Tim Coolbear

The role of autolysis of lactic acid bacteria in the ripening of cheese

Abstract The importance of autolysis of lactic acid bacteria in cheese ripening is evident from the literature. However, the mechanisms and the consequences still require investigation. The consequences of autolysis of mesophilic starters in Cheddar cheese are discussed and highlights from current physiological and genetic studies on starter autolysis are presented. The relative merits of measuring starter autolysis in cheese by viable starter cell densities, electron microscopic observations and assay of cell-free cytoplasmic enzymes are discussed for cheese studies using different starter strains and added phage to achieve different levels of autolysis. The balance of both the intact and autolysed starter cells in young curd appear to be important in cheese ripening. The intact cells are necessary for physiological reactions such as lactose fermentation and oxygen removal and possibly for a number of flavour reactions. In contrast, the main consequence of autolysed cells in cheese is to accelerate the peptidolytic reactions. The possible influences of autolysis of adventitious lactic acid bacteria during cheese ripening are discussed.

Detection and impact of protease and lipase activities in milk and milk powders

Freshly drawn milk contains indigenous enzymes, including proteases and lipases. During handling and processing, milk acquires contaminating bacteria that produce further proteases and lipases, adding to the enzyme loading in the milk. Even when several heat treatment steps are used to prepare milk products, these will not be sufficient to inactivate all of the enzymes. Adverse effects of heat on the product limit the extent of heat treatments that can be used. The activities of proteinases and lipases that survive the heat treatments may cause changes in functionality and flavour of milk products, including milk powders, during storage. This review focuses on the characteristics of proteases (specifically proteinases) and lipases in milk and milk powders, detection methods for such enzymes and the effects of these enzymes on milk and milk powders during storage.

The influence of phage-assisted lysis of Lactococcus lactis subsp. lactis ML8 on cheddar cheese ripening

Cheddar cheese was made with Lactococcus lactis subsp. lactis strain ML8 as starter and two levels of rennet and three levels of homologous phage. The use of the different phage levels in cheese milk resulted in various degrees of starter lysis early in the ripening process. The levels of activity of two starter cytoplasmic enzymes, lysylaminopeptidase and FBP-aldolase, in the cheese matrix were used as a direct measure of lysis and increased with the level of phage added. Elevated starter lysis was associated with an increased rate of formation of amino acids and ammonia but the removal rate of lactose in the cheese was decreased. The relative levels of hydrophobic and hydrophilic peptides in aqueous cheese extracts were also influenced by the extent of starter lysis. Bitter flavour was prominent in cheese with high rennet concentration, but not when there was also high starter lysis. The results suggest that a balance of lysed and intact cells is important for control of cheese ripening; enzymes released on cell lysis accelerate the rate-limiting peptidolytic steps and removal of some bitter peptides, while intact cells are required for lactose removal. The consequences of the relative levels of intact and lysed cells on substrate-enzyme interactions, enzyme stability and flavour profiles are discussed.

Purification of tributyrin esterase from Lactococcus lactis subsp. cremoris E8

A tributyrin esterase was purified from Lactococcus lactis subsp. cremoris E8 using FPLC chromatography. This was the major esterase activity observed in strain E8 and was associated with a single protein with a subunit molecular mass of 29 kDa and a holoenzyme of molecular mass 109 kDa. The enzyme was active against tributyrin and p-nitrophenyl butyrate. The N-terminal sequence of the enzyme was determined. The enzyme had a pH optimum in the neutral range, was stable on freezing at -20 degrees C, and had a half life of 1 h at 50 degrees C.

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 2. The levels and subcellular distributions of peptidase and esterase activities

Abstract The levels and subcellular distributions of various peptidase and esterase activities in a range of lactococcal and Streptococcus thermophilus strains were investigated. There was no correlation between the levels of the enzymes in the different strains and the ability of the strains to produce acid when grown in milk. While considerable differences between individual strains were apparent, average levels of X-prolyldipeptidyl aminopeptidase, dipeptidase and tripeptidase were similar in the Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris strains studied, while that of lysylaminopeptidase (i.e. activity assayed using lysine p-nitroanilide as substrate) in the L. lactis subsp. cremoris strains was approximately double that in the L. lactis subsp. lactis strains. The average levels of lysylaminopeptidase and X-prolyldipeptidyl aminopeptidase in the S. thermophilus strains studied were similar to those in the L. lactis subsp. cremoris strains, while the average levels of dipeptidase and tripeptidase were considerably lower. All peptidases studied were recovered predominantly in the cytoplasmic fraction, although in a few strains there was some evidence to suggest that a part of the tripeptidase activity may be associated with cell structures comprising the particulate fraction. The levels of esterase activity in the strains were considerably different between strains. However, the average level of esterase activity detected in the two lactococcal subspecies was similar, while that in the S. thermophilus strains was more than double the lactococcal average. The subcellular distribution of the esterase in all strains studied showed that a significant proportion of the activity is located on the cell surface.

