Ross Holland

The potential of dairy lactic acid bacteria to metabolise amino acids via non-transaminating reactions and endogenous transamination

The metabolism of amino acids by 22 starter and 49 non-starter lactic acid bacteria (LAB) was studied in a system consisting of amino acids and non-growing cells without added amino acceptors such as alpha-ketoglutarate. There were significant inter- and intra-species differences in the metabolism of amino acids. Some amino acids such as alanine, arginine, aspartate, serine and branched-chain amino acids (leucine, isoleucine and valine) were utilised, whereas other amino acids such as glycine, ornithine and citrulline were produced. Alanine and aspartate were utilised by some LAB and accumulated during the incubation of other LAB. Arginine was degraded not only by Lactococcus lactis subsp. lactis (the lactococcal subspecies known to catabolise arginine), but also by pediococci, heterofermentative lactobacilli (Lactobacillus brevis and Lb. fermentum) and some unidentified homofermentative lactobacilli. Serine was utilised predominantly by homofermentative Lb. paracasei subsp. paracasei, Lb. rhamnosus and Lb. plantarum. Of the LAB studied, Lb. brevis and Lb. fermentum were the most metabolically active, utilising alanine, arginine, aspartate, glutamate and branched-chain amino acids. Leuconostocs were the least metabolically active, showing little potential to metabolise amino acids. The formation of ammonia and acetate from amino acid metabolism varied both between species and between strains within species. These findings suggest that the potential of LAB for amino acid metabolism via non-transaminating reactions and endogenous transamination will impact both on the physiology of LAB and on cheese ripening, especially when transamination is rate-limiting in the absence of an exogenous amino acceptor such as alpha-ketoglutarate.

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.

Serine metabolism in Lactobacillus plantarum

This study investigated the metabolism of (L-) serine by Lactobacillus plantarum B3089 isolated from cheese. Serine was deaminated by growing cells to ammonia with the corresponding formation of acetate and formate. Serine was also deaminated by non-growing cells to ammonia but with the formation of acetate only (no production of formate). Phosphoserine and threonine were not catabolised. It is proposed that serine was deaminated by serine dehydratase (deaminase) to ammonia and pyruvate. Pyruvate was further catabolised predominantly to acetate, carbon dioxide and formate in growing cells, catalysed by pyruvate-formate lyase and pyruvate oxidase; some of the pyruvate was converted to acetoin. In non-growing cells, however, pyruvate-formate lyase was inactive and pyruvate oxidase degraded the pyruvate to acetate and carbon dioxide. Serine dehydratase activity could not be detected in cell-free extracts, presumably because of enzyme instability. The growth of L. plantarum was neither enhanced nor stimulated by serine under the current conditions. Whereas there was little difference in serine utilisation between pH 7.0 and pH 5.8, serine utilisation was decreased by 30% at pH 5.0. NaCl of up to 4% (w/v) concentration had little effect on serine utilisation. Serine had no impact on lactose metabolism. Lactose was fermented mainly to lactate (73%) with the remainder converted to an unidentified polysaccharide (27%).

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.

Synthesis of ethyl butanoate by a commercial lipase in aqueous media under conditions relevant to cheese ripening.

A fruity flavour note is traditionally regarded as a defect in cheese varieties such as Cheddar (Bills et al. 1965; McGugan et al. 1975; Horwood et al. 1987). However, fruitiness is an attribute of other cheese varieties such as Parmesan and Parmigiano Reggiano (Dumont et al. 1974; Meinhart & Schreier, 1986). It is well accepted that esters such as ethyl butanoate and ethyl hexanoate cause the fruity flavour described as apple-like or pineapple-like in raw milk and cheeses (Bills et al. 1965; Engels et al. 1997; Friedrich & Acree, 1998). The development of fruity flavour is often attributed to the esterification of free fatty acids and ethanol by esterases from lactic acid bacteria and psychrotrophic pseudomonads (Hosono et al. 1974; Morgan, 1976).

Acetaldehyde Metabolism by Leuconostoc mesenteroides subsp. cremoris under stress conditions

Abstract Non-growing cells of Leuconostoc mesenteroides subsp. cremoris converted acetaldehyde to ethanol and acetate. Acetaldehyde utilisation and the formation of ethanol and acetate were influenced by pH, salt and water activity. Low pH, increased levels of salt and low water activity reduced the rates of acetaldehyde utilisation and the formation of ethanol and acetate. Almost all leuconostocs tested removed added acetaldehyde in broth co-cultures with strains of Lactococcus lactis subsp. cremoris; an exception was L. mesenteroides subsp. cremoris 60 in co-culture with Lc. lactis subsp. cremoris 2254. L. mesenteroides subsp. cremoris 253 removed acetaldehyde produced by lactococci in milk co-cultures and the removal rate depended on the concentration of the leuconostocs and salt.

Production of natural fruity flavour in dairy foods

Purpose – The purpose of this paper is to investigate in situ production of aroma‐active esters in dairy foods so as to improve flavour and to produce fruity flavour concentrate.Design/methodology/approach – Lipase, ethanol or bacterial cultures are added to dairy media (milk, cream or cheese) and incubated for a period of time (from hours to months). Samples are then taken and analysed for aroma‐active esters using gas chromatography (GC) or gas chromatography‐mass spectrometry (GC‐MS).Findings – Analyses of samples show that significant levels of ethyl esters of fatty acids are produced in milk, cream, enzyme‐modified cheese and natural cheese. All the dairy foods possess an intense pleasant fruity aroma.Originality/value – This is a natural way to generate fruity flavours in dairy foods to enhance flavour and thus, consumer acceptance. The fruity flavour concentrate can also be used as a flavouring ingredient in dairy and non‐dairy food applications. Natural pure esters may also be extracted, separated...