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

Abstract

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...

Antimicrobial activity of d-3-phenyllactic acid produced by fed-batch process against Salmonella enterica

“…1 shows a schematic overview of the metabolism. PPA can then be reduced by hydroxyl acid dehydrogenases (Broadbent et al, 2004;Gummalla & Broadbent, 2001), which results in the production of PLA, or decarboxylated into phenylacetaldehyde, which in turn can be converted to phenylethanol or phenylacetate (Vermeulen et al, 2006). Additionally, when PPA is not enzymatically converted, it may undergo a chemical oxidation to benzaldehyde (Groot & de Bont, 1998).…”

Survival of microencapsulated probiotic Lactobacillus paracasei LBC-1e during manufacture of Mozzarella cheese and simulated gastric digestion

“…paracasei LBC-1e, an erythromycin-resistant derivate (Broadbent et al, 2004) of the probiotic strain LBC-1 (Cargill, Waukesha, WI), was obtained from the culture collection of Jeffrey Broadbent (Utah State University, Logan). Streptococcus thermophilus starter culture TS-10C was donated by DSM Food Specialties USA Inc. (Eagleville, PA).…”

The d -2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

“…Secondly, in lactic acid bacteria, hydroxyacid dehydrogenases are believed to be negatively involved in flavor production, since they compete with other enzymes generating flavor compounds from 2-keto acids derived from amino acids (66), while 2-hydroxyacids are not aroma compounds or precursors of flavor compounds. Indeed, overexpression of the D-2-hydroxyisocaproate dehydrogenase (D-2-HicDH) of Lactobacillus casei has been shown previously to decrease the production of aroma compounds and to delay flavor formation in low-fat cheddar cheese (12). Additionally, hydroxyacid dehydrogenases are involved in the biopreservation properties of lactic acid bacteria, because certain 2-hydroxyacids exhibit antifungal and antilisterial activities (15,37,64).…”