Use of a free form of the Streptococcus thermophilus cell envelope protease PrtS as a tool to produce bioactive peptides Oun Ki Chang a, b, 1 , Émeline Roux a, b, * , Ahoefa Ablavi Awussi a, b , Laurent Miclo a, b , Julien Jardin c, d , Nawara Jameh a, b , Annie Dary a, b , Gérard Humbert a, b , Clarisse Perrin a, b a Université de Lorraine, Unité de Recherche «Animal & Fonctionnalités des Produits Animaux» (UR AFPA), Équipe «Protéolyse & Biofonctionnalités des Protéines et des Peptides» (PB2P), Vandœuvre-lès-Nancy F-54506, France b INRA, Unité de Recherche «Animal & Fonctionnalités des Produits Animaux» (UR AFPA), USC 340, Vandœuvre-lès-Nancy F-54506, France c INRA, UMR1253 Science et Technologie du Lait et de l’Œuf, F-35042 Rennes, France d Agrocampus Ouest, UMR1253 Science et Technologie du Lait et de l’Œuf, F-35042 Rennes, France article info Article history: Received 18 September 2013 Received in revised form 13 January 2014 Accepted 14 January 2014 abstract Bioactive peptides can be produced from milk proteins in fermented products by proteases of lactic acid bacteria. The cell envelope protease (PrtS) of Streptococcus thermophilus is anchored at the cell wall, but we recently discovered that the 4F44 strain produces a soluble form that can be recovered in medium su- pernatant. This work was aimed at optimising the production of bioactive peptides from bovine caseins. By growing S. thermophilus 4F44 in the newly designed YLUNi medium, a high quantity of the soluble form of PrtS could be produced that could be directly used as the proteolytic agent on sodium caseinate. Peptide production was monitored by reverse phase-high performance liquid chromatography and tandem mass spectrometry; of 247 peptides identified, 143 were derived from b-casein. Twenty-two peptides, already reported in the literature as bioactive, include ACE-inhibitory, antioxidant, immunomodulating, or anti- bacterial peptides; addition of such peptides could improve the health benefits of dairy products. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Milk proteins are a major source of bioactive peptides, which may exert diverse activities on cardiovascular, nervous, immune or digestive systems. Therefore, they could be used as dietary sup- plements or food ingredients to promote health (Korhonen, 2009). Bioactive peptides are contained within the sequence of food pro- teins and can be released through four ways: (1) in vitro enzymatic hydrolysis using purified proteases, (2) industrial food processing, (3) in vivo hydrolysis during digestion in the stomach (Boutrou et al., 2013), and (4) proteolytic digestion by lactic acid bacteria during fermentation. Streptococcus thermophilus is a bacterium extensively used for milk fermentation for its high capacity of lactic acid production. As S. thermophilus is auxotrophic for some amino acids (Letort & Juillard, 2001), its growth in milk, which contains very few free amino acids and small peptides, depends on the ca- pacity of its proteolytic system to generate peptides from milk ca- seins. This proteolytic system is composed of PrtS, a facultative extracellular protease, anchored at the cell wall (Fernandez-Espla, Garault, Monnet, & Rul, 2000), an ABC-transporter of oligopep- tides (Garault, Le Bars, Besset, & Monnet, 2002), and a pool of intracellular peptidases (Rul & Monnet, 1997). In a study where we tested 30 strains of S. thermophilus, only the 4F44 strain was found to produce a free extracellular form of the PrtS protease in addition to the anchored form (Chang et al., 2012). Miclo et al. (2012) have previously shown that proteolytic S. thermophilus strains generate peptides of various sizes from ca- seins (CN) and PrtS is the main actor responsible for the hydrolysis of b-, a S1 - and a S2 -CN. During milk fermentation by S. thermophilus, casein proteolysis can be prematurely stopped because of the drop of pH to the value of 4.6. This results from the excretion of lactic acid, which leads to the precipitation and the subsequent inacti- vation of PrtS (pI 4.6). This undesirable sharp pH drop can be addressed by an increase of the urease activity, which is present in most S. thermophilus strains (Mora et al., 2002, 2004). Ammonia * Corresponding author. Tel.: þ33 3 83 68 49 73. E-mail address: emeline.roux@univ-lorraine.fr (É. Roux). 1 Present address: Imported Food Analysis Division, Gwangju Regional Food and Drug Administration,176-13, Chomdankwagi-ro, Buk-gu, Gwangju, Jeonnam, South Korea. Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2014.01.008 International Dairy Journal xxx (2014) 1e12 Please cite this article in press as: Chang, O. K., et al., Use of a free form of the Streptococcus thermophilus cell envelope protease PrtS as a tool to produce bioactive peptides, International Dairy Journal (2014), http://dx.doi.org/10.1016/j.idairyj.2014.01.008