Research On The Preparation Of Antihypertensive Peptides Derived From Crude Protein In Abalones’ Viscus By Enzymatic Method

The content of protein which accounting for about 72% of abalone viscera (Dry basis) is high in abalone visceral. Abalone viscera is also rich in the essential amino acids and non-essential amino acids, such as Alanine, Valine, Isoleucine, Leucine, Tyrosine, Phenylalanine, Proline and other aromatic amino acids and hydrophobic amino acids, the content of which are also very high. But there is almost no report about the study of proteins in abalone viscera, and we haven’t found the effective way to use of them currently. Therefore, developing and using it as a biological resources of natural active peptide has very important significance. This research uses abalone viscera as raw materials, and by analyzing its physical properties and discussing on technology conditions of extraction, separation and purification for protein in abalone viscera, preparation of antihypertensive peptides from abalone visceral which can inhibit angiotensin-converting enzyme (ACE) activity was completed, which provide the basis for further processing and comprehensive utilization of abalone resources and laid the foundation for the development of natural health care products that can lower blood pressure. This study include:1.Determination of index of crude protein from abalone visceral, the result shows that the ash content of the crude protein is 7.53%, moisture 64.94%, protein 14.72%.2.1n this paper, the preparation of ACE inhibitory peptides was studied by enzyme method. Abalone viscera crude protein is hydrolysis by using five different enzyme to get the product, and then measuring its hydrolysis degree and the inhibition rate of ACE. By comparison, the inhibitory of ACE with 1.398 neutral protease is 84.93%, hydrolysis degree 8.37%, the pH value of crude protein is neutral. Therefore, all things considered,1.398 neutral protease is the optimal one.3.Vitality test shows that the activity of 1.398 neutral protease is 140,000U/g.4.By single factor experiment to optimize the process that preparing ACE inhibitory peptides of abalone visceral protein by using the 1.398 neutral protease. The optimum process condition is that while substrate concentration is 3%, adding 4500U/g neutral protease at 45 ℃ with pH 7.0 for 5h.5.After isolation and purification, we get polypeptide solution below 3000Da, and measure the content of polypeptide and its inhibition rate to ACE. The experiments show that the content of polypeptide is 10.25mg/mL, its inhibition rate is 52.94%.6. In order to improve and guarantee the safety of food, food safety experts set the new national standard of the amount of cadmium in food in 2006. And the amount of cadmium in shellfish seafood must be not more than 2mg/Kg. The result of the experiment is that the cadmium content in ACE inhibitory peptide powder is 0.89 mg/Kg, which meet the national standard.7.After long-term administration experiment, we found it has not had an appreciable effect on the weight of SHR rats and WKY rats by gavage in abalone viscera protein-peptide and captopril. Therefore, abalone visceral protein-peptide and captopril will barely affect the increase or decrease in weight of SHR rats and WKY. However, after once administration experiment, it revealed that both abalone visceral protein-peptide and captopril keep the antihypertensive activity in SHR rats within 2 to 6h. And both of them have the biggest decline of blood pressure of SHR after 4h, respectively 22.68mmHg and 61.33mmHg. Afterward, the blood pressure began to rise, until administered for 8h it returned to its original level. The results indicate ACE inhibitor has a short-term antihypertensive activity in SHR rats body.8.During long-term administration experiment, the blood pressure of SHR rats which were chronically administered with abalone viscera protein-peptide reach the lowest level of 26.40mmHg,while with captopril 35.00mmHg.But both of them did not work on blood pressure of WKY rats.9.After long-term administration experiment, the determination of serum indexes of MDA and SOD in rats indicates that although there is a certain amount of damage to both SHR rats and WKY rats by intragastric administration with abalone viscera protein-peptide or captopril, which is not significant (P>0.05).Therefore, the injuries to rats caused by abalone visceral protein-peptide and captopril can be neglected. In other words, abalone viscera protein-peptide prepared in this experiment have no harm to the rats.10O.After long-term administration experiment, removing the rat liver, making liver slices and observing under light microscope. We do not find difference of SHR rat liver slice between the observed group and blank control group. As a result, abalone viscera protein-peptide is rarely harmful for its rat liver. While in drug groups, the intercellular space in hepatic tissue is greater than the other two groups. That is to say, captopril has a certain degree of harm for its rat liver. The intercellular space of WKY rats hepatic tissue in observed group is greater than the one in blank control group. Thus, abalone viscera antihypertensive protein-peptides has a certain amount of damage to SHR rats while the captopril harms the WKY rats most.11.After long-term administration, measuring the weight of rats, calculating the heart weight index and analyzing the data. From which, we can see that ACE inhibitory peptides and captopril will not aggravate cardiomegaly,which will not increase the load on the heart. So the damage of ACE inhibitory peptide to rats hearts can be generally disregarded.