Anticoagulant activities of goby muscle protein hydrolysates Rim Nasri a , Ikram Ben Amor b , Ali Bougatef a , Naima Nedjar-Arroume c , Pascal Dhulster c , Jalel Gargouri b , Maha Karra Châabouni a , Moncef Nasri a,⇑ a Laboratoire de Génie Enzymatique et de Microbiologie – Ecole Nationale d’Ingénieurs de Sfax, B.P. 1173-3038 Sfax, Tunisia b Centre Régional de Transfusion Sanguine, Route el-Ain Km 0.5, CP 3003 Sfax, Tunisia c Laboratoire de Procédés Biologiques, Génie Enzymatique et Microbien, IUT A Lille I, B.P. 179, 59653 Villeneuve d’Ascq Cedex, France article info Article history: Received 8 November 2011 Received in revised form 10 January 2012 Accepted 26 January 2012 Available online 4 February 2012 Keywords: Goby Protein hydrolysates Anticoagulant peptides Activated partial thromboplastin time Thrombin time abstract The anticoagulant activities of protein hydrolysates prepared from goby muscle by treatment with var- ious bacterial alkaline proteases were investigated. All proteases exhibited varying degrees of hydrolysis (DH) and all goby protein hydrolysates (GPHs) caused a significant prolongation of both the thrombin time (TT) and the activated partial thromboplastin time (APTT). The hydrolysate generated by the crude protease from Bacillus licheniformis NH1 displayed the highest anticoagulant activity, and the higher TT (about 32 s) at a concentration of 5 mg/mL was obtained with hydrolysate having a DH of 8.86%. This hydrolysate was then fractionated by size exclusion chromatography on a Sephadex G-25 column into five major fractions (F1–F5). Fraction F2, which exhibited the highest anticoagulant activity, was then fractionated by reversed-phase high-performance liquid chromatography. The molecular masses and amino acid sequences of four peptides in peptide sub-fraction F2–6, which exhibited the highest antico- agulant activity, were determined using ESI-MS and ESI-MS/MS, respectively. The structures of these pep- tides were identified as Leu-Cys-Arg, His-Cys-Phe, Cys-Leu-Cys-Arg and Leu-Cys-Arg-Arg. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Coagulation is the second step of haemostasis. It helps stop the bleeding by the consolidation of the platelet aggregate obtained at the end of primary haemostasis (Allford & Machin, 2004). The coagulation system is triggered in response to endothelium rup- ture. It involves a cascade of enzymatic reactions involving clotting factors, many of which are serine proteases subjected to activation and inhibition (Davie & Ratnoff, 1964; Macfarlane, 1964). Human blood coagulation consists of an intrinsic and an extrinsic path- ways. The two pathways converge at the formation of factor Xa (FXa) by factor IXa (FIXa) in an intrinsic pathway and FVIIa in an extrinsic pathway. The final step is the conversion of soluble fibrin- ogen into fibrin filaments by the action of thrombin. An imbalance in this balance leads to either bleeding or the formation of a throm- bus (Allford & Machin, 2004). Accumulation of fibrin in blood ves- sels can interfere with blood flow and lead to a multitude of serious cardiovascular diseases. These are the leading causes of mortality. Indeed, studies conducted by the World Health Organisation in 2003, showed that heart attacks accounted for 30% of the total mortality rate in the world. To treat blood clots and prevent the damage they cause, doctors use anticoagulants, which are com- monly called blood thinners, to decrease the clotting power of the blood and prevent growth of a clot. The most common antico- agulants used today are unfractionated heparin, low molecular weight heparin, and warfarin. Therapy with heparin prevents the extension and subsequent growth of a developing thrombus (Chiu, Hirsh, Yung, Regoeczi, & Gent, 1977). However, it does not demon- strate complete efficacy in all patients (Agnelli, Pascucci, Cosmi, & Nenci, 1990). Therefore, research and development to find new antithrom- botic agents (or drugs, or molecules) with reduced side risks is nec- essary for the prevention of thromboembolic events. Several studies in the past few decades have established that bioactive peptides, beyond their nutritional value, exhibit biologi- cal activities, such as opioid (Bitri, 2004), antioxidant (Bougatef et al., 2010), antihypertensive (Balti, Nedjar-Arroume, Guillochon, & Nasri, 2010), etc., and may therefore serve therapeutic roles in the body (Erdman, Cheung, & Schröder, 2008). These peptides have a size of 2–20 amino acids (Meisel & FitzGerald, 2003). Based on the sequence of amino acids, these bioactive peptides exhibit di- verse activities on the digestive system, the body’s defences (e.g., antimicrobial or immunomodulatory effect), cardiovascular sys- tem (including antithrombotic and antihypertensive effects), and/ or nervous system (such as a sedative and analgesic effect of opi- oid-like peptides) by inhibition of the key factors of these systems (enzyme, coenzyme, etc.). Bioactive peptides, which are inactive within the sequence of the parent proteins, can be released by enzymatic hydrolysis, 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.101 ⇑ Corresponding author. Tel.: +216 74 274 088; fax: +216 74 275 595. E-mail addresses: moncef.nasri@enis.rnu.tn, mon_nasri@yahoo.fr (M. Nasri). Food Chemistry 133 (2012) 835–841 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem