ACE Inhibitory Peptides Derived From Soybean Protein

Enzymatic hydrolysis to produce bioactive peptides not only can make the most use of protein sources, but has broad appliance outlook in dietary and pharmacy areas. Among the plant sources, soybean is widely used to obtain safe and effectual antihypertensive peptides. In this paper, HMF (high-molecular-weight fraction) after hydrolysis of SPI was used as substrate to produce ACE inhibitory peptides.Initially, the conditions for HMF hydrolysis were optimized. And the results showed that microwave pretreatment could promote enzymatic hydrolysis compared with heat pretreatment. And the optimal reaction conditions for Protease N hydrolysis were: microwave power of 200W, microwave pretreatment time of 30s, substrate concentration of 5% (w/v), the ratio of enzyme of 2%( w/w), temperature of 55℃, pH 7.0 and hydrolysis time of 120min. The hydrolyte under the hydrolysis conditions boasted the highest inhibitory activity to ACE, which was 65.1%. Meanwhile, the research showed that the content of hydrophobic peptides in HMF was greater compared with SPI.After purification of the hydrolyte with ultrafiltration, the IC50 decreased 15.5% compared with the original hydrolyte. And the fractions were desalted by adsorption on a macroporous resin and then eluted with ethanol at different concentrations. The results showed that the highest antihypertensive activity fractions with an IC50 of 0.093 mg·mL-1were eluted with 75% ethanol. From digestive experiment in vitro, it could be found that ACE inhibitory activity of the eluted fractions remained almost the same after being digested by digestive enzymes.The eluted fractions with 75% ethanol were further separated by gel filtration chromatography with 0.2mol·L-1,PH=3.6 HAc-NaAc as a buffer. And two fractions with the most ACE inhibitory activity which had IC50 values of 0.041μmol?L-1 and 0.043μmol?L-1 respectively were collected. One fraction was further purified by semi-preparative reversed phase high performance liquid chromatography (RP-HPLC). And liquid chromatography / electrospray ionization mass spectrometry (LC/ESI-MS) showed that amino acid sequence of the purified fraction P6-Ⅳwas Ser-Trp probablely, which had the most ACE inhibitory activity with an IC50 of 41.1μmol?L-1. The probable components of the other fraction obtained after Sephadex G-15 were soy isoflavones through being analyzed by LC/ESI-MS.