Abdallah A.A. Magboul

Proteolysis in Cheese during Ripening

This chapter discusses proteolysis process in cheese during ripening. Proteolysis contributes to: (1) The development of cheese texture: via hydrolysis of the protein matrix of cheese; via a decrease in aw through changes to water binding by the new carboxylic acid and amino groups liberated on hydrolysis of peptide bonds. These groups are ionized at the pH of cheese and thus bind water; indirectly via an increase in pH caused by the liberation of ammonia from amino acids produced by proteolysis. (2) Flavor and perhaps the off-flavor of cheese, directly by the production of short peptides and amino acids, some of which have flavors; indirectly by the liberation of amino acids, which act as substrates for a range of catabolic reactions, which generate important volatile flavor compounds; by facilitating the release of sapid compounds from the cheese matrix during mastication. Proteolysis in cheese during ripening is catalyzed by proteinases and peptidases from six sources: (1) the coagulant—the enzymes involved depend on the type of coagulant used. (2) the milk—a number of indigenous proteinases are present in milk, the most important of which is plasmin, which is produced from an inactive precursor, plasminogen. (3) starter lactic acid bacteria (LAB) contain a cell envelope-associated proteinase, which contributes to ripening principally by hydrolyzing intermediate-sized and short peptides produced from the caseins by the action of chymosin or plasmin. The other three sources are nonstarter lactic acid bacteria (NSEAB), secondary starter ( Propionibacterium freudenreichii subsp, shermanii in Swiss-type cheese), and exogenous proteinases and peptidases.

Thermal inactivation kinetics of bovine cathepsin D

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6.7) and skimmed milk of 6.5 and 7.6 degrees C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 degrees C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.

Purification and characterization of an acid phosphatase from Lactobacillus plantarum DPC2739

Abstract An acid phosphatase was partially purified from a cell-free extract of Lactobacillus plantarum DPC2739 by a combination of anion-exchange chromatography on DEAE-Sephacel, hydrophobic interaction chromatography on Phenyl Sepharose, gel permeation chromatography on Sephacryl S200 and high performance anion-exchange chromatography on MonoQ. The native enzyme (∼110 kDa) was tetrameric with a subunit molecular mass of ∼27 kDa. The enzyme was heat-stable, retaining ∼60% of its activity after heating for 30 min at 70°C. It was optimally active in the pH range 3.5–5.0 and at 40°C. The enzyme was strongly inhibited by 0.5 mM sodium fluoride, and hexametaphosphate and by 5 mM orthophosphate, tripolyphosphate and pyrophosphate. It was insensitive to metal chelators (ethylenediaminetetraacetic acid and o -phenanthroline), ascorbic acid, sulphydryl blocking agents (e.g., N-ethylmaleimide), phenylmethylsulphonyl fluoride and divalent metal ions at 5 mM concentration. The enzyme appeared to be a non-specific phosphomonoesterase and hydrolysed a number of phosphate esters. The amino acid sequence of the first 20 residues was determined and showed some homology with mammalian, yeast and Escherichia coli acid phosphatases, phosphoglycerate mutases and phosphoglycerokinases with a common motif Arg-His-Gly. ©

Purification and characterization of a dipeptidase from Lactobacillus curvatus DPC2024

A dipeptidase was purified to homogeneity from a cell-free extract of Lactobacillus curvatus DPC2024 by chromatography on diethylaminoethyl-sephacel, phenyl sepharose, chelating sepharose fast flow and MonoQ. The purified dipeptidase was a monomer of ∼52 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme was optimally active at pH 8 and 50°C and retained ∼10% of its maximum activity after pre-heating for 10 min at 70°C. The enzyme was a metallopeptidase, strongly inhibited by 0.1 mM ethylenediaminetetraacetic acid and o-phenanthroline and reactivated by a number of divalent metal ions. The enzyme was also inhibited by p-chloromercuribenzoate and β-mercaptoethanol. The enzyme was a strict dipeptidase, capable of hydrolysing a range of dipeptides but not tri-, tetra- or pentapeptides, p-nitroanilide derivatives of amino acids nor N- and C-terminal-blocked dipeptides. The N-terminal amino acid sequence of the first 20 residues showed significant homology with dipeptidases from Lactobacillus delbrueckii subsp. lactis DSM 7290 and Lactococcus lactis subsp. cremoris MG1363.

Biochemistry of Cheese Ripening: Proteolysis

Abstract The enzymatic hydrolysis of the casein matrix is a major biochemical event that occurs during cheese ripening. Products of proteolysis are a very large number of peptides, together with free amino acids. The enzymes which mediate proteolysis originate from different sources: the milk, coagulant, starter lactic acid bacteria (LAB), and adjunct organisms, nonstarter bacteria and, rarely, exogenous proteinases and peptidases. The principal indigenous proteinase in milk, plasmin, hydrolyzes β- and αs2-caseins during ripening and is of most significance in high-cook cheeses and varieties in which the pH increases significantly during ripening. Residual coagulant trapped in the curd is the major source of proteinase activity in low to medium cooked cheeses. Residual coagulant and plasmin act directly on the caseins to form a number of large and intermediate-sized peptides. The peptides are then acted upon by the cell envelope-associated proteinase of the starter LAB to produce shorter peptides, which are then degraded by a wide range of peptidases to form free amino acids. Only certain regions of the caseins are degraded extensively, and perhaps 75% of the caseins in mature Cheddar cheese remain intact or are present as large polypeptides. Proteolysis results, however, in the formation of perhaps hundreds of peptides and a complement of free amino acids which can be converted to volatile flavor compounds via amino acid catabolism.