Citrate metabolism in Lactobacillus casei and Lactobacillus plantarum

Information on the factors influencing citrate metabolism in lactobacilli is limited and could be useful in understanding the growth of lactobacilli in ripening cheese. Citrate was not used as an energy source by either Lactobacillus casei ATCC 393 or Lact. plantarum 1919 and did not affect the growth rate when co-metabolized with glucose or galactose. In growing cells, metabolism of citrate was minimal at pH 6 but significant at pH 4·5 and was greater in cells co-metabolizing galactose than in those co-metabolizing glucose or lactose. In non-growing cells, optimum utilization of citrate also occurred at pH 4·5 and was not increased substantially by the presence of fermentable sugars. In both growing and non-growing cells, acetate and acetoin were the major products of citrate metabolism; pyruvate was also produced by non-growing cells and was transformed to acetoin once the citrate was exhausted. Citrate was metabolized more rapidly than sugar by non-growing cells; the reverse was true of growing cells. Citrate metabolism by Lact. plantarum 1919 and Lact. casei ATCC 393 increased six- and 22-fold, respectively, when the cells were pre-grown on galactose plus citrate than when pre-grown on galactose only. This was probably due to induction of citrate lyase by growth on citrate plus sugar. These results imply that lactobacilli, if present in large enough numbers, can metabolize citrate in ripening cheese in the absence of an energy source.     

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach

AIMS: To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese. METHODS AND RESULTS: A putative citric acid cycle (PCAC) for Lact. casei was generated utilizing the genome sequence, and metabolic flux analyses. Although it was possible to construct a unique PCAC for Lact. casei, its full functionality was unknown. Therefore, the Lact. casei PCAC was evaluated utilizing end-product analyses of citric acid catabolism during growth in modified chemically defined media (mCDM), and Cheddar cheese extract (CCE). Results suggest that under energy source excess and limitation in mCDM this micro-organism produces mainly L-lactic acid and acetic acid, respectively. Both organic acids were produced in CCE. Additional end products include D-lactic acid, acetoin, formic acid, ethanol, and diacetyl. Production of succinic acid, malic acid, and butanendiol was not observed. CONCLUSIONS: Under conditions similar to those present in ripening cheese, citric acid is converted to acetic acid, L/D-lactic acid, acetoin, diacetyl, ethanol, and formic acid. The PCAC suggests that conversion of the citric acid-derived pyruvic acid into acetic acid, instead of lactic acid, may yield two ATPs per molecule of citric acid. Functionality of the PCAC reductive route was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This research describes a unique PCAC for Lact. casei. Additionally, it describes the citric acid catabolism end product by this nonstarter lactic acid bacteria during growth, and under conditions similar to those present in ripening cheese. It provides insights on pathways preferably utilized to derive energy in the presence of limiting carbohydrates by this micro-organism.     

Alcohol production by selected yeast strains in lactase-hydrolyzed acid whey

Ethanol production by Kluyveromyces fragilis and Saccharomyces cerevisiae was studied using cottage cheese whey in which 80 to 90% of the lactose present had been prehydrolyzed to glucose and galactose. Complete fermentation of the sugar by K. fragilis required 120 hr at 30°C in lactase-hydrolyzed whey compared to 72 hr in nonhydrolyzed whey. This effect was due to a diauxic fermentation pattern in lactase-hydrolyzed whey with glucose being fermented before galactose. Ethanol yields of about 2% were obtained in both types of whey when K. fragilis was the organism used for fermentation. Saccharomyces cerevisiae produced alcohol from glucose more rapidly than K. fragilis, but galactose was fermented only when S. cerevisiae was pregrown on galactose. Slightly lower alcohol yields were obtained with S. cerevisiae, owing to the presence of some lactose in the whey which was not fermented by this organism. Although prehydrolysis of lactose in whey and whey fractions is advantageous in that microbial species unable to ferment lactose may be utilized, diauxie and galactose utilization problems must be considered.     

Bacterial growth on lactose: an experimental investigation

A wild-type strain of Klebsiella oxytoca growing aerobically in batch culture has exhibited intermittent or oscillatory growth while growing on lactose at concentrations on the order of 1 g/L or less. In two-substrate experiments, preferred growth on glucose followed by growth on lactose also produced oscillatory growth behavior during the lactose growth phase at lactose concentrations of 1 g/L or less. Only oscillations in cell density have currently been observed. Alkalinization of the medium during growth on lactose indicated the presence of lactose active transport. The observed intermittent growth was reduced or removed during growth on lactose after preferred growth on galactose or in a medium containing 50 mM NaCl. Results suggested that the presence of an intracellular energy source or a sufficient DeltapH buffer may alleviate growth inhibition when transport and growth processes compete for essential energy resources during growth on lactose.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.     

