Tryptophan Catabolism by Lactobacillus casei and Lactobacillus helveticus Cheese Flavor Adjuncts

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...

Section: Discussionmentioning

confidence: 99%

Phenotypic and Genotypic Analysis of Amino Acid Auxotrophy in Lactobacillus helveticus CNRZ 32

Christiansen¹,

Hughes

Welker

et al. 2008

“…Specifically, the Phe catabolites phenylacetaldehyde and 2-phenethyl alcohol have been shown to impart floral, rose-like off-flavors, and the Tyr catabolite p-cresol imparts barny, medicinal, or utensil-like offflavors (15,22,32). Mechanisms for the production of these compounds in cheese have not been conclusively established, but AAA catabolism by lactococci and lactobacilli under simulated cheese-ripening conditions is initiated by aminotransferase (ATase) enzymes that convert AAAs into corresponding ␣-keto acids (17,20,21,39). Moreover, the aromatic ␣-keto acids produced by these reactions can be nonenzymatically converted to benzaldehyde, phenylacetaldehyde, 2-phenethyl alcohol, and other aroma compounds (17,19,20,21,38).…”

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confidence: 99%

“…Mechanisms for the production of these compounds in cheese have not been conclusively established, but AAA catabolism by lactococci and lactobacilli under simulated cheese-ripening conditions is initiated by aminotransferase (ATase) enzymes that convert AAAs into corresponding ␣-keto acids (17,20,21,39). Moreover, the aromatic ␣-keto acids produced by these reactions can be nonenzymatically converted to benzaldehyde, phenylacetaldehyde, 2-phenethyl alcohol, and other aroma compounds (17,19,20,21,38). However, many lactic acid bacteria possess hydroxy acid dehydrogenases (HADH) such as D-hydroxyisocaproic acid dehydrogenase (D-Hic) that convert ␣-keto acids to ␣-hydroxy acids (7,23), and these enzymes are active in cells incubated under cheese-ripening conditions (20,21).…”

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confidence: 99%

“…Moreover, the aromatic ␣-keto acids produced by these reactions can be nonenzymatically converted to benzaldehyde, phenylacetaldehyde, 2-phenethyl alcohol, and other aroma compounds (17,19,20,21,38). However, many lactic acid bacteria possess hydroxy acid dehydrogenases (HADH) such as D-hydroxyisocaproic acid dehydrogenase (D-Hic) that convert ␣-keto acids to ␣-hydroxy acids (7,23), and these enzymes are active in cells incubated under cheese-ripening conditions (20,21). Since ␣-hydroxy acids do not make a significant contribution to flavor development (38), this class of enzymes could be useful in controlling off-flavor development via their ability to divert the spontaneous degradation of AAA-derived ␣-keto acids to more-stable enzymatic products.…”

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confidence: 99%

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Overexpression of Lactobacillus casei d -Hydroxyisocaproic Acid Dehydrogenase in Cheddar Cheese

Broadbent¹,

Gummalla²,

Hughes

et al. 2004

Self Cite

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that ␣-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable ␣-keto acids to more-stable ␣-hydroxy acids via the action of ␣-keto acid dehydrogenases such as D-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that ␣-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of D-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of ␣-keto acids, but sensory work indicated that this effect retarded cheese flavor development.Microbial catabolism of amino acids generated from the degradation of milk proteins during cheese maturation is an essential and rate-limiting step in the development of cheese flavor and aroma properties (34, 38). Many of these reactions impact cheese flavor in beneficial ways. For example, the conversion of Met to methional, dimethyl sulfide, methanethiol, and other sulfur-containing compounds is thought to be essential for aroma development in many cheese varieties (35). On the other hand, compounds derived from the catabolism of aromatic amino acids (AAAs) have been implicated in the development of cheese off-flavors. Specifically, the Phe catabolites phenylacetaldehyde and 2-phenethyl alcohol have been shown to impart floral, rose-like off-flavors, and the Tyr catabolite p-cresol imparts barny, medicinal, or utensil-like offflavors (15,22,32). Mechanisms for the production of these compounds in cheese have not been conclusively established, but AAA catabolism by lactococci and lactobacilli under simulated cheese-ripening conditions is initiated by aminotransferase (ATase) enzymes that convert AAAs into corresponding ␣-keto acids (17,20,21,39). Moreover, the aromatic ␣-keto acids produced by these reactions can be nonenzymatically converted to benzaldehyde, phenylacetaldehyde, 2-phenethyl alcohol, and other aroma compounds (17,19,20,21,38). However, many lactic acid bacteria possess hydroxy acid dehydrogenases (HADH) such as D-hydroxyisocapr...

“…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.…”

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confidence: 99%

Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids

Hélinck

Bars

Moreau³

et al. 2004

105

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.