The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt ؉) and-negative (Prt ؊) strains of Lactococcus lactis. The Prt ؊ strains showed monophasic exponential growth, while the Prt ؉ strains grew in two phases. The first growth phases of the Prt ؉ and Prt ؊ strains were the same, and no hydrolysis of casein was observed. Also, the levels of consumption of amino acids and peptides in the Prt ؉ and Prt ؊ strains were similar. At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth. The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth. Oligopeptide transport-deficient strains (Opp ؊) of L. lactis were unable to utilize oligopeptides and grew poorly in milk. However, a di-and tripeptide transport-deficient strain (DtpT ؊) grew exactly like the wild type (Opp ؉ DtpT ؉) did. These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase. In the second phase of growth of Prt ؉ strains, milk proteins are hydrolyzed to peptides by the proteinase. Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E.

Summary -Camembert cheeses were made at 3 different times with milk containing initially 10', 103 or 105 Listeria monocytogeneslml.A nisin-producing starter composed of a pair of isogenic protease positive and protease negative strains of Lactococcus lactis subsp lactis was used to inhibitNisin concentration in curd and in cheese paralleled the growth of lactococci. Maximum nisin concentration of ca 700 IU/g was obtained in curd at 9 h, then nisin concentrations decreased slowly during 9-24 h and dramatically during ripening. In the presence of nisin, the numbers of L monocytogenes decreased rapidly from 6 h to 24 h. This inhibitory effect continued until the end of the second week of ripening in the core of Camembert cheeses, leading to a reduction of 3.3 log Listerialg (average from 3 experiments) compared to the initial level in cheese milk. Thereafter, regrowth occurred in Camembert cheeses, sooner on the surface than in the interior. However, a difference of 2.4 log CFU/g between numbers of Listeria in cheese made with Nis+ and Nis-starter cultures was maintained throughout ripening (6 weeks). Nisin was particularly effective when milk contained 10 1 or 10 3 L monocytogeneslml.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di-and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-␤-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the ⌬dtpT strain as a genetic background. This mutant is unable to transport di-and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di-and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.For optimal growth in milk, lactococci depend on the presence of a proteolytic system which consists of a cell envelopelocated proteinase, several peptidases, and amino acid and peptide transport systems (20). At present, two peptide transport systems have been characterized for Lactococcus lactis (6,10,11,29). The oligopeptide transport system (Opp) mediates the ATP-driven transport of peptides with four to at least eight residues. It plays a central role in the proteolytic pathway of L. lactis, as it is essential for the accumulation of all ␤-caseinderived amino acids (10). The level of activity in the Opp system is sufficiently high to support maximal growth rates on ␤-casein, provided that leucine and histidine are present in the medium as free amino acids. The di-and tripeptide transport system (DtpT) is unique among bacterial peptide transporters, as it is encoded by a single gene and uses the proton motive force to drive the transport of relatively hydrophilic di-and tripeptides (6). Spontaneous mutations which inactivate the dtpT gene lead to defective growth of L. lactis in chemically defined medium (CDM) supplemented with a mixture of caseins as the sole source of nitrogen (24).On the basis of the observation that the transport of some di-and tripeptides is totally abolished in alanyl-␤-chloroalanine-resistant mutants of L. lactis whereas other peptides are still taken up at significant rates (26), the utilization of di-and tripeptides in mutants lacking either Opp or DtpT or both peptide transport systems was investigated. In this report, we present evidence for a third peptide transport system in L. lactis with specificity for relatively hydrophobic di-and tripeptides. MATERIALS AND METHODSBacterial strains, culture conditions, and growth media. L. lactis subsp. lactis MG1363 wild-type and isogenic mutants, i.e., the di-and tripeptide transport mut...

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A chemically defined medium for the growth of Leuconostoc mesenteroides was developed. This medium contained lactose, Mn(sup2+), Mg(sup2+), 12 amino acids, eight vitamins, adenine, uracil, and Tween 80. We showed the beneficial effect of aerobic conditions on growth and that potassium phosphate (135 mM) is a suitable buffer. The growth rate in this medium was 0.85 (plusmn) 0.10 h(sup-1) for the six strains examined, and cell densities up to 3.5 x 10(sup9) CFU/ml were attained.