Comparative study of methods for the isolation and purification of bovine κ-casein and its hydrolysis by chymosin

kappa-Casein was purified from a single batch of whole acid casein (kappa-A variant) using different methods in order to compare their merits in producing a purified material with a carbohydrate and phosphate heterogeneity representative of the whole kappa-casein complement in milk. Ion-exchange methods of purification gave products of higher purity than precipitation techniques involving final purification by ethanol fractionation, but all methods resulted in kappa-caseins of apparently similar heterogeneity and chemical composition. The purified kappa-caseins were hydrolysed with chymosin and the derived macropeptides isolated. These were all virtually identical as determined by reversed-phase chromatography and gel electrophoresis. Some observations on chymosin hydrolysis of kappa-casein were made. In addition to formation of the major para-kappa-casein (Glu1-Phe105) and macropeptide (Met106-Val169), chymosin hydrolysis at pH 6.6 also resulted in two minor para-kappa-caseins with N-termini corresponding to Phe18 and Ser33 of kappa-casein. At pH 5.5 and 4.5 para-kappa-casein was rapidly hydrolysed into at least six fragments, one of which had an N-terminus corresponding to Trp76 of kappa-casein. At pH 6.6, 5.5 and 4.5 the kappa-casein macropeptide was stable to chymosin, but at pH 2.3 it was hydrolysed by chymosin into fragments with N-termini corresponding to Met106, Ile125, Ala138, Val139, Thr145 and Glu147 of kappa-casein.

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 1. Resistance to cell lysis and levels and cellular distribution of proteinase activities

Abstract By reference to subcellular fraction markers, the resistance to lysis of 23 strains of Lactococcus lactis subsp. cremoris , 30 strains of L. lactis subsp. lactis and five strains of Streptococcus thermophilus and the levels and distribution of proteinase activity in the strains were determined. Strains of L. lactis subsp. cremoris were readily lysed by transfer to hypotonic buffer after treatment with lysozyme alone, whilst strains of L. lactis subsp. lactis and S. thermophilus could be efficiently lysed in this way only after treatment with a combination of lysozyme and mutanolysin. With a few notable exceptions, those strains which gave the fastest rates of acid production also generally presented higher levels of cell surface proteinase, as determined by activity on fluorescein isothiocyanate-labelled β-casein. The highest levels of cell surface proteinase detected were found for strains of L. lactis subsp. cremoris . However, the levels of total proteinase activity in the lactococcal strains did not correlate with the rate of acid production in milk, some slow acid-producers yielding similar or greater total proteinase levels than fast acid-producers. Homology to DNA probes for the lactococcal cell surface proteinase gene and to the conserved region encoding the serine proteinase active site was shown by the fast acid-producing lactococcal strains, but not by most of the slow acid-producing lactococcal strains or by the strains of S. thermophilus . A significant proportion of the total proteinase activity was recovered in the subcellular fractions in which high levels of cytoplasmic marker enzyme activity were found. The total proteinase levels detected in strains of L. lactis subsp. lactis showed a greater range of variation than in the strains of L. lactis subsp. cremoris . High levels of total proteinase activity were found in the slow acid-producers despite the strains having been grown in the presence of yeast extract. For many of the strains, the levels of proteinase released from the cell surface during cell wall degradation with lytic enzyme treatment were higher than those found using whole cells, suggesting that a significant amount of proteolytic activity was either inaccessible to substrate or present in an inactive form.

