Antithrombotic and angiotensin-converting enzyme inhibitory properties of peptides released from bovine casein by Lactobacillus casei Shirota Rebeca Rojas-Ronquillo a , Alma Cruz-Guerrero a , Angélica Flores-Nájera a , Gabriela Rodríguez-Serrano a , Lorena Gómez-Ruiz a , Juan Pablo Reyes-Grajeda b , Judith Jiménez-Guzmán a , Mariano García-Garibay a, c, * a Departamento de Biotecnología, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Unidad Iztapalapa. Av. San Rafael Atlixco 186, Colonia Vicentina CP 09340, Distrito Federal, Mexico b Instituto Nacional de Medicina Genómica, Periférico Sur 4809, Colonia Arenal Tepepan CP 14610, Distrito Federal, Mexico c Departamento de Ciencias de la Alimentación, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Unidad Lerma. Av. Hidalgo Poniente 46, Colonia La Estación, CP 52006, Lerma de Villada, Edo. de México, Mexico article info Article history: Received 6 January 2012 Received in revised form 4 May 2012 Accepted 15 May 2012 abstract Inhibition of angiotensin-converting enzyme (ACE) and antithrombotic properties of peptides released from bovine caseins during fermentation by Lactobacillus casei Shirota and Streptococcus thermophilus were determined. Both species released ACE inhibitory peptides, whereas only Lb. casei Shirota produced antithrombotic activity. The highest thrombin inhibitory activity, 80.7%, was observed at 27 h of fermentation by Lb. casei Shirota. Active peptides were purified by HPLC; seven peptides were obtained, and their resistance to digestive enzymes, pepsin and trypsin, was evaluated. The most active peptide even after the hydrolysis was identified as YQEPVLGPVRGPFPIIV (fragment 193e209 of b-casein): it had an inhibition efficiency ratio for ACE of 0.14%/peptide concentration (mg mL 1 ), and a thrombin inhibition efficiency ratio of 4.6%/peptide concentration (mg mL 1 ). This peptide had been previously reported as an ACE-inhibitor, but this is the first time that it is evaluated for its thrombin inhibition activity. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Fermented milk has been associated with health benefits for a long time. In the last three decades, much research has been done attributing health benefits to probiotic lactic acid bacteria used in milk fermentation. These benefits are primarily related to survival and implementation of probiotic bacteria in the gastrointestinal tract; however, beyond these benefits, others could arise from indirect effects such as the production of bioactive peptides released by lactic acid bacteria from milk proteins. Milk proteins have been widely studied as a source of bioactive peptides (Erdmann, Cheung, & Schröder, 2008; Korhonen & Pihlanto, 2006; Muro-Urista, Álvarez- Fernández, Riera-Rodriguez, Arana-Cuenca, & Téllez-Jurado, 2011). In particular, caseins contain within their structure a variety of peptides that can affect different systems: nervous, immune, nutritional and cardiovascular (Silva & Malcata, 2005); furthermore, there are sequences with two or more functions (Meisel, 2004). These peptides may be released via milk fermentation by lactic acid bacteria (Möller, Scholz-Ahrens, Roos, & Schrezenmeir, 2008), including probiotic bacteria (Shah, 2007). Lactobacillus casei is a probiotic (Perdigón, Alvarez, & Pesce de Ruiz Holgado,1991) widely recognized and used in fermented milk products; in addition to this property, human trials have demonstrated the hypotensive ability of milk fermented by this species (FitzGerald & Murray, 2006). González-González, Tuohy, and Jauregi (2011) found that milk fer- mented by Lb. casei YIT 9029 is capable of inhibiting angiotensin- converting enzyme (ACE); this is explained by the production of bioactive peptides. Rokka, Syväoja, Tuominen, and Korhonen (1997) reported bioactive peptides in milk fermented by Lactobacillus GG, and expressed that certain probiotic properties attributed to this strain could be partially explained by these peptides. Muguerza et al. (2006) reported the antihypertensive activity of milk fermented by Enterococcus faecalis, which produced peptides with angiotensin- converting enzyme inhibitory activity. The main bioactive peptides studied are those with antihyper- tensive activity (Korhonen & Pihlanto, 2006), which act as inhibi- tors of ACE. Most antihypertensive peptides are released from caseins (Saito, 2008; Silva & Malcata, 2005). Those with the highest antihypertensive activity are the tripeptides IPP and VPP, derived from b-casein, which have been found in milk fermented by * Corresponding author. Tel.: þ52 728 282 2785. E-mail address: jmgg@xanum.uam.mx (M. García-Garibay). Contents lists available at SciVerse ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj 0958-6946/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2012.05.002 International Dairy Journal 26 (2012) 147e154