Bioactive peptides from caseins released by cold active proteolytic enzymes from Arsukibacterium ikkense Cristian De Gobba a,⇑ , Gorazd Tompa b , Jeanette Otte a a Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg C, Denmark b Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, 1230 Domz ˇale, Slovenia article info Article history: Received 25 November 2013 Received in revised form 7 April 2014 Accepted 15 May 2014 Available online 23 May 2014 Keywords: Caseins Hydrolysates ACE-inhibitory activity Antioxidant Cold active enzymes abstract Proteolytic enzymes secreted by the cold-adapted microorganism Arsukibacterium ikkense were tested for their ability to degrade caseins at low temperature and produce bioactive peptides. The caseins were extensively degraded (90%) after 24 h of hydrolysis at 5 °C and completely degraded at 25 °C, and many novel peptides were formed. The most hydrolysed sample showed high angiotensin I converting enzyme (ACE)-inhibitory and antioxidant activity, and a number of potent ACE-inhibitory and antioxidant pep- tides were identified. The presence of tyrosine seemed fundamental for both ACE-inhibitory and antiox- idant activity, while phenylalanine seemed to potentiate the antioxidant activity. The novel peptide YPELF was found to have strong radical scavenging and lipid oxidation inhibitory activities, with IC 50 for both around 3.5 lM. None of the hydrolysates showed antimicrobial activity. Secreted enzymes from cultures of A. ikkense could thus be a valuable enzyme preparation for inexpensive, energy-efficient pro- duction of potent bioactive peptides from caseins in milk at low temperatures. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Food proteins not only provide amino acids for growth and maintenance, they are also a source of physiologically active pep- tide sequences, which, when liberated, exert beneficial effects in the gastrointestinal tract or, after absorption, at target sites in the body (Möller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008). Among the many food proteins shown to be precursors of bioactive peptides, milk proteins are the most well studied (Nagpal et al., 2011). Peptides with activities such as anti-hypertensive, opioid, immune-modulatory, antimicrobial and antioxidant have been released from caseins and whey proteins through microbial fer- mentation or enzymatic hydrolysis in vitro, or during digestion of such (partly hydrolysed) proteins (Contreras, Carron, Montero, Ramos, & Recio, 2009; Hernández-Ledesma, García-Nebot, Fernández-Tomé, Amigo, & Recio, in press; Mao, Cheng, Wang, & Wu, 2011; Nagpal et al., 2011; Phelan, Aherne, FitzGerald, & O’Brien, 2009). Caseins have been shown to be the overall best milk protein substrate for release of peptides inhibiting angiotensin I converting enzyme (ACE), an enzyme involved in hypertension (Espejo-Carpio, De Gobba, Guadix, Guadix, & Otte, 2013; Otte, Shalaby, Zakora, Pripp, & El-Shabrawy, 2007). Use of proteolytic enzyme preparations to improve or modify food is an energy-efficient biotechnological tool. In vitro enzymatic hydrolysis of milk proteins with commercial proteolytic enzymes, from plant, animal and bacterial sources, such as papain, trypsin, pepsin and subtilisin, have been performed for up to 24 h at 30–65 °C according to the optimum activity of the enzyme (Espejo-Carpio et al., 2013; Jiang, Chen, Ren, Luo, & Zeng, 2007; Otte et al., 2007). These conditions lead to risk of spoilage due to bacterial growth, creating concern on food safety for consumers and food producers. Moreover, these reactions demand high energy input in order to keep high temperatures, increasing the cost of the overall process. Therefore, it would be a great advantage to have access to enzymes that are active at lower temperatures. Interest in cold-active enzymes is increasing not only within the food industry, but also within the biotechnological and pharma- ceutical industries. Cold-active protease producing microorganisms have been iso- lated from different cold locations on earth, such as mountain soil, glacier ice and deep sea (Joshi & Satyanarayana, 2013). Enzymes secreted from such microorganisms generally exhibit high catalytic efficiency at low temperature and are easily inactivated by a moderate temperature increase (Kim et al., 2010). The submerged ikaite columns in the Ikka Fjord in Greenland represent one such http://dx.doi.org/10.1016/j.foodchem.2014.05.082 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +45 353 31432; fax: +45 353 33344. E-mail address: gobba@life.ku.dk (C. De Gobba). Food Chemistry 165 (2014) 205–215 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem