Preparation And Physicochemical Properties Of ACE-inhibitory Peptides From Sweet Sorghum Protein

ACE-inhibitory peptide derived from food has the obvious advantages, such aslowering blood pressure, non-toxic side effects and so on. Therefore, ACE-inhibitorypeptide is one of the hot areas in finding antihypertensive drug. In this paper, ACEinhibitory peptide of sweet sorghum was prepared by enzymatic method. Taking ACEinhibitory activity as index, the suitable protease was choosen. The hydrolysis process wasoptimized by Plackett-Burman design, steepest ascent test and Box-Benhken design. Thekinetic model of sweet sorghum proteolysis was analyzed. Then the sweet sorghum ACEinhibitory peptide was separated and purified by ultrafiltration, ion exchangechromatography, gel chromatography. In addition, the structure of purified peptide wasidentified by liquid chromatography-mass spectrometry/mass spectrometry （LC-MS/MS）.Experimental results showed the content of crude protein in sweet sorghum （Dry base）was about13.27%. The content of hydrophobic amino acid was37.92%in total of aminoacids of the crude protein. Thus, sweet sorghum protein was ideal protein material toprepare ACE inhibitory peptides. Differential scanning calorimetry （DSC） results showedthat, the heat thermal denaturation temperature of sweet sorghum protein was62.78℃.Alcalase was chosen from four proteases （Alcalase, α-chymotrypsin, trypsin andpapain） to prepare ACE-inhibitory peptides from sweet sorghum protein. ThePlackett-Burman tests showed that the importance of three factors affecting activities ofACE-inhibitory peptide was hydrolysis temperature, pH and Alcalase dose.The optimizedprocessing parameters were as follows: pH8.0, hydrolysis temperature56℃and Alcalasedose5200U/g.The kinetic model of hydrolysis rate （R） was R=（50.103E₀-0.4234S₀） exp [-0.206（DH）]. The kinetic model of degree of hydrolysis （DH） was DH=4.854ln[1+（10.321E₀/S₀-0.087） t]. Stability tests showed that the sweet sorghum ACE-inhibitory peptides has goodacid, alkaline, thermal stability and resistance to intestinal enzymes digestion.After ultrafiltration, the ACE inhibitory activities of sweet sorghum protein hydrolysatefractions from high to low were <1kDa fractions (IC₅₀=0.116mg/mL),1⁵kDa fractions(IC₅₀=0.305mg/mL),5¹0kDa fractions (IC₅₀=1.012mg/mL) and>10kDa fractions(IC₅₀=1.225mg/mL) in turn. The Lineweaver–Burk curve revealed that the1–5kDa and<1kDa fractions was shown to be a competitive inhibition pattern, the5¹0kDa and>10kDa fractions was shown to be a non-competitive inhibition pattern. In order to obtain the highest ACE inhibitory activity components Ⅰb-2, the sweet sorghum ACE inhibitorypeptide was separated and purified by732cation exchange chromatography, SephadexG-50and Sephadex G-15gel chromatography sequentially. After the LC-MS/MS analysis,the sequence of the polypeptide which has the highest ACE inhibitory activity and contentin Ⅰb-2was Thr-Ile/Leu-Ser.