An assay for catheptic activity of lamb gastric tissue extract has been proposed which involves the use of a pH 3.5 hemoglobin substrate following activation of zymogens at pH 2.0, 25C for 30 min.; and inactivation at pH 8.0, 40C for 30 min thereby eliminating the effects of pepsin and rennin. Cathepsin was isolated and purified by ammonium sulfate fractionation, acetone precipitation and gel viii filtration. The purified cathepsin represented approximately 50 fold increase in specific activity over the original extract and a recovery of 15% of the original activity. Degree of purity was monitored by isoelectric focusing in polyacrylamide gels. Some characteristics of the cathepsin were determined. The purified cathepsin hydrolyzed urea-denatured hemoglobin readily at pH 3.5, but it had no activity on substrates specific for cathepsins A, B or c. a-N-benzoyloxycarbonyl- L-gutamyl-L-tyrosine, a-N-benzoyl-L-argininamide hydrochloride and a-N-acetyl-L-tyrosinamide. Approximate isoelectric point was pH 5.6. The purified enzyme was similar to cathepsin D. Parmesan, Romano, and Cheddar cheese manufactured with lamb pregastric esterase and gastric extracts added to the curd or milk were superior in flavor when both were employed, and either extract alone made better cheese than the uninoculated control.

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Amino acids derived from the degradation of casein in cheese serve as precursors for the generation of desirable and undesirable flavor compounds. Microbial degradation of aromatic amino acids is associated with the formation of aroma compounds that impart putrid-fecal, barny-utensil, and floral off-flavors in cheese, but pathways for their production had not been established. This study investigated Tyr and Phe catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under simulated Cheddar cheese-ripening conditions (pH 5.2, 4% NaCl, 15°C, no sugar). Enzyme assays of cell-free extracts and micellar electrokinetic capillary chromatography of supernatants indicated that L. casei and L. helveticus strains catabolize Tyr and Phe by successive transamination and dehydrogenation reactions. Major products of Tyr and Phe catabolism included off-flavor compounds formed by chemical degradation of the α-keto acids, produced by transamination, and aromatic α-hydroxy acids derived from α-keto acids by α-hydroxy acid dehydrogenases. Action of Lacrococcus lactis aminotransferase enzymes on Trp, Tyr, and Phe also leads to the formation of α-keto acids, but unlike lactobacilli, the former bacteria do not express dehydrogenase activity under cheese-like conditions (pH 5.2, 4% NaCl, 15°C, no sugar). Since aromatic α-keto acids may degrade spontaneously into undesirable flavor compounds, α-hydroxy acid dehydrogenases may be useful in controlling off-flavor development via diversion of chemically labile α-keto acids to more stable a-hydroxy acids. To test this hypothesis, we investigated the effect of D-hydroxyisocaproate dehydrogenase overexpression by a L. casei adjunct on chemical and sensory properties of reduced-fat Cheddar cheese made with and without addition of 20 mM α-ketoglutarate. The D-hydroxyisocaproic acid dehydrogenase gene (D-HicDH) was cloned into a high copy number vector pTRKH2 and transformed into L. casei ATCC334. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter + L. casei ATCC334 with pTRKH2, and starter + L. casei ATCC334 with pTRKH2: D-HicDH, and then volatile analysis was performed by gas chromatography and mass spectrometry. Statistical analysis of volatile data after 3 mo of ripening at 7°C showed profiles of ketones, aldehydes, alcohols, esters, sulfur compounds, and benzaldehyde were significantly altered by culture treatments and α-ketoglutarate addition, and these treatments also affected sensory flavor attributes of experimental cheeses. Results also indicated overexpression of D-hydroxyisocaproic acid dehydrogenase can divert labile α-keto acids into more stable compounds, but the overall effect seemed to diminish both beneficial and detrimental flavor notes.