Optimization Of Protein Hydrolysate Preparation And Their Angiotensin I-Converting Enzyme Inhibitory Activity From Shiitake Mushrooms Lentinula Sp.

Abstract

Angiotensin-I converting enzyme (ACE) is a key enzyme involved in regulating blood pressure. ACE converts angiotensin-I into angiotensin-II, which results in raised blood pressure. Inhibition of ACE leads to a decreased concentration of angiotensin-II and lowering of blood pressure. Food protein-derived ACE inhibitory peptides are considered to be a safer alternative to antihypertensive drugs. Shiitake mushrooms, one of the most consumed mushrooms in Thailand, are recognized as a nutritive food with health-stimulating properties. The aim of this study was to optimize the preparation of a shiitake-protein hydrolysate and investigate the ACE inhibitory activity of the hydrosylate. Shiitake mushrooms were hydrolyzed by Alcalase using response surface methodology. The degree of hydrolysis (DH) and ACE inhibitory activity were monitored. The optimum conditions, under which the DH reached a maximum of 28.88%, were 50.2 °C, a hydrolysis time of 3 h, and an enzyme/substrate ratio of 1.16. However, the optimum conditions under which the hydrolysate exhibited the highest ACE inhibitory activity (IC50 = 0.33 μg/mL) were 47 °C, a hydrolysis time of 3 h 28 min, and an enzyme/substrate ratio of 0.59%. After fractionation into five ranges of molecular weight, the fraction < 0.65 KDa showed the highest activity with an IC50 value of 0.23 μg/mL. This fraction was purified by RP-HPLC and the peak with a retention time of 3.96 min had high ACE inhibitory activity (IC50 = 0.03287 μg/mL). The amino acid sequence of this peak, identified by mass spectrometry, was Lys-Ile-Gly-Ser-Arg-Ser-Arg-Phe-Asp-Val-Thr (KIGSRSRFDVT), with a molecular weight of 1,265.43 Da. The ACE inhibitory activity (IC50) of the synthesized version of this peptide was 0.3714 μM. The peptide KIGSRSRFDVT was found to act as a non-competitive inhibitor from analysis of the Lineweaver-Berk plot. A docking study confirmed that the peptide binds at an ACE non-active site. These results indicate that peptides from shiitake mushrooms may be beneficial ingredients in pharmaceuticals, or functional foods, to treat hypertension.