| ACE inhibitory peptides were prepared from loach protein through controlled enzymatic technology. Separation and isolation of the activity peptides from the loach protein hydrolysate(LPH) by a series of purification technology, including ultrafiltration, gel filtration chromatograghy and reversed-phase high performance chromatography. The sequence of the active peptide was identified by mass spectrum technology. The antihypertensive effect of LPH were evaluated in vitro and in vivo. It provided a theoretical basis of functional food development. Model of QSAR was proposed for tetra-peptide inhibitors of ACE based on z-scales. It provided a theoretical basis of new compounds design.The basic nutritional components of loach were analyzed first. Its nutritional value was assessed from the point of amino acid analysis. The chemical composition of loach was analyzed as follows:77.2%moisture,1.3%ash,1.5%fat,17.4%protein. It was a typical high protein food. Most of the essential amino acid contents were very close to FAO/WHO mode, except to methionine and cysteine which was the first limiting amino acids. The total content of essential amino acids reached42.5%, higher than the WHO recommended ratio of36%for measuring ideal protein resources. Efficiency ratio was PER I of2.9, PERⅡ of2.9and PER Ⅲ of2.3. The high content of hydrophobic amino acids (47.9%), aromatic amino acids (13.3%) and branched chain amino acids (17.7%) were the excellent material source of ACE inhibitory peptides by enzymolysis.A wide range of proteases were selected from plant, animal sources to microbial origin including pepsin, acidic protease, α-chymotrypsin, protamex, bromelain, papain, alcalase, trypsin and neutrase.9different proteases were applied to hydrolyze the loach protein, and bromelain was chosen to be the best one for the enzymatic hydrolysis through the comparison of half inhibitory concentration IC50of hydrolysates. On the basis of single-factor test, taking ACE inhibitory rate as response value, the effect of enzymatic temperature, the ratio of enzyme to substrate([E]/[S]) and hydrolysis time were optimized with Box-Behnken design and response surface methodology(RSM). The results showed that the optimum conditions were as follows:[E]/[S]4.9‰, substrate concentration30%, pH6.5, enzymatic temperature at54.4℃for5.9h. The two multiple regression equation for ACE inhibitory rate (Y) were as follows:Y=82.17+4.81A+5.57B+3.86C-4.16B2-3.97AB+4.27AC, where the [E]/[S](A), temperature (B) and hydrolysis time (C) were independent variables. In optimum conditions, hydrophobic amino acids, aromatic amino acids and branched chain amino acids content in hydrolysates were increased significantly, and the ACE inhibitory rate reached88.9%.Loach peptides were isolated and purified by ultrafiltration, gel filtration chromatography Sephadex G-15and a two-step reverse high-performance liquid chromatography(RP-HPLC) according to the molecular size and hydrophobicity differences step by step. The purified antihypertensive peptide was identified as Ala-His-Leu-Leu (452.2Da) using electrospray ionization(ESI) mass spectrometer. The purified peptide with IC50value of18.2±0.9μ.g/mL showed a33.7-fold higher ACE inhibitory activity compared with the crude loach peptides, and protein recovery was5.2%. The purified peptide is the first tetrapeptide with significant angiotensin-Ⅰ-converting enzyme inhibitory activity to be found.LPH had nice water-soluble ability with NSIs of70%above over a wide rang of pH(3-9). After the hydrolysis of loach protein, foaming and foam stability were decreased by15.8%and15.5%. Loach peptides exhibited good emulsifying capacity and emulsion stability, which were60.8%and68.6%respectively at pH7.0. The antioxidant ability increases significantly (P<0.05). DPPH·and ABTS·+removal rate of LPH were increased6.1-fold and2.7-fold. The stability experiment showed that ACE inhibitory activity didn’t decrease after2h heating below80℃, and it still maintained80%after2h heating at100℃. Loach peptides remained at high active stability from pH2.0to8.0. ACE inhibitory activity of loach peptides decreased with pH rise above8.0. Based on evaluation of ACE inhibitory activity, freeze drying was better than spray drying. Mg2+promoted the angiotensin-Ⅰ-converting enzyme inhibitory activity, while Zn2+and Mn2+inhibited the angiotensin-Ⅰ-converting enzyme inhibitory activity with metal ion concentration of5mmol/L for2h at room temperature. With the increase of glucose concentration, the ACE inhibition activity gradually decreased and browned after the reaction at121℃for20min. Sodium chloride had no significant effect on antihypertensive activity. When its concentration was more than9%, the ACE inhibitory activity and sodium chloride concentrations showed a negative correlation.Loach peptide exhibited the ACE inhibitory activity because that it competed with the substrate binding ACE enzyme activity sites. Thus, it belonged to competitive inhibition. In simulated gastrointestinal digestion system, peptides hydrolyzed by pepsin exhibited higher angiotensin-I-converting enzyme inhibitory activity than before. But it was lower after further hydrolyzed by trypsin. Loach peptides belonged to the substrate type of ACE inhibitor.Hypertension animal model SHR was used to evaluate the antihypertensive effect of the peptides, systolic blood pressure (SBP) of the animal were test by a tail cuff blood pressure analyzer through acute and long-term animal studies. In the acute experiment, the maximal decrease of29mmHg in SBP caused by30mg/kg BW of LPH was observed after14h post-administration. In the eight months animal administration of LPH, there was no effect on heart rate. Body weight of SHR was significantly lower than that of control group (P<0.05). After4weeks administration, the SBP of the high doze group showed the maximal reduction from195.0mmHg to165.1mmHg. The reductions of systolic blood pressure(SBP) after8weeks were24.3mmHg and10.5mmHg for high doze group and low doze group respectively. Compared to the model control group, abdomen aorta/body weight ratio increased significantly (P<0.05), heart/body weight ratio decreased significantly (P<0.05), loach peptides played a positive role of organ protection. Serum triglyceride concentration, sodium concentration and MDA concentration in serum and abdomen aorta were reduced significantly(P<0.05) in high doze group after2months administration. Loach peptides contributed to easing lipid peroxidation of some organ or serum. The total free amino acid content in serum of high doze SHR group was higher than that of the blank control group and positive control group, in which His, Ala, Glu, Leu, Ile were significantly increased (P<0.05). LHP exerted its antihypertensive effect on the abdomen aorta, and the activity of the tissue was significantly decreased17.6%(P<0.05).The traditional model can well explain the ACE inhibition of the di-peptides or the tri-peptides. But the QSAR of tetra-peptides or above still need further research. A tetra-peptide was prepared from loach protein, the QSAR of the tetra-peptides was further explored in this paper. Using z-scales descriptor,92tetra-peptides was encoded. Through partial least squares regression analysis of multivariate data by the Matlab software, the QSAR model of ACE inhibitory tetra-peptides was established as follows:y=2.1519+0.0704x1+0.0443x2+0.0166x3+0.0302x4-0.0758x5-0.0084x6-0.031x7+0.1372x8-0.0629x9+0.0256x10+0.0396x11+0.035x12. The correlation coefficient (R) was0.7489, cross-validation correlation coefficient (Rcv2) was0.5956. The model was validated with tetra-peptide AHLL from loach protein hydrolysates. The results showed that the QSAR model constructed in this paper exhibited a good generalization performance, which could predict the activity of ACE inhibitory tetra-peptide exactly. |
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