Preparation Of Antihypertensive Peptides From Litopenaeus Vannamei Head Via Autolysis Approach

Hypertension is one of the most popular cardiovascular diseases with high incidence, many complications, high mortality and low cure rate. At present many synthetic Angiotensin I-converting Enzyme (ACE) inhibitors are available for clinical use. They have some undesirable side effects. Naturally ACE inhibitory peptides derived from food proteins are a sort of bioactive peptides which exert inhibition in ACE. These peptides have no known side effects and have become a potential alternative for the synthetic ACE inhibitors.Shrimp head is the main by-product in shrimp processing. It is estimated that the output of shrimp head in China is up to 200,000 tons annually, seventy percent of which is from Litopenaeus vannamei. Shrimp head is a rich source of protein, nutritive components and endogenous enzymes. It is susceptible to autolysis at certain conditions. The protein in it is degraded into soluble protein, peptides and amino acids. The preliminary study indicated that the autolysis hydrolysate of shrimp head by its endogenous proteases exerted potent ACE inhibitory activity and had high peptide content. This paper systematically studies the process of preparing antihypertensive peptides from Litopenaeus vannamei head by autolysis method, separation and purification the ACE inhibitory peptides in the autolysis hydrolysate and characterize their amino acid sequences. In the meanwhile, the antihypertensive effects of the hydrolysate were investigated in SHR, and preliminary discussion on its antihypertensive mechanism of action. The outcome of this study laid the foundation of research and development of antihypertensive peptides,health food and medicine, and has lessons of higher value application for cheap aquatic product and its by-products.The main results were as following:(1) The study was conducted to investigate the effect of temperature, pH and substrate concentration on the autolysis process of Litopenaeus vannamei head and the change law of the hydrolysate during the autolysis. The rules of the hydrolysate releasing during the first five hours fitted the first order reaction kinetics: Y = 39.496^-0.3913x, KP = -1.1464Y+59.506, Pe = -0.7167Y+32.551. It showed a good linear relativity between total protein (KP), autolysis hydrolysate under 5000Da and residual protein. In the course of autolysis, the important factors were temperature, pH and substrate concentration which affect shrimp head autolysis rate. During the autolysis, the value of Ka increased with temperature rising from 40℃to 50℃and reached a peak at 50℃, while the value of Ka reduced with temperature rising from 50℃to 60℃; the effect of pH and substrate concentration on Ka did not show regularity, the value of Ka reaches a peak at pH9 and substrate concentration at 1∶3 (shrimp head: water), respectively. The optimized condition for the autolysis of shrimp head was obtained: temperature at 50℃, pH at 9 and substrate concentration at 1∶3 (shrimp head: water).(2) Shrimp head was autolyzed under the optimum condition. ACE inhibitory activity of the autolysis hydrolysates at different autolysis time was studied to determinate the optimum autolysis time. The results showed that 3 hr yielded a hydrolysate with the ACE inhibitory activity being up to 44.23%, 66.80% of which were components under 5000Da. The total content of branched chain amino acid(BCAA) and aromatic amino acid(AAA) was up to 24.78%.(3)Separation and purification were performed on the ACE inhibitory peptides in the hydrolysate autolysis of 3 hr autolysis to characterize their structure. Firstly, the hydrolysate was subject to primary ultrafiltration of different molecular weight cut-off. It showed the ACE inhibitory activity and peptide contents of the product ultrafiltered by a 3000Da cut-off membrane were significantly enhanced. The IC₅₀ value decreased from 1.61mg/mL to 0.61mg/mL. 61.83% of the ultrafiltered product was below 3000Da. The content of BCAA and AAA increased from 24.78% to 25.17%.(4)The product ultrafiltered by a 3000Da cut-off membrane was further subjected to gel chromatography using a Sephadex G-25 column. The chromatography yielded four absorbance peaks. The no. 2 peak exerted the most potent ACE inhibitory activity with the IC₅₀ being 0.27mg/mL. The content of BCAA and AAA was up to 28.53%. Then no. 2 peak was further subjected to a SP Sephadex C-25 column and yielded three absorbance peaks, peak A, B and C. They were desalted by a Sephadex G-15 column and then determined the ACE inhibitory activity and peptide content. Peak B exhibited the most potent ACE inhibitory activity with an IC₅₀ value of 0.19mg/mL, and had the highest peptide content in all the fractions. This active peak was further separated by RP-HPLC, two peptides were isolated and characterized to be Tyr-Pro and Leu- Pro/ Ile -Pro by ESI-MS/MS, respectively.(5) Antihypertensive effects of the product ultrafiltered by a 3000Da cut-off were investigated in Spontaneously Hypertensive Rat (SHR). The results of short term trials (24h) showed that the product ultrafiltered by a 3000Da cut-off exhibited potent antihypertensive effects. The reactive time of recorded Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DBP) is at 2h after orally administration the trail sample at a dose of 900mg/kg body weight. The peak time was at 10h. The recorded SBP reduced 44mmHg and the recorded DBP reduced 41mmHg, and the effects were maintained for 10h. The long term trials (8 weeks) showed that the product ultrafiltered by a 3000Da cut-off exerted reposeful antihypertensive effects during the experiment. The recorded SBP decreased at two week after orally administration product under 3000Da at a dose of 900mg/kg body weight, while DBP at one week. In eight week, the recorded SBP reduce 35mmHg, and the recorded DBP reduce 35mmHg. Short term and long term trials showed the product ultrafiltered by a 3000Da cut-off had no significant effects on the heart rate of SHR.(6) The organs weight of SHR after orally administration the product ultrafiltered by a 3000Da cut-off for eight weeks and partial blood index were investigated. The security of ACE inhibitory peptides and the mechanism of antihypertensive activity were discussed. The results indicated that the organ weight (heart, liver, kidney, lung) of SHR showed significantly different or very significantly different between orally administration the product ultrafiltered by a 3000Da cut-off with the captopril group and the control group. It indicated that the product ultrafiltered by a 3000Da cut-off could slowdown the disease condition of SHR. The content of renin, angiotensin and aldosterone of SHR were at high levels before orally administration. It indicated that the imbalance of Renin-Angiotensin System (RAS) maybe result in high blood pressure of SHR. The levels of renin activity, angiotensin and aldosterone of SHR of the control group showed significantly lower than the groups orally administered the product ultrafiltered by a 3000Da cut-off and captopril, indicating that the antihypertensive activity of the ACE inhibitory peptides we extracted was maybe connected with its inhibition on hormones such as renin, angiotensin and aldosterone.