| Abalone,a species of primitive marine biology,which is known for its high nutritional value and medicinal value,contains the most comprehensive amino acid,the lowest cholesterol and fat among marine biology. According to some research, abalone viscera is rich in protein, polysaccharides, celluLose and other active substances,and for the connective tissue of abalone viscera,collagen is considered to be the most abundant protein. Only the body of abalones used as food resuorse or processed deeply, abalone viscera,30%-40% of the abalone gross weight,mostly will be discarded, few used to be processed as animal feed, so the resource will be utilizated lowly. Our aim was to reuse the connective tissue of abalone viscera to prepare the collagen ACE inhibitory polypeptides from abalone viscera by enzymatic method, and to study on the antihypertensive effect in vivo through the animal experiments.First of all, the analysis of the main components of abalone viscera connective tissue showed that it was a kind of value marine products processing wastes which was high in protein(12.54%), but low in fat (2.04%). And the content of initial collagen was 33.77%(dry basis). Additionaly, the analysis of the amino acid composition showed that, many kinds of amino acid of abalone visceral connective tissue coincided with amino acid composition of the ACE inhibitory peptide model.also,hydrophobic amino acids, aromatic amino acids, alkalineeach and proline amino acids of the total amino acids respectively accounted for 32.46%,4.89%,12.88% and 5.47%.therefore, abalone viscera connective tissue can be used for the preparation of collagen ACE inhibitory polypeptides.Secondly, by comparing the enzymolysis effect of trypsin, AS.1398 neutral protease, papain, alkaline protease and pepsin on abalone viscera connective tissue,comparing the collagen extraction effect of glacial acetic acid and citric acid combined with protease on abalone viscera connective tissue,and comparing the pulverizing pretreatment effect of juicing machine, colloid mill on abalone viscera connective tissue, The colloid mill pulverizing pretreatment and pepsin combined with glacial acetic acid were selected to prepare the collagen solution. In order to look for the optimal hydrolysis conditions of pepsin, the single factor and orthogonal experiment were conducted. The results showed that the optimal hydrolysis conditions were ascertained:hydrolysis temperature of 4 ?,hydrolysis time of 48h, the enzyme concentration of 240U/g, the substrate proportion of 1:12. unde the hydrolysis conditions, the abalone visceral collagen extraction ratio was 23.25%.Thirdly, trypsin, AS.1398 neutral protease, papain, alkaline protease, pepsin were used to hydrolyze the collagen solution respectively,And AS.1398 neutral protease was selected to prepare collagen ACE inhibitory polypeptides. In order to look for the optimal hydrolysis conditions of AS.1398 neutral protease, the single factor and the basis of 4 factor quadratic composite rotation experiment were conducted. The results showed that the optimal hydrolysis conditions were ascertained:hydrolysis time of 6.29h, the enzyme concentration of 12405.29U/g, pH of 7.07, hydrolysis temperature of 52.04?. The hydrolyte under the hydrolysis conditions boasted the highest inhibitory activity to ACE, which was 67.92%.Meanwhile, by comparing the desalting effect of the DA201-C, DA201-B, DA201-C macroporous adsorption resin on the hydrolysis solution and comparing the peptides desorption effect of 25%,50%,75%,95%,100% ethanol solution on the macroporous resin, DA201-C macroporous resin was used to adsorp the hydrolysis solution staticly for 12h and 75% ethanol was selected to desorp for 24h. unde these conditions, the ACE inhibitory rate of the freeze-dried products in vitro was 67.89%.Finally, to stuy on the change tendencies of rats weight, systolic blood pressure, the content of serum SOD, the content of serum MDA and the cardiac mass index, the Spontaneously Hypertensive Rat(SHR)andWKY(normal control)were taken as model of animal experiments in vivo. The abalone viscera collagen ACE inhibitory polypeptides in different doses(200mg/kg/d,400mg/kg/d and 600mg/kg/d) were conducted by short-term administration and long-term administration. The result of the single administration experiment showed that the blood pressure of SHR, compared with the base blood pressure,dropped for 14.79mmHg (P>0.05),16.43mmHg (P<0.05) and 17.35mmHg (P<0.05),respectively, after the administration for 3.25h. However, the SHR blood pressure of captopril group(10mg/kg/d),compared with the base blood pressure,dropped for 61.67mmHg (P<0.05), after the administration for 3.25h. Meanwhile,the blood pressure of WKY group remained stable,and the blood pressure decreased of the WKY group was not significant (P>0.05). The result of the long-term administration experiment showed that the blood pressure of SHR group increased with the time of administration. the blood pressure of SHR,compared with the blood pressure of SHR blank group, dropped for 4.19 mmHg (P>0.05),12.80 mmHg (P<0.01) and 17.95 mmHg (P<0.01), respectively, after the administration for 30d. However, the SHR blood pressure of captopril group(10mg/kg/d), compared with the blood pressure of SHR blank group,dropped for 25.79mmHg (P<0.01), after the administration for 30d. Meanwhile, the blood pressure of WKY group remained stable after the administration for 30d,and the blood pressure decreased of the WKY group was not significant (P>0.05). In addition,rats administrated by abalone viscera collagen ACE inhibitory polypeptides were found to have no significant influence on rats weight, the content of serum SOD, the content of serum MDA and the cardiac mass index.To sum up, abalone viscera collagen ACE inhibitory polypeptides not only had no bad effect on the normal growth of SHR, WKY rats, but also controlled the lower blood pressure in vivo. And the hypotensive mechanism of abalone viscera collagen ACE inhibitory polypeptides in vivo may be associated with the regulation of renin-angiotensin aldosterone system. |
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