Cloning and Expression of an Oligopeptidase, PepO, with Novel Specificity from Lactobacillus rhamnosus HN001 (DR20)

ABSTRACT Oligopeptidases of starter and nonstarter lactic acid bacteria contribute to the proteolytic events important in maturation and flavor development processes in cheese. This paper describes the molecular cloning, expression, and specificity of the oligopeptidase PepO from the probiotic nonstarter strain Lactobacillus rhamnosus HN001 (DR20). The pepO gene encodes a protein of 70.9 kDa, whose primary sequence includes the HEXXH motif present in certain classes of metallo-oligopeptidases. The pepO gene was cloned in L. rhamnosus HN001 and overexpressed in pTRKH2 from its own promoter, which was mapped by primer extension. It was further cloned in both pNZ8020 and pNZ8037 and overexpressed in Lactococcus lactis subsp. cremoris NZ9000 from the nisA promoter. The purified PepO enzyme demonstrated unique cleavage specificity for αs1-casein fragment 1–23, hydrolyzing the bonds Pro-5-Ile-6, Lys-7-His-8, His-8-Gln-9, and Gln-9-Gly-10. The impact of this enzyme in cheese can now be assessed.

Comparison of subcellular fractionation methods for lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

Abstract Cells of Lactococcus lactis subsp. lactis proved to be resistant to cell wall digestion by lysozyme using 0·6 M glycylglycine/10 mM MgCl 2 as stabilizing agent, this procedure having been described recently as suitable for subcellular fractionation of cells of L. lactis subsp. cremoris (Coolbear et al. (1992). Int. Dairy J. , 2, 213-32). A procedure has now been developed for L. lactis subsp. lactis , based on the use of a combination of lysozyme and mutanolysin to digest the walls of cells stabilized in 24% sucrose/10 mM MgCl 2 . Although the transfer of cell wall-depleted cells to hypotonic buffer resulted in lysis of most of the cells, a proportion of the cells remaining were permeable to small molecules (but not to proteins). The proportions of permeabilized cells, intact cells and cell wall-membrane complexes in the particulate fraction depended on both the lactococcal strain and the actual protocol used in the fractionation. Further, the concentration of N -acetylglucosamine in the subcellular fractions showed considerable variation between strains and the distribution of N -acetylglucosamine and another cell wall marker, rhamnose, in the fractions did not correlate. Lysylaminopeptidase activity was distributed between subcellular fractions in a similar manner to aldolase, but some evidence was obtained to suggest that a proportion of the activity may be associated with the cell membrane. For the four strains studied in detail (two strains each of L. lactis subsp. lactis and L. lactis subsp. cremoris ), nearly half of the total proteinase and esterase activities were associated with the cell surface. The origin of the remainder of the activities was unclear.

Parameters affecting the release of cell surface components and lysis of Lactococcus lactis subsp. cremoris

Abstract Studies have been undertaken on the effects of a number of parameters, including MgCl2 concentration, temperature, stabilizing buffer concentration and growth conditions on the response of cells of Lactococcus lactis subsp. cremoris strain E8 to subcellular fractionation procedures. Optimum stabilization of cells during partial cell wall digestion with lysozyme was obtained using 0·6 m glycylglycine, pH 7·5, containing 10 m m MgCl2, Concentrations of glycylglycine below 0·4 m severely reduced stabilization. Cooling of cell-wall-depleted cells below 20°C caused considerable lysis; separation of these sensitive cells from solubilized cell wall material necessitated centrifugation at room temperature. Subsequent transfer of lysozyme-treated cells to ice-cold Tris-HCl, pH 7·5, achieved virtually complete lysis as determined using aldolase as a cytoplasmic marker. Increasing the MgCl2 concentration above 10 m m in the stabilizing buffer subsequently resulted in decreased lysis of protoplasts in the hypotonic buffer due, in part, to carry-over of MgCl2. MgCl2 could be substituted by CaCl2 with respect to stabilization, but proteinase distribution profiles between subcellular fractions were altered. KCl substituted only poorly for MgCl2. Inclusion of NaCl at even low concentrations in the hypotonic buffer decreased the levels of cell lysis. The distribution of two cell wall components, rhamnose and N-acetylglucosamine, between fractions did not correlate and responded differently to variations in MgCl2 concentration. Rhamnose remained almost entirely associated with the particulate material remaining after cell lysis. Two pools of N-acetylglucosamine were evident: a proportion of this monosaccharide could be readily released from the cell surface without loss of cell integrity, further release requiring more severe conditions and being accompanied by cell lysis. Cells grown in peptide-rich broth were more resistant to lysis after lysozyme treatment than when grown in reconstituted skim milk (RSM) and were almost completely resistant to lysis after mutanolysin treatment under the conditions used, whilst the RSM-grown cells were extremely susceptible to mutanolysis-induced lysis.