Bacterial metabolism of Tyr and Phe has been associated with the formation of aromatic compounds that impart barny-utensil and floral off-flavors in cheese. In an effort to identify possible mechanisms for the origin of these compounds in Cheddar cheese, we investigated Tyr and Phe catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under simulated Cheddar cheese-ripening (pH 5.2, 4% NaCl, 15 degrees C, no sugar) conditions. Enzyme assays of cell-free extracts indicated that L. casei strains catabolize Tyr and Phe by successive, constitutively expressed transamination and dehydrogenation reactions. Similar results were obtained with L. helveticus strains, except that the dehydrogenase enzymes were induced during incubation under cheese-ripening conditions. Micellar electrokinetic capillary chromatography of supernatants from L. casei and L. helveticus strains incubated under simulated cheese-ripening conditions confirmed that Tyr and Phe transamination and dehydrogenation pathways were active in both species and also showed these reactions were reversible. Major products of Tyr catabolism were phydroxy phenyl lactic acid and p-hydroxy phenyl acetic acid, while Phe degradation gave rise to phenyl lactic acid, phenyl acetic acid, and benzoic acid. However, some of these products were likely formed by nonenzymatic processes, since spontaneous chemical degradation of the Tyr intermediate p-hydroxy phenyl pyruvic acid produced p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, and p-hydroxy benzaldehyde, while chemical degradation of the Phe intermediate phenyl pyruvic acid gave rise to phenyl acetic acid, benzoic acid, phenethanol, phenyl propionic acid, and benzaldehyde.

Microbial degradation of Trp is thought to promote the formation of aromatic compounds that impart putrid, fecal, or unclean flavors in cheese, but pathways for their production have not been established. This study investigated Trp catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under carbohydrate starvation (pH 6.5, 30 or 37°C, no sugar) and near cheese-ripening (pH 5.2, 4% NaCl, 15°C, no sugar) conditions. Enzyme assays of cell-free extracts indicated that both species of Lactobacillus catabolized Trp to indole-3-lactic acid, and micellar electrokinetic capillary chromatography of culture supernatants showed this reaction occurred via successive transamination and dehydrogenation reactions. Tryptophan decarboxylase activity was also detected in all Lactobacillus cell-free extracts, but tryptamine was not detected in culture supernatants. Micellar electrokinetic capillary chromatography showed that Trp metabolism in Lactobacillus casei LC301 and LC202 was similar under both incubation conditions and that those catabolic reactions were reversible (i.e., conversion of indole-3-lactic acid to Trp). In contrast, Trp catabolism by Lactobacillus helveticus LH212 was only detected under near cheese ripening conditions. Cells of Lactobacillus helveticus CNRZ32 did not catabolize Trp in either condition but did convert indole-3-pyruvic acid to Trp in carbohydrate starvation medium and to Trp and indole-3-lactic acid under near cheese ripening conditions. (Key words: Lactobacillus, amino acid catabolism, tryptophan, cheese flavor) Abbreviation key: A = absorptance (used with number indicating wavelength), AAA = aromatic AA, ATase = aminotransferase, CDM = chemically defined AA medium, CFE = cell-free extract, CS = carbohydrate starvation conditions, DCOOHase = decarbox- Received February 10, 1999. Accepted May 24, 1999 ylase, IAA = indole-3-acetic acid, ILA = indole-3-lactic acid, ILDHase = indole-3-lactic acid dehydrogenase, IPyA = indole-3-pyruvic acid, MECC = micellar electrokinetic capillary chromatography, NADH = reduced NAD, NCR = near cheese-ripening conditions.

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...

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Food processors face serious challenges due to Listeria monocytogenes contamination. Environmental monitoring is used to control L. monocytogenes from the processing environment. Although frozen foods do not support the growth of L. monocytogenes, the moist and cold conditions in frozen food production environments are favorable for growth of L. monocytogenes. The purpose of the study was to determine the current state of awareness and practices applied across a variety of frozen food facilities related to environmental monitoring for Listeria. A survey tool was created to elicit information on existing environmental monitoring programs within the frozen food industry. The topics included cleaning and sanitizing applications and frequency, microbiological testing, and environmental areas of concern. The survey was reviewed by academic and industry experts with knowledge of microbiology and frozen food processing and was field tested by industry personnel with extensive knowledge of environmental monitoring. The survey was distributed and analyzed electronically via Qualtrics among 150 frozen food contacts. Data were gathered anonymously with a response rate of 31% (n = 46). The survey indicated that facilities are more likely to test for Listeria spp. in environmental monitoring zones 2 to 4 (nonfood contact areas) on a weekly basis. The major areas of concern in facilities for finding Listeria-positive results are floors, walls, and drains. At the time of the survey, few facilities incorporated active raw material and finished product testing for Listeria; instead, programs emphasized the need to identify presence of Listeria in the processing environment and mitigate potential for product contamination. Recognition of environmental monitoring as a key component of a comprehensive food safety plan was evident, along with an industry focus to further improve and develop verification programs to reduce prevalence of L. monocytogenes in frozen food processing environments. HIGHLIGHTS