Effect of angiotensin I converting enzyme inhibitory peptide purified from skate skin hydrolysate

Abstract

Our objective was to evaluate the angiotensin I converting enzyme (ACE) inhibitory activity of skate skin protein hydrolysates and its corresponding fractions. The skate skin hydrolysates were obtained by enzymatic hydrolysis using alcalase, α-chymotrypsin, neutrase, pepsin, papain, and trypsin. Amongst the six hydrolysates, the α-chymotrypsin hydrolysate had the highest ACE inhibitory activity compared to other hydrolysates. The amino acid sequences of the purified peptides were identified to be Pro–Gly–Pro–Leu–Gly–Leu–Thr–Gly–Pro (975.38 Da), and Gln–Leu–Gly–Phe–Leu–Gly–Pro–Arg (874.45 Da). The purified peptides from skate skin had an IC₅₀ value of 95 μM and 148 μM, respectively, and the Lineweaver–Burk plots suggest that they act as a non-competitive inhibitor against ACE. Our study suggested that novel ACE inhibitory peptides derived from skate skin protein may be beneficial as anti-hypertension compounds in functional foods.

Introduction

Hypertension is related to the incidence of coronary heart disease and its treatment is effective in reducing the risk of cardiovascular disease and associated diseases (Collins et al., 1990). The renin–angiotensin system is amongst the most important vasoconstrictor and vasodilator mechanisms regulating blood pressure in human physiology. The angiotensin I converting enzyme (ACE, peptidyldipeptide hydrolyase, EC 3.4.15.1) acts to produce the vasoconstrictor angiotensin II by removing the C-terminal dipeptide (His–Leu) from the precursor decapeptide angiotensin I (Skeggs, Marsh, Khan, & Shumway, 1954) and also degrades the vasodilator bradykinin (Yang, Erdos, & Levin, 1970). Due to the biological activity of ACE, which belongs to a class of zinc proteases that require zinc and chloride for their activation (Tadasa, Shimoda, & Kayahara, 1992), ACE inhibitors, such as captopril and enalapril, have been used to lower blood pressure in hypertensive subjects in both in vivo and clinical studies (Hata et al., 1996, Maria et al., 2009). However, the subjects were presented with side effects, such as hypotension, headache, insomnia, and fever. Recently, many inhibitory peptides against ACE are reported as natural alternative biofunctional peptides that are safer than the artificiality synthesised ACE inhibitory drugs. Various ACE inhibitory peptides have been isolated from food proteins, such as the tuna back bone (Lee, Qian, & Kim, 2010), canola meal (Jianping, Rotimi, & Alister, 2009), algae waste (I-Chuan, Fang, & Wu, 2009), sardinelle (Sardinella aurita) (Ali et al., 2008), and Alaska–Pollack skin (Byun & Kim, 2001). Improved nutritional and potent biofunctional peptides have been produced by enzymatic protein digestion (Fahmi et al., 2004, Lahl and Braun, 1994). While these peptides are inactive within the sequence of the parent protein, biofunctional peptides can be released by enzymatic digestion or food processing during the food manufacturing process (Korhonen, Pihlanto, Rantamaki, & Tupasela, 1998). In addition, protein hydrolysates are also a source of bioactive peptides, which are short peptides released from food proteins by hydrolysis and have certain biological activities that may be beneficial for the organism (Je, Park, Byun, Jung, & Kim, 2005). Bioactive peptides with activities involved in the regulation of the gastrointestinal, nervous, cardiovascular and immune systems have been described (Korhonen & Pihlanto, 2006).

Skates are a popular food in South Korea. Approximately 30% of processing waste from skate consists of skin and bone, which contain high protein concentrations (Nam & Lee, 1995). In addition, the demand for skate has greatly increased, due to its unique taste, flavour and health benefits (Nam & Lee, 1995). Its popularity has recently promoted the large demand for fermented skate resulting in increased production and consequently generating a large volume of skin as a by-product (Nam & Lee, 1995). Therefore, there is a greater necessity and interest to investigate a potential application to utilise this by-product. Previously, studies with skate skin included the production of collagen and the purification of an antimicrobial peptide (Cho, Michael, & Eun, 2004). However, there is currently no report available on the ACE inhibitory peptides of skate skins. For effective usage of fishery by-products, our study proposed that bioactive material can be isolated from the skate skin by-product. Hence, we investigated the ACE inhibitory activity of skate skin’s protein hydrolysate. The objective of this study was to isolate ACE inhibitory peptides from skate skin protein hydrolysate and identify the purified peptide with regards to the ACE inhibitory activity. Furthermore, we also investigated the inhibition pattern of the isolated peptide on ACE.