Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine ␣ S1 -casein (␣ S1 -CN) 24-47 fragment (f24-47), f169-193, and ␤-CN f58-76; ovine ␣ S1 -CN f1-6 and ␣ S2 -CN f182-185 and f186-188; caprine ␤-CN f58-65 and ␣ S2 -CN f182-187; buffalo ␤-CN f58-66; and a mixture of three tripeptides originating from human ␤-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 g/ml (100 mol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC 50 ) of some of the crude peptide fractions was very low (16 to 100 g/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC 50 s were confirmed. An antibacterial peptide corresponding to ␤-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 g/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.

Gram-positive bacteria secrete a variety of peptides that are often subjected to posttranslational modifications and that are either antimicrobials or pheromones involved in bacterial communication. Our objective was to identify peptides secreted by Streptococcus thermophilus, a nonpathogenic bacterium widely used in dairy technology in association with other bacteria, and to understand their potential roles in cell-cell communication. Using reverse-phase liquid chromatography, mass spectrometry, and Edman sequencing, we analyzed the culture supernatants of three S. thermophilus strains (CNRZ1066, LMG18311, and LMD-9) grown in a medium containing no peptides. We identified several peptides in the culture supernatants, some of them found with the three strains while others were specific to the LMD-9 strain. We focused our study on a new modified peptide secreted by S. thermophilus LMD-9 and designated Pep1357C. This peptide contains 9 amino acids and lost 2 Da in a posttranslational modification, most probably a dehydrogenation, leading to a linkage between the Lys2 and Trp6 residues. Production of Pep1357C and transcription of its encoding gene depend on both the medium composition and the growth phase. Furthermore, we demonstrated that transcription of the gene coding for Pep1357C is drastically decreased in mutants inactivated for the synthesis of a short hydrophobic peptide, a transcriptional regulator, or the oligopeptide transport system. Taken together, our results led us to deduce that the transcription of the Pep1357C-encoding gene is controlled by a new quorum-sensing system.

A great number of milk-derived peptides have been shown to exhibit angiotensin converting enzyme (ACE) inhibitory properties and thus potential utility in the regulation of blood pressure. The present work aimed to investigate the effects of 2 milk trypsin hydrolysates from alpha(S1)- and alpha(S2)-casein (CH1 and CH2, respectively) on ACE activity evaluated in human umbilical vein endothelial cells (HUVEC) in vitro, rat aortic tissues ex vivo, and renovascular hypertensive rat in vivo. Incubation of HUVEC and rat aortic tissues with CH1 or CH2 induced a concentration-dependent inhibition of hydrolysis of the ACE substrate hippuryl-histidyl-leucine (HHL), the hydrolysates being much less potent than perindopril (an ACE inhibitor). However, in contrast to perindopril, CH1 and CH2 failed to modify angiotensin I-induced aortic ring vasoconstriction. The HPLC profiles of rat plasma after intragastric administration were variable among individuals but none of the observed peaks corresponded to peptides comprising CH1 or CH2 or to fragments of these peptides. During 4 wk of cardiovascular monitoring, in hydrolysate-fed renovascular hypertensive rats, systolic blood pressure weakly decreased compared with the control group. However, the CH1-fed hypertensive rats exhibited a decrease of heart rate during the nocturnal period of activity. To conclude, our results show that CH1 and CH2 inhibited ACE activity in HUVEC and rat aortic tissue but failed to antagonize the aortic-constricting effects of the natural agonist angiotensin I. Moreover, we demonstrated that CH1, to a greater extent than CH2, can slightly affect cardiovascular parameters although the ingested bioactive peptides could not be detected in the blood.

-The proteolytic system of lactic acid bacteria has been characterised in detail and numerous modified strains with null or increased specific proteolytic activities have been constructed or identified among natural strains. Based on this knowledge, our objective was to ferment milk with modified strains and produce mixtures of peptides with specific features corresponding to potential bio-activities. We used a collection of Lactococcus lactis negative mutants for peptidase activities available in the laboratory to ferment the milk. In particular, we focused our work on mutants lacking either aminopeptidase N, X-prolyl dipeptidyl aminopeptidase or tripeptidase in order to test their ability to form peptides with immunomodulating or antihypertensive activities. At the end of fermentation, supernatants were fractionated by RP-HPLC. Each fraction collected was analysed by Maldi-Tof MS and sequencing. We observed that mutants accumulated specific peptides consistent with the specificity of the missing peptidases. Some of the peptides identified present similarities with peptides having immunomodulating or anti-hypertensive effects. One of these was quantified. At the same time, we observed that the inhibition of angiotensin converting enzyme was stronger in supernatants obtained with mutant strains than in supernatant obtained using the wild-type strain. In conclusion, we showed that in some cases, modifications to the proteolytic system of Lactococcus lactis gave rise to significant differences in the mixtures of peptides produced during milk fermentation. The differences in bio-activity of these peptide mixtures were only partially determined in vitro and evidently need to be demonstrated in vivo. Exploitation of the biodiversity of the proteolytic system of lactic acid bacteria may enable a direct application of this work and undoubtedly a promising means of directly producing natural bio-active peptides in fermented milk products. Milk fermentation / Lactococcus lactis / bio-active peptide / proteolysis / mutantRésumé -Conséquences de modifications du système protéolytique de Lactococcus lactis sur l'accumulation de peptides potentiellement bio-actifs pendant la fermentation du lait. Le système protéolytique des bactéries lactiques a été caractérisé en détail et de nombreuses souches aux activités protéolytiques modifiées ont été construites ou identifiées parmi des souches sauvages. Notre objectif est de produire des mélanges de peptides ayant des activités biologiques, en utilisant ces souches modifiées pour fermenter le lait. Pour cela, nous avons utilisé une collection de mutants de Lactococcus lactis dont certaines activités peptidasiques ont été supprimées. En particulier, nous nous sommes intéressés à des souches n'ayant plus l'aminopeptidase N, la X-prolyl-dipeptidyl aminopeptidase, ou la tripeptidase pour tester leur aptitude à former des peptides ayant des activités * Corresponding author: algaron@jouy.inra.fr 116 F. Algaron et al.