Preparation Of Angiotensin I-converting Enzyme Inhibitory Peptides From Cobia Heads By Enzymatic Hydrolysis

Hypertension is one of the most important risk factors that cause human death. Antihypertensive peptides derived from food proteins have been huge applied foreground in market due to high safety and easy absorbance. The technological optimum conditions and methods of extracting antihypertensive peptides of cobia heads were studied by enzymatic hydrolysis, ultrafiltration, gelatin filtration and high performance liquid chromatography. Resulting peptides were tested by model animal in vivo verified significant anti hypertensive effect. Main results are as follow:1. Nutrient component of cobia heads were determined. The results showed that the crude protein contents were 14.75%, fatty contents were 12.68%, non-protein nitrogen contents were 161 mg/100g; unsaturated fatty acid contents were 56%, the contents of EPA and DHA were high; total amino acid contents were 14274mg/100g sample, free amino acid contents were 293mg/100g sample, essential amino acid contents were 4418 mg/100g sample,the contents of aromatic amino acid, branched chain amino acid and Pro were 5.62%,14.46%,7.85% to their structural amino acid respectively. The first limited amino acid were methionine, amino acid score was 84. The results indicated that cobia heads was suitable to material for antihypertensive peptidesby enzymatic hydrolysis because of high contents of protein, high nutritive value of protein.2. The optimum conditions for ACE inhibitory peptides from the protein of cobia heads by enzymatic hydrolysis were determined. Cobia heads hydrolyzed proteins were prepared using single enzymatic hydrolysis by Papain, Neutrase, Alcalase, Trypsin and Pepsin.With ACE inhibitory activity being index, five commercial proteases hydrolysate were used to select the optimum enzyme at different hydrolysis time. The results indicated that the hydrolysate (2h) by pepsin exerted the most active ACE inhibition ratio 82.5%. Combing the DH at different time, the optimum conditions were determined by central composite design which be selected temperature, enzyme dosage , pH as three independent variables. The resulting condition was that ratio of matieral to water1:2, temperature 72℃, enzyme dosage2300U/g stuff, pH6.0, hydrolysis time 4h. Under the conditions, the ACE inhibition ratio was 82.63% and the DH 17.21%.3. The enzymatic hydrolysate was separated to four kinds of components with 8KDa, 5KDa and 3KDa ultrafiltration membrane. Ultrafiltrated components were studied with ACE inhibitory activity, molecular weight distribution and digestion tolerance. The results indicated that ACE inhibitory activity of four kinds of components were reduced with increasing molecular weight, ACE inhibitory activity of ultrafiltrated componentsⅣ(< 3KDa ) was strongest, IC50 1.17mg/mL,its soluble protein concentration to hydrolysate was 70.47%. Ultrafiltrated componentsⅣwas digested by Pepsin, Trypsin, Pepsin and Trypsin, the molecular weight of hydrolysate after digestion became smaller, their IC50 all fell. Ultrafiltrated componentsⅣshowed good resistance tolerance to the enzymes in simulated gastrointestinal digestion4. Ultrafiltrated componentsⅣwas filtered to three absorption peak with a Sephadex G-25 gel column, chromatography components were studied with ACE inhibitory activity, heating stability. The results were: component B showed the strongest inhibitory activity, its IC50 was 0.004 mg/mL. The enzymatic hydrolysate conteined pro-drug type peptides of ACE, component conteined inhibitor type peptides of ACE, chromatography components conteined substrate type peptides. In componentⅣand B, the summation of branched chain amino acid and contents of aromatic amino acid and Pro were 29.66% and 45.14% to their structural amino acid respectively. These results showed that ACE inhibitory peptides were enriched by polydextran gel chromatography.5. ComponentⅣwas evaluated for antihypertensive activity by animal experiment. Changes of SBP were observed after oral administration of lyophilizated componentⅣin 24h. It showed that the recorded SBP mostly reduced 32mmHg at 4 h after orally administration at a low dose of 150mg/kg body weight, less than positive control (captopril) group. The recorded SBP mostly reduced 44 mmHg at 6 h after orally administration at a average dose of 600mg/kg body weight, similar as positive control group. The recorded SBP mostly reduced 57mmHg at 6 h after orally administration at a high dose of 1200mg/kg body weight, better than positive control group.in addition, componentⅣhad no significant effects on the heart rate of SHR and normotensive WKY rates.