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Continuous Preparation Of Corn Peptide With Potent ACE-Inhibitory Activity By Using Enzyme Membrane Coupling Technology

Posted on:2012-07-16Degree:MasterType:Thesis
Country:ChinaCandidate:W H HuangFull Text:PDF
GTID:2211330344452243Subject:Food Science
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In order to utilize the plant protein and enhance the added value of byproduct of starch industry-- corn gluten meal (CGM), the corn proteins were extracted from CGM. The corn peptide (CP) with potent angiotensinⅠ- converting enzyme (ACE) inhibitory activity were prepared by continuous enzyme membrane coupling technology. First the preparation processes of CP with potent ACE inhibitory activity were optimized. Second, the effect of the treatment by different molecular mass cut off (MmCO) ultrafiltration membranes on ACE inhibitory activity of CP and the desalting of CP by nanofiltration (NF) was investigated. Third, the stability of corn-protein-derived ACE inhibitory peptides was studied. Furthermore its antihypertensive effect and mechanism of action were researched according to their activities in spontaneously hypertensive rats (SHRs). The last, the amino acid sequences of peptides were analyzed by HPLC-MS/MS, the structure-effect of corn peptides was discussed. The main results were shown as follows:1. Study on the hydrolysis process of corn peptides with high ACE inhibiting activity in enzymatic membrane reactorThe optimum conditions were as follows:circulating speed = 5 L/min, [E]/[S] = 1.5%(w/w), material to water ratio = 3% and T = 45℃. Under these conditions, the optimized values were 89.82%,73.48% and 83.52% for ACE inhibitory rate, protein recovery rate, and average membrane flux. The ACE-inhibitory activity of CP improved 20% compared to batch preparation processes.2. Fractionating CP by ultrafiltration membranes and desalting by nanofiltration (NF)The Mm <3 kDa fraction exhibited the lowest IC5o value (IC50= 0.29 mg peptides/ml) among all fractions (Mm<1 kDa, Mm <3 kDa, Mm <5 kDa), with the IC50 value decreasing fourfold of the Mm <5 kDa fraction. The optimum conditions of desalting were as follows:P = 8 bar, cycle-index = 4, pH = 9. Under these conditions, the optimized values were 86.77% and 89.72% for ACE inhibitory rate and desalting rate. The ACE activities of CP had no evident change when subjected to NF.3. Kinetic model for continuous enzymatic hydrolysis of AlcalaseBy making some suitable hypothesis, the equation of degree of hydrolysis (DH) with substrate concentration and hydrolysis time was built Referring to the equation, we could predict the variation of hydrolysis degree at other substrate concentration with time. Further more, the kinetic model for continuous enzymatic hydrolysis corn protein of Alcalase was built Km of 22.34 gL-1 and vmax of 0.507 gL-1min-1, were obtained.4. The stability of corn peptide and its antihypertensive effects in vivo①All CP fractions of Mm< 1 kDa, Mm< 3 kDa and Mm< 5 kDa could withstand the temperature within 100℃, and they maintained stable ACE-inhibitory activity around the pH 8. At the same time gastrointestinal proteases had little effect on the ACE inhibitory activity of CP, in which Mm< 3 kDa CP fraction had a highest ACE inhibitory activity than the other two before and after simulating digestion, it remained the activity of more than 90% after gastrointestinal digestion.②In the acute oral administration experiment, the maximum systolic blood pressure (SBP) reductions levels of these treatment groups were, in descending order:CP high dose group (26.57 mmHg), CP middle dose group (19.57 mmHg) and CP low dose group (17.91 mmHg). The antihypertensive effects of these treatments were maintained for 5h. The best antihypertensive effect was observed in positive control group (Captopril). However, the significant reduction of SBP in CP high dose group (100 mg/kg-bw) could last for 5h that was longer than in Captopril group (2 mg/kg-bw).③In the long-term experiments, the SBP of all treatment groups were significantly lowered (P< 0.01) compared to that of the SHR model control group. The antihypertensive effect improved with oral administration times. After 30 days treatment, the maximal SBP reductions in all treatment groups were, in descending order:Captopril group (37.28 mmHg), CP high dose group (34.45 mmHg), CP middle dose group (30.95 mmHg) and CP low dose group (27.49 mmHg). Interestingly, the SBP of Wistar-Kyoto rats showed no significantly changes during long-term oral treatment with high dose CP.④CP and Captopril showed the antihypertensive effects through inhibiting the ACE activity in lung and testes, and it implied lung and testes were the target sites of CP and Captopril. The antihypertensive effects of CP at a dose of 100 mg/kg-bw was approximative to that of Captopril (2 mg/kg-bw) 5. Study on the Structure of ACE inhibitory corn peptidesThe primary structure of CP was analysed by HPLC-MS/MS, and the primary results were retrieved in the MS-MS database. Primary structure of three peptides was definited, which were:QQLLPF,QQFLPF and QLLPF. These short peptides are rich in hydrophobic amino acids, N-terminal and the first three C-terminal amino acid are same among them. The 3 kinds of peptides could play an important role in ACE inhibitory activities as the structure-activity relationship theory.
Keywords/Search Tags:corn peptide, ultrafiltration, nanofiltration, spontaneously hypertensive rats(SHRs), kinetic model, primary structure
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