The production of fermented foods is based on the use of starter cultures, for instance lactic acid bacteria that initiate rapid acidification of the raw material. Recently, new starter cultures of lactic acid bacteria with an industrially important functionality are being developed. The latter can contribute to the microbial safety or offer one or more organoleptic, technological, nutritional, or health advantages. Examples are lactic acid bacteria that produce antimicrobial substances, sugar polymers, sweeteners, aromatic compounds, vitamins, or useful enzymes, or that have probiotic properties.

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Lactic acid bacteria as functional starter cultures for the food fermentation industry

Bacterial volatiles: the smell of small organisms

Flavour development in dairy fermentations, most notably cheeses, results from a series of (bio)chemical processes in which the starter cultures provide the enzymes. Particularly the enzymatic degradation of proteins (caseins) leads to the formation of key-flavour components, which contribute to the sensory perception of dairy products. More specifically, caseins are degraded into peptides and amino acids and the latter are major precursors for volatile aroma compounds. In particular, the conversion of methionine, the aromatic and the branched-chain amino acids are crucial. A lot of research has focused on the degradation of caseins into peptides and free amino acids, and more recently, enzymes involved in the conversion of amino acids were identified. Most data are generated on Lactococcus lactis, which is the predominant organism in starter cultures used for cheese-making, but also Lactobacillus, Streptococcus, Propionibacterium and species used for surface ripening of cheeses are characterised in their flavour-forming capacity. In this paper, various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.

Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products

Cheese flavour formation by amino acid catabolism

Traditional cheeses: Rich and diverse microbiota with associated benefits

Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine gamma-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine beta-lyase and cystathionine gamma-lyase. The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making. Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine. Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium. Met aminotransferase activity was detected in all of the lactic acid bacteria tested and alpha-ketoglutarate was used as the amino group acceptor. In Lactococcus lactis subsp. cremoris S2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium. While no Met aminotransferase activity was detected in Brevibacterium linens BL2, it possessed high levels of L-methionine gamma-lyase that was induced by addition of Met to the growth medium. Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells. These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix.

Conversion of Methionine to Thiols by Lactococci, Lactobacilli, and Brevibacteria

Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest. Volatile sulfur compounds, specifically methanethiol, are correlated to desirable flavor in Cheddar cheese, but their definitive role remains elusive. Only recently have enzymes been found that produce this compound in bacteria associated with cheese making. Cystathionine β- and γ-lyase are found in lactic acid bacteria and are capable of producing methanethiol from methionine. Their primary function is in the metabolism of cysteine. Methionine γ-lyase produces methanethiol from methionine at a higher efficiency than the cystathionine enzymes. This enzyme is found in brevibacteria, bacilli, and pseudomonads. Addition of brevibacteria containing this enzyme improves Cheddar cheese flavor. Despite recent progress in sulfur metabolism more information is needed before cheese flavor associated with sulfur can be predicted or controlled.

Sulfur metabolism in bacteria associated with cheese

l-Methionine γ-lyase (EC 4.4.1.11) was purified to homogeneity from Brevibacterium linens BL2, a coryneform bacterium which has been used successfully as an adjunct bacterium to improve the flavor of Cheddar cheese. The enzyme catalyzes the α,γ elimination of methionine to produce methanethiol, α-ketobutyrate, and ammonia. It is a pyridoxal phosphate-dependent enzyme, with a native molecular mass of approximately 170 kDa, consisting of four identical subunits of 43 kDa each. The purified enzyme had optimum activity at pH 7.5 and was stable at pHs ranging from 6.0 to 8.0 for 24 h. The pure enzyme had its highest activity at 25°C but was active between 5 and 50°C. Activity was inhibited by carbonyl reagents, completely inactivated by dl-propargylglycine, and unaffected by metal-chelating agents. The pure enzyme had catalytic properties similar to those of l-methionine γ-lyase from Pseudomonas putida. Its Km for the catalysis of methionine was 6.12 mM, and its maximum rate of catalysis was 7.0 μmol min−1 mg−1. The enzyme was active under salt and pH conditions found in ripening Cheddar cheese but susceptible to degradation by intracellular proteases.

Purification and characterization of L-methionine γ-lyase from Brevibacterium linens BL2

L'influence du NaCl et d'une reduction du pH a ete determinee sur les aminopeptidases, les lipases/esterases, ainsi que l'aptitude a produire du methanethiol de bacteries lactiques et de brevibacteries selectionnees dans des conditions de fabrications fromageres simulees. Les observations, dans ces conditions, etaient confirmees, dans le cas du cheddar a teneur en matieres grasses reduites de 60 %. Les activites de chaque enzyme diminuaient avec l'addition de NaCl et lorsque le pH etait abaisse a des niveaux proches des conditions de fabrication du cheddar (NaCl 5 % et pH 5,2). L'activite aminopeptidasique intracellulaire residuelle etait dominee par l'activite aminopeptidasique generale (aminopeptidase N et/ou aminopeptidase C) au laboratoire, en conditions fromageres simulees, et dans un caille de cheddar a teneur en MG reduite de 60 %. Pendant le vieillissement des fromages, l'activite totale lipase/esterase atteignait un pic a 120 jours puis diminuait, meme si les populations de levains demeuraient elevees. L'aptitude a produire du methanethiol se manifestait en conditions fromageres simulees dans les cellules entieres, mais non dans les extraits depourvus de cellules. Les peptides Met et Met-contenant induisaient une aptitude a produire du methanethiol sur deux ou trois generations, et pouvaient etre reinduits ulterieurement dans le cycle de croissance de Brevibacterium linens BL2. L'activite aminopeptidase et lipase/esterase dans le fromage a teneur reduite en matiere grasse n'etait pas correlee avec une augmentation de la saveur de type cheddar, mais l'aptitude d'une culture a produire du methanethiol etait associee a des notes de preference plus elevees chez les consommateurs de fromages. Les resultats suggerent que l'utilisation de conditions fromageres simulees peut aider a la selection de cultures, pour augmenter les flaveurs souhaitees en fabrication de fromage a faible teneur en matiere grasse. De plus, les donnees suggerent que les cellules entieres sont importantes dans le developpement d'une flaveur correcte dans le cheddar a teneur en matiere grasse reduite de 60 %.

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Dias Benjamin E

Papers

Conversion of Methionine to Thiols by Lactococci, Lactobacilli, and Brevibacteria

Sulfur metabolism in bacteria associated with cheese

Purification and characterization of L-methionine γ-lyase from Brevibacterium linens BL2

Influence of NaCl and pH on intracellular enzymes that influence Cheddar cheese ripening

Production of volatile sulfur compounds in Cheddar cheese slurries