Original article Separation of angiotensin I-converting enzyme inhibitory peptides from bovine connective tissue and their stability towards temperature, pH and digestive enzymes Yu Fu, Jette F. Young, Trine K. Dalsgaard & Margrethe Therkildsen* Department of Food Science, Aarhus University, Blichers All  e 20, 8830 Tjele, Denmark (Received 8 October 2014; Accepted in revised form 19 January 2015) Summary Bovine collagen was isolated from connective tissue, a by-product in the meat processing industry and characterised by SDS-PAGE. Alcalase and papain were employed to generate collagen hydrolysates with different degree of hydrolysis (DH). In vitro angiotensin I-converting enzyme (ACE) inhibitory activities were evaluated and the two most potent hydrolysates from each enzyme were separated by two-step puri- fication. Both alcalase-catalysed and papain-catalysed hydrolysates exhibited strong ACE inhibitory capacities with IC 50 values of 0.17 and 0.35 mg mL À1 , respectively. Purification by ion-exchange chroma- tography and gel filtration chromatography revealed higher ACE inhibitory activities in one fraction from each enzyme with IC 50 values of 3.95 and 7.29 lg mL À1 . These peptide fractions were characterised as 6-12 amino acid residues by MALDI-TOF/MS. The peptides retained their activity (>90%) after exposure to processing temperature and pH and in vitro simulated gastrointestinal digestion. The present results demonstrated that collagen peptides can be utilised for developing high value-added ingredients, for exam- ple ACE inhibitory peptides. Keywords ACE inhibitory peptides, by-product, collagen hydrolysates, purification, stability. Introduction Hypertension, as a global risk factor in cardiovascular diseases, is deemed the world’s largest killer and an estimated one billion people around the world will be diagnosed by 2025 (Ahhmed & Muguruma, 2010). Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) plays a crucial role in the renin–angiotensin system as it catalyses the transition of decapeptide (angiotensin I) to vasoconstrictive octapeptide (angiotensin II) which is a key regulatory peptide in vasodilation. ACE also inactivates the vasodilatory bradykinin in the kal- likrein–kinin system, a peptide that helps reduce the blood pressure (Skeggs et al., 1956). Therefore, effec- tive inhibition of the ACE enzyme has been regarded as a therapeutic approach of hypertension (Ahhmed & Muguruma, 2010). Compared with some synthesised drugs, such as captopril, enalapril, lisinopril and ala- cepril for counteracting hypertension, the ACE inhibi- tory peptides from food-derived proteins are considered to be safer and milder (Webb et al., 2010). Some in vivo studies on spontaneously hypertensive rats or preclinical experiments have suggested that these biopeptides can reduce blood pressure remark- ably, by either oral or endovenous administration without exerting side effects on normotensive subjects (Fern andez-Musoles et al., 2013). Moreover, the clini- cal data demonstrated that oral administration of tri- peptides (e.g. IPP and VPP) can significantly reduce systolic blood pressure (SBP) and diastolic blood pres- sure (DBP) (Mart  ınez-Maqueda et al., 2012). In recent years, collagen or gelatine has been reported as good sources of ACE inhibitory peptides after enzymatic digestions. These potent ACE inhibitory peptides have been obtained not only from land-based sources such as porcine skin collagen (Ichimura et al., 2009), bovine skin gelatine (Kim et al., 2001) and chicken collagen (Onuh et al., 2013), but also from marine sources such as fish skin (Byun & Kim, 2001), fish cartilage (Nagai et al., 2006) and squid tunic (Alem an et al., 2011). The meat processing industry produces considerable quantities of slaughter by-products every year. They are highly underutilised and regarded as huge wastes and potential pollution to the environment (Mokrejs et al., 2009). Connective tissue, accounting for 9–12% *Correspondent: Fax: +45-87154891; e-mail: Margrethe.Therkildsen@food.au.dk International Journal of Food Science and Technology 2015, 50, 1234–1243 doi:10.1111/ijfs.12771 © 2015 Institute of Food Science and Technology 1234