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.

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SPECIFIC AIMSAccording to folk wisdom, milk intake improves sleeping or has a calming role, a belief supported by some scientific studies. As caseins contain peptides with physiological roles, we hypothesized that one of their peptides can carry this anxiolytic activity. In this study, ␣ s1 -casein tryptic hydrolysate (␣ s1 -CnTH) was tested in vitro on benzodiazepine (BDZ) receptors and checked in vivo for its anticonvulsant and anxiolytic effects in the rat. PRINCIPAL FINDINGS ␣ s1 -CnTH inhibits pentylenetetrazole (PTZ) -induced seizures in Wistar ratBovine ␣ s1 -CnTH was injected intraperitoneally (i.p.) in a Wistar rat 30 min before an i.p. injection of 60 mg/kg of PTZ to evaluate anticonvulsant activity. Crisis severity parameters, crisis latency, and clonus duration were determined after 45 min observation of behavior. The i.p. injection of 1 mg/kg of ␣ s1 -CnTH reduced crisis severity (PϽ0.02). Other parameters were not significantly different from the control. The i.p. injection of 3 mg/kg of ␣ s1 -CnTH increased crisis latency (PϽ0.005), decreased crisis severity (PϽ0.002), and decreased clonus duration (PϽ0.005) of PTZ-induced seizures. The anticonvulsant action of the ␣ s1 -CnTH was underestimated because of a sensitization of animals to PTZ during successive experiments. ␣ s1 -CnTH displays an anxiolytic activity in the elevated plus maze paradigmElevated plus maze test was used to evaluate the anxiolytic effect of the ␣ s1 -CnTH. Diazepam (1 mg/kg) and ␣ s1 -CnTH (3 mg/kg) enhanced (PϽ0.02) the percentage of entries in the open arms whereas general activity, represented by entries in closed arms, was not modified (Fig. 1). ␣ s1 -CnTH displays an anxiolytic activity in the conditioned defensive burying (CDB) experimentWhen a rat is shocked once through an electrical probe mounted on a wall of a test cage, it returns to the probe and buries it with bedding material from the floor of the cage. This CDB response is reduced by anxiolytic drugs. Some behavioral sequences such as exploratory approaches to the probe and escape movements from the probe are needed to differentiate agonists from partial inverse agonists. Similar to diazepam, the ␣ s1 -CnTH, administrated at 3 mg/kg i.p., decreased the duration of probe burying (PϽ0.005) (Fig. 2). The ratio of the retreats to the approaches decreased in rats treated with ␣ s1 -CnTH or diazepam. The CDB paradigm indicates that the ␣ s1 -CnTH exerts an anxiolytic activity over rats. A peptide (␣-casozepine) of the ␣ s1 -CnTH binds on the BDZ site of the GABA A receptorAffinity for the BDZ site of the GABA A receptor from bovine cerebral cortex membranes was measured in competition with [methyl-3 H]-flunitrazepam. ␣ s1 -CnTH competed with the radioligand for binding on the BDZ site of the GABA A receptor with an IC 50 of 72 1 To read the full text of this article, go to http://www. fasebj.org/cgi/doi/10.1096/fj.00 -0685fje; to cite this article, use FASEB J.

Angiotensin-I-converting enzyme (ACE) inhibitory activity of a tryptic digest of bovine

The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, presented strong identities with regions of alpha(s1)-CN primary structures of other species. In particular, 51, 48, 43, and 40% identities were obtained with corresponding regions of sow, dromedary, cow, and human alpha(s1)-CN, respectively. On the other hand, trace amounts of equine gamma-CN-like and proteose peptone component 5-like peptides were found in the whole CN. They were identified by microsequencing and corresponded to beta-CN peptides generated by plasmin action on the whole CN. The equine alpha(s1), beta-, and kappa-CN were separated by bidimensional electrophoresis in numerous isoelectric variants with apparent isoelectric points distributed between pH 4.4 to 6.3, 4.4 to 5.9, and 3.5 to 5.5, respectively. The beta- and kappa-CN displayed a more acidic character in the mare than in the cow.

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beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.