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Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an ␣-keto acid such as ␣-ketoglutarate (␣-KG) as the amino group acceptor, and produced ␣-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces ␣-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without ␣-KG addition. In the presence of ␣-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced ␣-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from ␣-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an ␣-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the ␣-keto acid conversion to acids in L. helveticus and S. thermophilus is an ␣-keto acid dehydrogenase that produces acyl coenzymes A.Amino acid catabolism by the microflora is a major process for the formation of a large number of key aroma compounds in Swiss-type cheeses such as gruyere and emmental (7,14,17). However, the amino acid catabolic pathways in the different bacteria present in these cheeses are not well known. This knowledge could lead to the development of cultures with optimized aromatic properties.Amino acid catabolism by Lactococcus lactis and mesophilic lactobacilli has been extensively studied recently (2,8,11,19,26; S. Gummalla and J. R. Broadbent, Abstract, J. Dairy Sci. 79(Suppl.

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.

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The functions necessary for bacterial growth strongly depend on the features of the bacteria and the components of the growth media. Our objective was to identify the functions essential to the optimum growth of Streptococcus thermophilus in milk. Using random insertional mutagenesis on a S. thermophilus strain chosen for its ability to grow rapidly in milk, we obtained several mutants incapable of rapid growth in milk. We isolated and characterized one of these mutants in which an amiA1 gene encoding an oligopeptide-binding protein (OBP) was interrupted. This gene was a part of an operon containing all the components of an ATP binding cassette transporter. Three highly homologous amiA genes encoding OBPs work with the same components of the ATP transport system. Their simultaneous inactivation led to a drastic diminution in the growth rate in milk and the absence of growth in chemically defined medium containing peptides as the nitrogen source. We constructed single and multiple negative mutants for AmiAs and cell wall proteinase (PrtS), the only proteinase capable of hydrolyzing casein oligopeptides outside the cell. Growth experiments in chemically defined medium containing peptides indicated that AmiA1, AmiA2, and AmiA3 exhibited overlapping substrate specificities, and that the whole system allows the transport of peptides containing from 3 to 23 residues.

Sugar fermentation was long considered the sole means of energy metabolism available to lactic acid bacteria. We recently showed that metabolism of Lactococcus lactis shifts progressively from fermentation to respiration during growth when oxygen and heme are available. To provide insights into this phenomenon, we compared the proteomic profiles of L. lactis under fermentative and respiratory growth conditions in rich medium. We identified 21 proteins whose levels differed significantly between these conditions. Two major groups of proteins were distinguished, one involved in carbon metabolism and the second in nitrogen metabolism. Unexpectedly, enzymes of the proteolytic system (PepO1 and PepC) which are repressed in rich medium in fermentation growth were induced under respiratory conditions despite the availability of free amino acids. A triple mutant (dtpT dtpP oppA) deficient in oligopeptide transport displayed normal respiration, showing that increased proteolytic activity is not an absolute requirement for respiratory metabolism. Transcriptional analysis confirmed that pepO1 is induced under respiration-permissive conditions. This induction was independent of CodY, the major regulator of proteolytic functions in L. lactis. We also observed that pepO1 induction is redox sensitive. In a codY mutant, pepO1 expression was increased twofold in aeration and eightfold in respiration-permissive conditions compared to static conditions. These observations suggest that new regulators activate proteolysis in L. lactis, which help to maintain the energetic needs of L. lactis during respiration.

SummaryA proteinase was purified from a cell wall extract of a culture ofStreptococcus lactisNCDO 763 grown in skim milk. Being active at a low pH (at pH 4·8 on haemoglobin and pH 6·–6·5 on casein) and completely inhibited by diisopropylfluorophosphate, it was considered to be a serine proteinase partly inhibited by EDTA; the mol. wt was ∼ 80000.

Aims:  To verify whether diacetyl can be produced by Lactococcus lactis via amino acid catabolism, and to investigate the impact of the pH on the conversion. Methods and Results:  Resting cells of L. lactis were incubated in reaction media at different pH values, containing l‐aspartic acid or l‐alanine as a substrate. After incubation, the amino acid and metabolites were analysed by HPLC and GC/MS. At pH 5 about 75% of aspartic acid and only 40% of alanine was degraded to pyruvate via a transamination step that requires the presence of α‐ketoglutarate in the medium, but diacetyl was only produced from aspartic acid. Three per cent of pyruvate was transformed to acetolactate of which 50% was converted into diacetyl. At pH 5·5 and above the pyruvate conversion into acetolactate was less efficient than at pH 5, and acetolactate was mainly decarboxylated to acetoin. Conclusions:  Acetoin and diacetyl can be formed as a result of aspartate or alanine catabolism by L. lactis in the presence of α‐ketoglutarate in the medium. Significance and Impact of the Study:  Lactic acid bacteria exhibiting both glutamate dehydrogenase activity and high aspartate aminotransferase activity are expected to be good diacetyl producers during cheese ripening at pH close to 5.