Peptide Accumulation and Bitterness in Cheddar Cheese Made Using Single-Strain Lactococcus lactis Starters with Distinct Proteinase Specificities

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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...

Section: Chemicalsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

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

Broadbent¹,

Gummalla²,

Hughes

et al. 2004

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“…The purpose of this study and another study (10) was to identify and characterize an L. helveticus endopeptidase(s) involved in the hydrolysis of ␤-CN(f193-209). Additionally, the accumulation of ␣ s1 -CN(f1-9) has been associated with bitterness (4,5,15); therefore, the hydrolysis of this peptide was also examined.…”

mentioning

confidence: 99%

Identification and Characterization of Lactobacillus helveticus PepO2, an Endopeptidase with Post-Proline Specificity

Chen¹,

Christensen

Broadbent

et al. 2003

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A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-␤-casein-(f203-209)--nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as ␤-casein(f193-209).The proteolytic systems of dairy lactic acid bacteria (LAB) have received extensive research attention due to their importance in the physiology of these organisms and in cheese flavor development. LAB are fastidious microorganisms with multiple amino acid auxotrophies (18). During growth in milk, LAB rely on their proteolytic systems to obtain essential amino acids from caseins (CNs), the most abundant proteins in milk (9, 17). Additionally, proteolytic enzymes from LAB produce flavor compounds and precursors that are essential for cheese flavor development (9, 23).The proteolytic systems of LAB can be functionally divided into three components: (i) cell envelope-associated proteinases which hydrolyze CNs to oligopeptides; (ii) peptide transport systems, of which the oligopeptide transport system is the most important in milk and cheese; and (iii) numerous intracellular peptidases (9, 17). The intracellular peptidases of LAB include both endopeptidases and aminopeptidases. Endopeptidases, due to their ability to hydrolyze peptide bonds within a peptide, are of particular interest in targeting peptides for rapid hydrolysis. In Lactococcus lactis, the best-characterized LAB, the endopeptidases that have been identified include PepO, PepO2, PepF1, and PepF2. All of these enzymes are metalloproteases, and PepO, PepF1, and PepF2 are encoded in operons (9, 17). The physiological roles of these endopeptidases remain unclear; however, PepF appears to be important for protein turnover during nitrogen starvation (24). To date, one metalloendopeptidase, designated PepO (8), and a thiol-dependent endopeptidase, designated PepE (13), have been characterized from Lactobacillus helveticus.The ability of L. helveticus CNRZ32 to ...

“…Brevibacteria accelerate flavor development in low-fat cheddar cheese when added with the starter culture via the production of sulfur and FAs (1,10,20). This change is associated with the later stages of cheese ripening, which correspond to lactose depletion in cheese.…”

Section: Discussionmentioning

confidence: 99%

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Ganesan

Seefeldt²,

Weimer

2004

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Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and ␣-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of ␣-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and ␣-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from ␣-keto acids only. BL2 also converted ␣-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and ␣-keto acids and that carbon metabolism is important in regulating this event.