Characterization of a Lactobacillus strain producing white crystals on cheddar cheese

From an enrichment culture of white-crystal deposits from aged Cheddar cheese, an atypical Lactobacillus strain was characterized. The new isolate is facultatively heterofermentative, has a G + C content of 40 mol%, and produces D and L isomers of lactic acid. The strain had a limited ability to ferment carbohydrates. It utilized fructose, galactose, glucose, lactose, maltose, mannose, and ribose but was negative for esculin, gluconate, citrate, and several other carbon sources. The isolate also had low DNA-DNA homologies with strains of Lactobacillus casei and Lactobacillus plantarum. Cheese prepared with milk containing the isolate developed white crystals during curing. Formation of copious D-lactate from unknown substrates during curing probably caused the white-crystal deposits. The strain has been deposited in the American Type Culture Collection (ATCC 49178).     

Characterization of a Lactobacillus strain producing white crystals on cheddar cheese.

From an enrichment culture of white-crystal deposits from aged Cheddar cheese, an atypical Lactobacillus strain was characterized. The new isolate is facultatively heterofermentative, has a G + C content of 40 mol%, and produces D and L isomers of lactic acid. The strain had a limited ability to ferment carbohydrates. It utilized fructose, galactose, glucose, lactose, maltose, mannose, and ribose but was negative for esculin, gluconate, citrate, and several other carbon sources. The isolate also had low DNA-DNA homologies with strains of Lactobacillus casei and Lactobacillus plantarum. Cheese prepared with milk containing the isolate developed white crystals during curing. Formation of copious D-lactate from unknown substrates during curing probably caused the white-crystal deposits. The strain has been deposited in the American Type Culture Collection (ATCC 49178).     

Citrate metabolism by Enterococcus faecium and Enterococcus durans isolated from goat's and ewe's milk: influence of glucose and lactose

Citrate metabolism by Enterococcus faecium ET C9 and Enterococcus durans Ov 421 was studied as sole energy source and in presence of glucose or lactose. Both strains utilized citrate as the sole energy source. Enterococcus faecium ET C9 showed diauxic growth in the presence of a limiting concentration of glucose. Neither strain used citrate until glucose was fully metabolized. The strains showed co-metabolism of citrate and lactose. Lactate, acetate, formate, and flavour compounds (diacetyl, acetoin, and 2,3-butanediol) were detected in both strains. The highest production of flavour compounds was detected during growth of E. durans Ov 421 in media supplemented with citrate-glucose and citrate-lactose. Citrate lyase was inducible in both strains. Acetate kinase activities presented the highest values in LAPTc medium, with E. faecium ET C9 displaying a specific activity 2.4-fold higher than E. durans. The highest levels of alpha-acetolactate synthase specific activity were detected in E. durans grown in LAPTc+g, in accordance with the maximum production of flavour compounds detected in this medium. Diacetyl and acetoinreductases displayed lower specific activity values in the presence of citrate. Enterococcus faecium and E. durans displayed citrate lyase, acetate kinase, alpha-acetolactate synthase, and diacetyl and acetoin reductase activities. These enzymes are necessary for conversion of citrate to flavour compounds that are important in fermented dairy products.     

Solubility and selective crystallization of lactose from solutions of its hydrolysis products glucose and galactose

A high degree of conversion is desired when lactose is hydrolyzed to glucose and galactose. This produces, however, a high concentration of galactose, which is inhibitory for the enzyme catalyst (beta-galactosidase). The inhibition can be reduced by limiting the conversion per pass over the enzyme (e.g. to ca. 50%), separating unconverted lactose from the reactor effluent, and recycling it to the reactor inlet. (This allows the overall conversion to be raised to ca. 80-90%). The solubilities of lactose, glucose, and galactose have been determined at various temperatures and for sugar mixtures having different concentrations and degrees of hydrolysis. Various cooling crystallizations have defined convenient and simple processes for the selective separation of lactose from its hydrolysis products.     

Sorbitol synthesis by an engineered Lactobacillus casei strain expressing a sorbitol-6-phosphate dehydrogenase gene within the lactose operon

Sorbitol is claimed to have important health-promoting effects and Lactobacillus casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing L. casei strain might therefore be of considerable interest in the food industry. A recombinant strain of L. casei was constructed by the integration of a d-sorbitol-6-phosphate dehydrogenase-encoding gene (gutF) in the chromosomal lactose operon (strain BL232). gutF expression in this strain followed the same regulation as that of the lac genes, that is, it was repressed by glucose and induced by lactose. (13)C-nuclear magnetic resonance analysis of supernatants of BL232 resting cells demonstrated that, when pre-grown on lactose, cells were able to synthesize sorbitol from glucose. Inactivation of the l-lactate dehydrogenase gene in BL232 led to an increase in sorbitol production, suggesting that the engineered route provides an alternative pathway for NAD(+) regeneration.     

