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

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 γ-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 β-lyase and cystathionine γ-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 α-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 γ-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.The primary classes of compounds that contribute to cheese flavor include amino acids and their degradation products, peptides, carbonyl compounds, and fatty acids. These partition primarily into the aqueous fraction of cheese (3). The volatile fraction of cheese has sulfur-containing compounds such as methanethiol, methional, dimethyl sulfide, dimethyl tetrasulfide, carbonyl sulfide, and hydrogen sulfide (28), and they contribute to the aroma of cheese (7). Methanethiol has been associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2), and it is also implicated as an influential aroma and flavor compound in many foods, including surface-ripened cheeses that use brevibacteria (17). However, methanethiol, when present alone, does not contribute to typical Cheddar-like flavor notes in cheese (17).Production of methanethiol is important in cheese, but the Met biosynthetic and catabolic pathways vary among bacteria (23). The mechanisms involved and amounts of methanethiol produced during cheese ripening also vary. In an effort to increase and accelerate the development of typical Cheddar cheese flavor, adjunct bacteria have been used during the manufacture of low- and full-fat cheese. Initial selection of flavor adjunct cultures focused on those bacteria used to accelerate flavor development in full-fat cheese, which are typically lactobacilli because they dominate (10⁷ to 10⁹ CFU/g of cheese during storage at 8°C) the microflora during aging (15). The Lactobacillus genus is considered to be a member of the nonstarter lactic acid bacteria subgroup because it is not added with the starter culture for Cheddar cheese. In addition to lactobacilli, micrococci and pediococci have been used as adjunct bacteria to aid in flavor development (20). Brevibacteria, which are normally found on the surfaces of Limburger and other Trappist-type cheeses, are not traditionally used as flavor adjuncts in Cheddar cheese. One advantage these organisms have over other adjuncts is their profuse production of methanethiol (8). Weimer et al. (30) successfully used Brevibacterium linens as an adjunct to improve the flavor of low-fat Cheddar cheese.The mechanism for the production of methanethiol in cheese by bacteria can be a result of the direct catabolism of Met or it can arise from inadvertent catalysis by other enzymes (1, 6, 17). The most direct route to methanethiol is the conversion of Met to methanethiol, ammonia, and α-ketobutyrate (Fig. ​(Fig.1).1). This transformation is catalyzed by inducible Met γ-lyase, a pyridoxal phosphate (PLP)-dependent enzyme (24) which has been purified to homogeneity from Pseudomonas putida (14, 26), Aeromonas spp. (27), and Clostridium sporogenes (16) and partially purified from B. linens (6). Open in a separate windowFIG. 1Metabolic pathways for Met interconversion. The primary intermediates and enzymes are listed. Enzyme 1 is cystathionine γ-lyase, enzyme 2 is cystathionine β-lyase, enzyme 3 is cystathionine β-synthase, enzyme 4 is homocysteine methyltransferase, enzyme 5 is aromatic aminotransferase (tyrB) or transaminase B (ilvE), enzyme 6 is amino acid oxidase, enzyme 7 is Met adenosyltransferase, and enzyme 8 is Met γ-lyase (adapted from reference 18 and 23).Pathways leading away from Met are important to consider because this amino acid is central to many other critical metabolic functions (Fig. ​(Fig.1).1). Utilization of Met for other metabolic functions would lower the pool of Met available for conversion to methanethiol. Methionine adenosyltransferase (S-adenosylmethionine [SAM] synthetase) converts Met into SAM at the expense of one ATP. SAM, one of the major methylating agents in a cell, is also important in the regulation of several of the Met-biosynthetic enzymes (22). Reduced SAM synthetase activity leads to low intracellular levels of SAM, resulting in the induction of the Met-biosynthetic pathway (32).Another mechanism that directs Met away from methanethiol is the deamination reaction to form α-keto γ-methyl thiobutyrate (KMTB). This conversion can be catalyzed by various aminotransferases (33) or amino acid oxidases (21). These enzymes are common in bacteria and are usually the last step in amino acid synthetic pathways (13). Amino acid oxidase activity is a possible route for KMTB production, and it is a possible route for subsequent methanethiol production in cheese, but this is unlikely because cheese tends to be anaerobic. Evidence for the conversion of KMTB to methanethiol is lacking for bacteria; however, this reaction has been shown to take place enzymatically in fungal species (23).When the catabolic pathways for Met are considered, the enzymes involved in the biosynthesis of Met must also be included. Although the principal reactions that these enzymes catalyze are involved in the synthesis of Met, they also coincidentally catalyze catabolic reactions that lead to the production of methanethiol and possibly other cheese flavor compounds. For example, cystathionine β-lyase, which primarily catalyzes the conversion of cystathionine to homocysteine, a reaction involved in the synthesis of Met (29), also catalyzes the conversion of Met to methanethiol, ammonia, and α-ketobutyrate but with 100 times less efficiency than that of its conversion to homocysteine in Lactococcus lactis subsp. cremoris S2 (1). This enzyme was purified from lactococci and has been implicated in the generation of methanethiol in Cheddar cheese (1). Cystathionine γ-lyase catalyzes the α,γ elimination of cystathionine to produce cysteine (Cys), α-ketobutyrate, and ammonia (19). A cystathionine γ-lyase purified from L. lactis subsp. cremoris is capable of catalyzing the α,γ elimination of Met to produce methanethiol at an efficiency much lower than that of the primary reaction it catalyzes (4). These enzymes may be present in the cells and liberated when the cells die and lyse during cheese storage, as occurs in Cheddar cheese ripening (10). With these observations in mind, the objective of this study was to determine the conversion pathways of Met to free thiols under laboratory and cheese-like conditions in bacteria used as starter cultures and flavor adjuncts in Cheddar cheese.