions that could produce protein hydrolysates with the highest scavenging activity on hydroxyl free radical for using trypsin were enzyme content 0.25% (w/w), time duration 60min, pH 8.0, temperature 55℃ and substance concentration 1:2, and that the highest scavenging activity (SA) was 84.2%. The fractionated active peptide, which separated from the Hypophthalmichthys molitrix protein hydrolysates produced by papain, had the highest scavenging activity, SA=95.1%, on hydroxyl free radical and its molecular weights was 2.2 kDa. And the fractionated peptides, which separated from the Hypophthalmichthys molitrix protein hydrolysates produced by trypsin had the strong scavenging activity, SA=89.6%, on hydroxyl free radical, and its molecular weight was 14.2 kDa.