Purification and identification of hypocholesterolemic peptides from freshwater clam hydrolysate with in vitro gastrointestinal digestion

The muscles of freshwater clams were extracted separately using hot water. Subsequently, the edible muscle part was freeze-dried, hydrolyzed at 50°C using Protamex to obtain the freshwater clam hydrolysate (PX), and then digested with pepsin. The bile-acid-binding capacity and inhibition of cholesterol micelle formation were subsequently investigated using pepsin-digested PX (PXP) through ultrafiltration (UF) fractionation or size exclusion chromatography. After the UF treatment, soluble protein (171.0 mg/g) and peptide (109.4 mg/g) contents in fraction III were found to be higher than those of all other membrane fractions. Assuming that bile acid binds to PXP at 100%, the relative bile-acid-binding capacities of fractions I, II, and III were 161.2%, 64.3%, and 55.1%, respectively. Fraction f showed the highest inhibitory efficiency ratio (IER), and its inhibition-peptide content percentage was 831.5% mg/mL. The amino acid sequences of two hypocholesterolemic peptides were Val–Lys–Pro and Val–Lys–Lys, with IERs of 64.8% and 10.2% mg/mL, respectively. Practical applications The edible muscle part of freshwater clam after hot water extraction can be recovered as value-added by-product. After hydrolysis by commercial and digestive proteases, the edible muscle part of clams, including the viscera, showed higher bile acid binding capacity and exhibited in vitro inhibition percentages of cholesterol micellar solubility. Novel hypocholesterlemic peptides (Val-Lys-Pro and Val-Lys-Lys) were identified from the freshwater clam hydrolysate. Notably, this hydrolysate was confirmed to have hypocholesterolemic and hypolipidemic effects in vivo. Additionally, in vivo antihypertensive effects were confirmed in spontaneously hypertensive rats. This freshwater clam hydrolysate is expected to be a useful ingredient in physiologically functional foods for the prevention of hypertension and hypercholesterolemia.