Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses L. Ong, N.P. Shah * School of Molecular Sciences, Victoria University, Werribee Campus, P.O. Box 14428, Melbourne, Victoria 8001, Australia Received 5 June 2007; received in revised form 5 November 2007; accepted 6 November 2007 Abstract The aim of the study was to examine the release of angiotensin-converting enzyme (ACE)-inhibitory peptides in Cheddar cheeses made with starter lactococci and Bifidobacterium longum 1941, B. animalis subsp. lactis LAFTI Ò B94, Lactobacillus casei 279, Lb. casei LAFTI Ò L26, Lb. acidophilus 4962 or Lb. acidophilus LAFTI Ò L10 during ripening at 4 and 8  C for 24 weeks. ACE-inhibitory activity of the cheeses was max- imum at 24 weeks. Cheeses made with the addition of Lb. casei 279, Lb. casei LAFTI Ò L26 or Lb. acidophilus LAFTI Ò L10 had significantly higher (P < 0.05) ACE-inhibitory activity than those without any probiotic adjunct after 24 weeks at 4 and 8  C. The IC 50 of cheeses ripened at 4  C was not significantly different (P > 0.05) to that ripened at 8  C. The lowest value of the IC 50 (0.13 mg mL 1 ) and therefore the highest ACE-inhibitory activity corresponded to the cheese with the addition of Lb. acidophilus LAFTI Ò L10. Several ACE-inhibitory peptides were identified as k-CN (f 96e102), a s1 -CN (f 1e9), a s1 -CN (f 1e7), a s1 -CN (f 1e6), a s1 -CN (f 24e32) and b-CN (f 193e209). Most of the ACE-inhibitory peptides accumulated at the early stage of ripening, and as proteolysis proceeded, some of the peptides were hydrolyzed into smaller peptides. Ó 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Angiotensin-converting enzyme; Cheddar cheese; Probiotic bacteria; Proteolysis 1. Introduction Bioactive peptides have been defined as ‘peptides with hormone- or drug-like activity that eventually modulate physiological function through binding interactions to specific receptors on target cells leading to induction of physiological responses’ (FitzGerald & Murray, 2006). Milk protein is a rich source of biologically active peptides such as antihypertensive- , antithrombotic-, opioid-, immune-stimulating, antimicrobial-, mineral carrying- and cholesterol lowering-peptides (Shah, 2000). Most of these peptides are hidden in the inactive state in the original parent protein structure and may be released through (a) hydrolysis by digestive enzymes such as trypsin and pepsin (FitzGerald, Murray, & Walsh, 2004; Korhonen & Pihlanto, 2006), (b) food processing (Van-Beresteijn et al., 1994) and (c) through hydrolysis by proteolytic microorganisms or through the action of proteolytic enzymes (FitzGerald & Murray, 2006). Proteolysis is one of the major biochemical events which takes place during cheese ripening. It is caused by enzymes contained in milk (plasmin), from rennet (pepsin and chymo- sin) or released by microorganisms. These enzymes break down casein (a s1- , a s2- , b- and k-caseins) into smaller peptides and amino acids, which are important for the development of Cheddar flavour (Cliffe, Marks, & Mulholland, 1993; Lynch, Muir, Banks, McSweeney, & Fox, 1999). Several peptides from Cheddar cheeses have been isolated and identified Abbreviations: ACE, angiotensin-converting enzyme; CAF, chemically as- sisted fragmentation; CN, casein; HHL, hippurylehistidyleleucine; IC 50 , 50% inhibitory concentration; MALDI-TOF-MS, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; TFA, trifluoroacetic acid; WSE, water- soluble extract. * Corresponding author. Tel.: þ61 3 9919 8289; fax: þ61 3 9919 8284. E-mail address: nagendra.shah@vu.edu.au (N.P. Shah). 0023-6438/$34.00 Ó 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2007.11.026 Available online at www.sciencedirect.com LWT - Food Science and Technology 41 (2008) 1555e1566 www.elsevier.com/locate/lwt