The conversion of amino acids into volatile and nonvolatile compounds by lactic acid bacteria in cheese is thought to represent the rate-limiting step in the development of mature flavor and aroma. Because amino acid breakdown by microbes often entails the reversible action of enzymes involved in biosynthetic pathways, our group investigated the genetics of amino acid biosynthesis in Lactobacillus helveticus CNRZ 32, a commercial cheese flavor adjunct that reduces bitterness and intensifies flavor notes. Most lactic acid bacteria are auxotrophic for several amino acids, and L. helveticus CNRZ 32 requires 14 amino acids. The reconstruction of amino acid biosynthetic pathways from a draft-quality genome sequence for L. helveticus CNRZ 32 revealed that amino acid auxotrophy in this species was due primarily to gene absence rather than point mutations, insertions, or small deletions, with good agreement between gene content and phenotypic amino acid requirements. One exception involved the phenotypic requirement for Asp (or Asn), which genome predictions suggested could be alleviated by citrate catabolism. This prediction was confirmed by the growth of L. helveticus CNRZ 32 after the addition of citrate to a chemically defined medium that lacked Asp and Asn. Genome analysis also predicted that L. helveticus CNRZ 32 possessed ornithine decarboxylase activity and would therefore catalyze the conversion of ornithine to putrescine, a volatile biogenic amine. However, experiments to confirm ornithine decarboxylase activity in L. helveticus CNRZ 32 by the use of several methods were unsuccessful, which indicated that this bacterium likely does not contribute to putrescine production in cheese.Flavor development in Cheddar and other bacterium-ripened cheeses is a dynamic and complex biochemical process that requires lactic acid bacteria (LAB) and enzymes. The LAB that contribute to this process include deliberately added starter cultures and adjunct cultures as well as nonstarter LAB that enter the cheese through the milk or the processing environment. Collectively, these microbes influence flavor development through several basic mechanisms that include lactose fermentation, conversion of milk proteins (primarily caseins) into peptides and free amino acids, catabolism of amino acids into volatile aroma compounds, lipase/esterase activity, and citrate catabolism (6). In particular, the conversion of amino acids into volatile and nonvolatile compounds by LAB in cheese is thought to represent the rate-limiting step in the development of mature flavor and aroma (28). The conversion of amino acids into volatile cheese flavor compounds in cheese may be catalyzed by starter, adjunct, and nonstarter cultures of LAB and may also occur via biochemical interactions between different bacteria (13). The basis for culture interactions is not fully understood, but LAB are typically auxotrophic for several amino acids, and amino acid breakdown by LAB often involves the reversible action of enzymes involved in anabolic pathways (28). Thus...
Catabolism of sulfur-containing amino acids plays an important role in the development of cheese flavor. During ripening, cystathionine -lyase (CBL) is believed to contribute to the formation of volatile sulfur compounds (VSCs) such as methanethiol and dimethyl disulfide. However, the role of CBL in the generation of VSCs from the catabolism of specific sulfur-containing amino acids is not well characterized. The objective of this study was to investigate the role of CBL in VSC formation by Lactobacillus helveticus CNRZ 32 using genetic variants of L. helveticus CNRZ 32 including the CBL-null mutant, complementation of the CBL-null mutant, and the CBL overexpression mutant. The formation of VSCs from methionine, cystathionine, and cysteine was determined in a model system using gas chromatography-mass spectrometry with solid-phase microextraction. With methionine as a substrate, CBL overexpression resulted in higher VSC production than that of wild-type L. helveticus CNRZ 32 or the CBL-null mutant. However, there were no differences in VSC production between the wild type and the CBL-null mutant. With cystathionine, methanethiol production was detected from the CBL overexpression variant and complementation of the CBL-null mutant, implying that CBL may be involved in the conversion of cystathionine to methanethiol. With cysteine, no differences in VSC formation were observed between the wild type and genetic variants, indicating that CBL does not contribute to the conversion of cysteine.Lactobacillus helveticus CNRZ 32 is used as a starter and an adjunct bacterium to enhance cheese flavor development and reduce bitterness (15). Catabolism of amino acids, including sulfur-containing amino acids, by lactic acid bacteria is a major contributor to the development of flavor compounds in cheese during ripening (8). Sulfur-containing amino acids, namely, methionine, are precursors of aroma-active volatile sulfur compounds (VSCs) such as methanethiol, dimethyl disulfide, and dimethyl trisulfide (12, 16). There are two different microbial pathways potentially leading to amino acid conversion into flavor compounds: one is initiated by a transamination reaction while the other is initiated by an elimination reaction (29). The transamination pathway is catalyzed by aminotransferases, which transfer the amino acid amino group to an ␣-keto acid, while the elimination reaction-based pathway is catalyzed by the activity of amino acid lyases which cleave amino acid side chains (29).Cystathionine -lyase (CBL) (EC 4.4.1.8) is a pyridoxal-5Ј-phosphate (PLP)-dependent enzyme and was purified and cloned from Lactococcus lactis (1,14). CBL is involved in the ␣,-elimination of cystathionine to form homocysteine, pyruvate, and ammonia. CBL from L. lactis can also catalyze the conversion of methionine into methanethiol (1). A CBL overexpression variant of L. lactis has been shown to produce higher quantities of VSCs with methionine as a substrate compared with a wild strain (14), suggesting a possible mechanism to increase VSC product...
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