Characterization of an antihypertensive peptide from an Alfalfa white protein hydrolysate produced by a continuous enzymatic membrane reactor Romain Kapel, Elhassan Rahhou, Didier Lecouturier, Didier Guillochon, Pascal Dhulster * Laboratoire de Technologie des Substances Naturelles, IUT, Polytech’lille, Aile C, 59655 Villeneuve d’Ascq, France Received 6 September 2005; received in revised form 10 March 2006; accepted 10 April 2006 Abstract An industrial Alfalfa white protein concentrate hydrolysed at pilot plant scale by Delvolase 1 in a continuous enzymatic membrane reactor leads to a hydrolysate exhibiting a high ACE inhibition activity (8.8 mg/ml). This hydrolysate, fractionated by two chromatography steps (exclusion size fast protein liquid chromatography and reversed-phase high performance liquid chromatography) revealed that around 15% of the total ACE inhibition activity is due to a single ACE inhibitory peptide. This peptide identified by electrospray mass spectrometry as VW, a potent antihypertensive peptide in vivo, contained four times of that in the main Alfalfa white protein (RuBisCO). # 2006 Elsevier Ltd. All rights reserved. Keywords: Alfalfa white proteins; RuBisCO; Continuous membrane reactor; Enzymatic proteolysis; ACE inhibitory peptides; Antihypertensive peptides 1. Introduction For the last decade, many bioactive peptides derived from various food proteins were characterized. Main biological activities reported were immunomodulating [1], opioid like [2], mineral-carrying [3], diazepam like [4], antimicrobial [5] and antihypertensive activities [6]. These bioactive peptides are latent in the primary sequences of proteins and may be released during gastro-intestinal like proteolysis [7] or food processing such as cheese ripening [8] and fermentation [9]. Hydrolysates containing bioactive peptides may also be generated by controlled hydrolysis carried out in enzymatic reactor, intending to produce nutraceuticals or ingredients suitable for functional foods [10]. Food protein hydrolysis is widely performed at industrial scale by conventional batch processes. However, batch hydrolysis processes are expensive and not very productive due to the single use of high cost enzyme and enzyme inhibition by end products [11]. To overpass these disadvantages, proteolysis may be coupled with an ultrafiltration membrane. The ultrafiltration step allows separation of small peptides from high molar mass residues and retention of enzyme. Then, in continuous enzymatic membrane reactors, enzyme can be repeatedly used and the molar masses of peptides extracted are standardized by ultrafiltration membrane [12]. Furthermore, continuous enzymatic membrane reactor can be considered as a convenient process to produce nutraceuticals from food protein since the generation of bioactive peptides as opioid [13], ACE inhibition [10] or mineral carrier [14] were successfully reported in such processes. The angiotensin I-converting enzyme (E.C. 3.4.15.1; ACE) is the key regulation enzyme of arterial blood pressure since it both converts inactive angiotensin I into potent vasoconstrictor octapeptide angiotensine II and degrades the vasodilatator peptide bradykinin. Chemical ACE inhibitors as Captopril 1 , were then designed to be used as antihypertensive drugs. Such treatments are now widely administrated with success, but these chemical molecules may provoke undesirable side effects such as cough, lost of taste and renal impairment. For one decade, many in vitro ACE inhibitory peptides were characterized in various food protein hydrolysates. Interest- ingly, some hydrolysates derived from plant food proteins as rapeseed [6], soy [15] or chickpea legumin [16] proteins were reported to contain ACE inhibitory peptides. The plant origin, nutritional properties and biological activities taken all together, make these hydrolysates potentially suitable for a nutraceutical used to prevent hypertension. www.elsevier.com/locate/procbio Process Biochemistry 41 (2006) 1961–1966 * Corresponding author. E-mail address: pascal.dhulster@univ-lillel.fr (P. Dhulster). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.04.019