S.-Q. Liu

Practical implications of lactate and pyruvate metabolism by lactic acid bacteria in food and beverage fermentations

This article reviews the metabolism of pyruvate and lactate by lactic acid bacteria (LAB) involved in food and beverage fermentations with an emphasis on practical implications. First, the formation of pyruvate and lactate from a range of substrates, including carbohydrates, organic acids and amino acids, is briefly described. The catabolism of pyruvate and lactate by LAB is then reviewed. This is followed by a discussion of lactate degradation and racemisation by LAB from specific fermented foods and beverages. Finally, the impact of environmental factors and metabolic engineering on pyruvate and lactate metabolism by LAB is evaluated with regard to practical significance.

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 and properties of intracellular esterases from Streptococcus thermophilus

Abstract Two of the three intracellular esterases identified in Streptococcus thermophilus were purified to homogeneity using ammonium sulphate fractionation and three chromatographic steps: anion exchange, hydrophobic interaction and gel filtration. The subunit molecular masses of esterases I and II were ∼34 and ∼60xa0kDa, respectively. The holoenzyme molecular masses of esterases I and II were ∼50 and ∼60xa0kDa, respectively, indicating that esterase I could be a dimer and that esterase II was a monomer. Phenylmethylsulphonyl fluoride inhibited the activity of both esterases, but to different degrees. Dithiothreitol, N -ethylmaleimide and EDTA strongly inhibited the activity of esterase I but significantly enhanced the activity of esterase II. Esterase I was active on p -nitrophenyl esters of the short-chain fatty acids from C 2 to C 10 and esterase II was active on p -nitrophenyl esters of the C 2 –C 6 fatty acids. For both enzymes, maximum activity was obtained with p -nitrophenyl butyrate (C 4 ). The K m values of esterase I on p -nitrophenyl esters of C 2 –C 8 fatty acids ranged from 6.7 to 0.004xa0m m and the corresponding V max values ranged from 8.12 to 1.12xa0μmolxa0min −1 xa0mg −1 protein. The N-terminal amino acid sequences of the two esterases also differed. The major esterase (I), accounting for ∼95% of the total esterase activity, was further characterized. Esterase I was also active against tributyrin (C 4 ), dicaproin (C 6 ) and monoglycerides of up to C 14 with maximum activity on monocaprylin (C 8 ). Decreasing pH (from 8.0 to 5.5), temperature (from 37° to 25°C) or water activity (from 0.99 to 0.80) considerably reduced the activity of esterase I, whereas increasing NaCl concentration up to 7.5% (w/v) markedly enhanced the activity of this enzyme. Esterase I may play a role in the development of cheese flavour with respect to lipolysis.

Ester synthesis in an aqueous environment by Streptococcus thermophilus and other dairy lactic acid bacteria

Abstract The ability of Streptococcus thermophilus ST1 and 19xa0other dairy lactic acid bacteria (LAB) to synthesize esters was investigated in an aqueous environment. These LAB were able to synthesize esters from alcohols and glycerides via a transferase reaction (alcoholysis) in which fatty acyl groups from glycerides were transferred to alcohols. S. thermophilus ST1 was active on tributyrin and on di- or monoglycerides of up to C10 with ethanol as the acyl acceptor. This strain was also active on a diglyceride of C6 and monoglyceride of C8 with 2-phenyl ethanol as the acyl acceptor. Alcoholysis occurred preferentially over hydrolysis. S. thermophilus ST1 had an apparent Km value of 250xa0mM for ethanol and an apparent Km value of 1.3xa0mM for tributyrin, measured against whole cells. Around 80% of both the transferase activity and the esterase activity were detected in the cell-free extract (CFE) of strain ST1. Both activities in the CFEs of five LAB tested were, to a similar degree, enhanced slightly by growth in the presence of ethanol and tributyrin. Using tributyrin and ethanol as substrates, the transferase activities ranged over 0.006–1.37xa0units/mg cell dry weight among the LAB tested and were both species- and strain-dependent.

Ethyl Butanoate Formation by Dairy Lactic Acid Bacteria

Abstract The formation of ethyl butanoate by non-growing cells of 22 starter and 49 non-starter dairy lactic acid bacteria (LAB) varied widely (0.4–310xa0units 100xa0mg-1 dry weight cells) and was both species and strain dependent. Strains of the thermophilic starter Streptococcus salivarius subsp. thermophilus produced the highest levels of ethyl butanoate (an average of 156 units 100xa0mg-1 dry weight cells), while strains of the mesophilic starters Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis gave a more moderate production of the ester (averages of 39 and 27 units 100xa0mg-1 dry weight cells, respectively). Non-starter LAB (lactobacilli, pediococci and leuconostocs) and propionibacteria varied widely in their ability to produce ethyl butanoate (0.4–160xa0units 100xa0mg-1 dry weight cells). The effect of physicochemical factors (pH, NaCl, water activity (aw) and temperature) on ethyl butanoate production was also investigated using Lactococcus lactis subsp. cremoris 2272 and Lactobacillus rhamnosus 2615. For both strains, ester formation increased by up to 175% when the pH was lowered from 5.8 to 4.0, but was drastically inhibited by up to 99% in the presence of NaCl and at reduced aw. Lowering the temperature from 30 to 13°C decreased ester production by 25% for both strains. The findings suggest that the normal cheese pH (∼5.0) and ripening temperature (∼13°C) are not critical factors in ester formation, but NaCl concentration and aw level are pivotal in determining ester formation.

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%).

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.