Variability of Hydrolysis of β-, α s1 -, and α s2 -Caseins by 10 Strains of Streptococcus thermophilus and Resulting Bioactive Peptides Laurent Miclo, ∥ E ́ meline Roux, ∥ Magali Genay, E ́ milie Brusseaux, Chantal Poirson, Nawara Jameh, Clarisse Perrin, and Annie Dary* Unite ́ de Recherche “Animal et Fonctionnalite ́ s des Produits Animaux” (UR AFPA), E ́ quipe “Prote ́ olyse et Biofonctionnalite ́ s des Prote ́ ines et des Peptides” (PB2P), Nancy-Universite ́ , 54506 Vandœuvre-le ̀ s-Nancy, France ABSTRACT: Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the cell envelope proteinase, PrtS, were incubated with α s1 -, α s2 -, or β-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the β- casein was preferentially hydrolyzed, followed by α s2 -casein and then α s1 -casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1−23 of α s1 -casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from β-casein, 5 from α s2 -casein, and 2 from α s1 -casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors. KEYWORDS: Streptococcus thermophilus, bioactive peptides, cell envelope proteinase, casein hydrolysis ■ INTRODUCTION In recent years, a link between the prevalence of certain diseases commonly encountered in Western countries, such as cardiovascular diseases, obesity, diabetes, cancer, and dietary factors, has been emphasized. The engineering and manufactur- ing of functional ingredients and foods is emerging to counter these pathologies. Among them, fermented dairy products have received much attention because they contain several compounds, such as bioactive peptides, obtained by milk protein hydrolysis. 1,2 Bioactive peptides are encrypted within many dietary protein sequences and, when released by various proteases, exercise physiological functions in the human body with a wide range of potential applications (heart, bone, dental, and digestive health; weight management; immunomodulation; mood swings, memory, and stress control). 3 Bioactive peptides described in the literature have been released from dietary proteins either by enzymes of the digestive tract such as trypsin or by the proteolytic system of microorganisms during food manufacture. 3,4 Numerous studies deal with the capacity of lactococci or lactobacilli to generate bioactive peptides from milk proteins during fermentation. 3 These studies were carried out on pure cultures of species such as Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. cremoris, Lb. acid- ophilus, Lb. casei, Lb. jensenii, Lb. reuteri, Lb. rhamnosus, Lc. lactis ssp. lactis, Lc. raffinolactis, Leuconostoc mesenteroides ssp. cremoris, and numerous strains of Lb. helveticus or cocultures with industrial yogurt starters (Lb. delbrueckii ssp. bulgaricus and Streptococcus thermophilus). 5−9 Although it belongs to the Streptococcus genus, S. thermophilus is classified as “Generally Recognized As Safe”. 10 It is the second lactic acid bacterium (LAB), after Lc. lactis, in terms of industrial use. 11 S. thermophilus, having strong acidifying capacities, plays an important role in the initial stages of dairy product manufacture. To our knowledge, very few studies have proved the ability of this bacterium to produce bioactive peptides from milk proteins, perhaps because its proteolytic capacity was considered to be very weak. In particular, 16 peptides were characterized in a study on milk proteolysis by pure or mixed cultures of Lb. delbrueckii subsp. bulgaricus and S. thermophilus. 12 Two of these peptides correspond to potential angiotensin-converting enzyme inhib- itors. The proteolytic system of S. thermophilus consists of a facultative cell envelope proteinase (CEP) named PrtS, an ABC-transporter of oligopeptides and di/tripeptide transporter, and a pool of intracellular peptidases. 13 It has long been believed that the high cellular density of S. thermophilus in milk depends upon its cocultivation with other bacteria such as Lb. bulgaricus during the manufacture of fermented dairy products; 14 with this assumption in mind, the screening of 97 strains of the collection of the Institut National de la Recherche Agronomique (INRA, France) showed that only 3 strains were PrtS + and probably able to grow at high density in milk because they were able to strongly acidify it during their growth. 15 However, recent publications report the presence of the CEP PrtS in numerous strains. 16−18 In a previous work, we clustered into 3 groups 30 strains of our laboratory collection on the basis of their ability to acidify milk during their growth: high (H)-, low (L)-, and medium (M)-acidifying strains. 16 We showed that all of the H-strains possess the prtS gene (prtS + genotype) and expressed it (PrtS + phenotype), whereas the L- strains were prtS − . For the M-strains, the situation was more complex. Indeed, 62% had a prtS + genotype and displayed a Received: June 1, 2011 Revised: November 20, 2011 Accepted: November 21, 2011 Published: November 21, 2011 Article pubs.acs.org/JAFC © 2011 American Chemical Society 554 dx.doi.org/10.1021/jf202176d | J. Agric.Food Chem. 2012, 60, 554−565