V.L. Crow

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.

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%).

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.

Exchanging lactocepin plasmids in lactococcal starters to study bitterness development in Gouda cheese: a preliminary investigation

Most fast-milk-coagulating Lactococcus lactis starter strains contain a plasmid-encoded cell envelope proteinase (lactocepin; EC 3.4.21.96) having PI-type (lactocepin I) or PI/III intermediate-type (lactocepin I/III) specificity (those with PIII-type specificity are rare). We assessed the effect of lactocepin I and lactocepin I/III on peptide accumulation and bitterness development in dry-salted Gouda cheese by exchanging lactocepin plasmids between two L. lactis starter strains low in autolysis, thus eliminating the effects of other starter variables. Gouda was made using starter strains, either L. lactis subsp. cremoris HP (lactocepin I) or L. lactis subsp. lactis U (lactocepin I/III), or using constructs of these two strains with exchanged lactocepin plasmids. Bitterness in Gouda was consistently greater when the starter strains contained lactocepin I. Following ripening, cheese peptide profiles (determined by high-performance liquid chromatography) were found to vary more in relation to starter lactocepin specificity than in relation to starter strain background or sub-species (lactis or cremoris).

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...

Chapter 33 – Cheddar Cheese and Related Dry-Salted Cheese Varieties

This chapter describes the manufacture of Cheddar cheese and related dry-salted cheese varieties. Curd formation, whey separation, cheddaring, milling, salting, and pressing are described in detail, including an illustration of how the microstructure of this cheese develops. The chapter also explains the main factors that determine Cheddar cheese quality including the chemical composition, texture, and flavour of the cheese. It summarizes the role of lipolysis, proteolysis, starter, nonstarter lactic acid bacteria, or adjunct cultures in the development of Cheddar flavor during ripening. Although there is no universal standard for measuring Cheddar quality, different grading and assessments of Cheddar cheese are also presented including sensory evaluation and the use of instrumental analysis to measure key aspects of Cheddar cheese ripening.