No abstract

Fermented milk (FM) with putative antihypertensive effect in humans could be an easy applicable lifestyle intervention against hypertension. The mode of action is supposed to be through active milk peptides, shown to possess in vitro ACE-inhibitory effect. Blood pressure (BP) reductions upto 23 mm Hg have been reported in spontaneously hypertensive rats fed FM. Results from human studies of the antihypertensive effect are inconsistent. However, many studies suffer from methodological weaknesses, as insufficient blinding and the use of office BP measurements. We conducted a randomised, double-blind placebo-controlled study of the antihypertensive effect of Lactobacillus helveticus FM in 94 prehypertensive and borderline hypertensive subjects. The participants were randomised into three treatment groups with a daily intake of 150 ml of FM, 300 ml of FM or placebo (chemically acidified milk). The primary outcome was repeated 24-h ambulatory BP measurements. There were no statistically significant differences in the outcome between the groups (systolic BP (SBP), P ¼ 0.9; diastolic BP (DBP), P ¼ 0.2). However, the group receiving 300 ml FM had reduced BP across the 8-week period in several readings, which could be compatible with a minor antihypertensive effect. Heart rate and lipids remained unchanged between groups. Hence, our study does not support earlier studies measuring office BP-measurements, reporting antihypertensive effect of FM. Based on straight performed 24-h ambulatory BP measurements, milk fermented with Lactobacillus helveticus does not posses significant antihypertensive effect.

No abstract

Despite a known ACE inhibitory effect in vitro and in animals, milk fermented with Lb. helveticus did not inhibit ACE in humans. Our results suggest that the intake of fermented milk decreases sympathetic activity, although not to an extent mediating reductions of blood pressure and heart rate in borderline-hypertensive subjects.

The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards ␤-casein was investigated. ␤-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from ␤-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.Due to their limited capacity for synthesizing amino acids (3), lactococci have to utilize exogenous nitrogen sources for optimal growth. The amino acid requirements appear to be strain specific, but most Lactococcus lactis strains need at least Leu, Ile, Val, and His for growth (15,28). In milk, the concentrations of several essential amino acids, especially those of Ile and Leu (less than 1 mg liter Ϫ1 ), are very low (10, 18). In addition, milk peptides are a poor source of Leu and Met (16). Thus, for optimal growth in milk, lactococci depend on utilization of casein (18,29). A complex proteolytic system is needed for casein hydrolysis. According to proposed models, lactocepin (EC 3.4.21.96; previously named cell envelope proteinase PrtP) (34) is involved in the first step of casein degradation. Only some of the oligopeptides released by lactocepin are taken up by the oligopeptide transport system (Opp) and subsequently cleaved into amino acids by intracellular peptidases (for recent reviews, see references 17 and 22).Two different types of lactocepins (lactocepin I and lactocepin III) have been identified in lactococci on the basis of their specificity for caseins (44). Lactocepin I cleaves ␤-casein preferentially and -casein to a lesser extent. In contrast, lactocepin III cleaves ␤-, -, and ␣ s1 -caseins. There are only 44 differences in the amino acid sequences of lactocepins I and III, and 5 of them are responsible for the differences in specificity between the two lactocepins (7, 40).Lactocepin purification requires release of the enzyme from the cell (30), which results from autoproteolysis of the protein in a Ca 2ϩ -free buffer (24, 25). Autoproteolysis takes place in the C-terminal part of the protein, presumably in the B domain of the protein, and results in a 145-kDa enzyme, compared to the 186-kDa cell-anchored lactocepin III (1). The action of purified lactocepins towards caseins has been studied extensively (32,33,37,38). In particular, most, if not all, of the peptides released from ␤-casein by purified lactocepin I have been identified by on-line coupling of liquid c...

The influence of the type of cell envelope-located proteinase (P I versus P III) on the associative growth of Lactococcus lactis in milk was studied. Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study. An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the P I-type proteinase strain, whereas that of the P III-type proteinase strain remained unaffected. The reduction in proteolytic activity of the P I-type proteinase strain (presumably resulting from an inhibition of the synthesis of the enzyme) due to the peptides released by the P III-type proteinase was found to be partly responsible for this interaction. Extension of the study to wild-type proteinase-positive L. lactis strains showed a systematic imbalance of the mixture of the two strains in favor of the P III-type proteinase strain.

-Incidence of nutritionaI factors on the interaction between mesophilic lactic acid bacteria during growth in milk. The importance the nutritional factors have on the interactions between mesophilic lactic acid bacteria during associative growth in milk is demonstrated by analyzing three examples of mixed cultures used in cheese technology. (i) The interaction between proteinase-positive and proteinase-negative strains of Lactococcus lactis results from a competition for the utilization of casein-derived peptides. The extent of the competition depends on the balance between the two types of cells, the proteolytic activity of the proteinase-positive strain, and the type ofproteinase the strain produces. (ii) The interaction between proteinase-positive strains of L. lactis is influenced by the type of proteinase (P 1 -or Pm-type) the strains produce. A competition for the use of casein-derived peptides, and a reduction in proteolytic activity of the Pctype proteinase strain were presumably responsible for the interaction. (iii) Finally, the effects of co-culturing lactococci and leuconostocs are apparently strain-dependent. Nevertheless, a competition between both species for peptide utilization seems to be a frequent feature of such mixed cultures. These three examples emphasize the major role of the proteolytic activity of L. lactis when co-cultured in milk. © InrafElsevier, Paris. lactococci / proteinase / leuconostoc / milk / interaction Résumé -Trois exemples, inspirés des problèmes rencontrés par la technologie fromagère, illustrent l'importance des facteurs nutritionnels dans les interactions entre bactéries lactiques méso-philes constitutives d'un levain. L'interaction entre souches protéolytiques et variants non protéolytiques de lactocoques résulte d'une compétition pour les produits de dégradation des caséines, compétition dont l'intensité dépend de la proportion relative des deux types de cellules, du niveau d'activité protéolytique de la souche protéase-positive et du type de protéase qu'elle synthétise. L'interaction qui se produit entre souches protéolytiques de lactocoques est également régie par le type de protéase de paroi synthétisée. Elle semble résulter d'une compétition pour l'utilisation des produits de protéolyse, au détriment de la soucheayant une protéase de type PI' et d'une inhibition de la synthèse de cette protéase par les peptides accumulés par l'autre type de protéase pendant la culture. Enfin, contrairement aux deux exemples précédents, les interactions entre lactocoques et leuconostocs semblent dépendre des souches en présence. Néanmoins, l'existence d'une compétition entre les deux espèces pour l'utilisation des nutriments azotés est fréquente. L'ensemble de ces résultats souligne l'importance de l'activité protéolytique des lactocoques dans les phénomènes d'interactions lorsque ces microorganismes sont cultivés en association dans du lait. © Inra/Elsevier, Paris. lactocoque / protéase / leuconostoc / lait / interaction 92 V. Juillard et al.

The ability of caseins to fulfill the amino acid requirements ofLactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing αs1-, β-, and κ-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124–2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.