Escherichia coli growth on lactose requires cycling of beta-galactosidase products into the medium

Growth on lactose was found to be restricted in an Escherichia coli strain deficient in its ability to transport glucose and galactose. If the latter sugars were removed from the medium as they were being produced, a wild-type strain grew only poorly, while the transport-deficient strain did not grow at all. These results suggested that all of the products of beta-galactosidase action on lactose are released into the medium before being metabolized. This contention was strongly supported by the finding that the appearance of products in the medium was equal to lactose disappearance at three limiting lactose concentrations and by an experiment which showed that essentially all of the label from added lactose ( [1-14C]glucose) was found in the medium as glucose when chased with unlabelled lactose.     

Stimulation of Lactic Streptococci in Milk by β-Galactosidase

Acid production in milk by lactic streptococci was stimulated by added beta-galactosidase. Both glucose and galactose accumulated rapidly in the presence of this enzyme. Glucose accumulation ceased as the culture entered the most rapid period of acid production, whereas galactose accumulation continued. In cultures without added beta-galactosidase, a low concentration of galactose accumulated in the milk, whereas glucose was not detected after 2 hr of incubation. Cultures grew and produced acid faster in broth containing glucose rather than galactose or lactose. These observations suggest that the lactic streptococci do not metabolize the lactose in milk efficiently enough to permit optimum acid production and that a phenomenon such as catabolite repression functions to allow for a preferential use of glucose over either galactose or lactose. In addition to providing the culture with a more readily available energy source, it is possible that the culture produced more acidic metabolites as a result of preferentially utilizing the glucose released by the action of the beta-galactosidase.     

Mixed cultures: Commensalism and competition with Proteus vulgaris and Saccharomyces cerevisiae

Several modes of interaction are demonstrated and modeled in the Proteus vulgaris–Saccharomyces cerevisiae system, in particular, commensalism (citrate present. glucose limiting, the nicotinic acid required by the bacterium supplied by the yeast), and commensalism and competition [citrate absent, both organisms compete for the same limiting carbon source (glucose) and the yeast supplies the nicotinic acid for the bacterium]. By varying the medium component concentrations, competition (citrate absent, glucose limiting, nicotinic acid not limiting), mutualism (citrate excess, glucose limiting, nicotinic acid absent), and neutralism (citrate limiting, glucose limiting, and nicotinic acid excess) could also be created. Kinetic models for commensalism and commensalism plus competition were developed to describe cell growth, substrate utilization, and nicotinic acid production. Good agreement with experiment was obtained for the commensalism case.     

Carbon Source Requirements for Exopolysaccharide Production by Lactobacillus casei CG11 and Partial Structure Analysis of the Polymer

Exopolysaccharide production by Lactobacillus casei CG11 was studied in basal minimum medium containing various carbon sources (galactose, glucose, lactose, sucrose, maltose, melibiose) at concentrations of 2, 5, 10, and 20 g/liter. L. casei CG11 produced exopolysaccharides in basal minimum medium containing each of the sugars tested; lactose and galactose were the poorest carbon sources, and glucose was by far the most efficient carbon source. Sugar concentrations had a marked effect on polymer yield. Plasmid-cured Muc^- derivatives grew better in the presence of glucose and attained slightly higher populations than the wild-type strain. The values obtained with lactose were considerably lower for both growth and exopolysaccharide yield. The level of specific polymer production per cell obtained with glucose was distinctively lower for Muc^- derivatives than for the Muc⁺ strain. The polymer produced by L. casei CG11 in the presence of glucose was different from that formed in the presence of lactose. The polysaccharide produced by L. casei CG11 in basal minimum medium containing 20 g of glucose per liter had an intrinsic viscosity of 1.13 dl/g. It was rich in glucose (76%), which was present mostly as 2- or 3-linked residues along with some 2,3 doubly substituted glucose units, and in rhamnose (21%), which was present as 2-linked or terminal rhamnose; traces of mannose and galactose were also present.     

Regulation and characterization of the galactose-phosphoenolpyruvate-dependent phosphotransferase system in Lactobacillus casei.

Cells of Lactobacillus casei grown in media containing galactose or a metabolizable beta-galactoside (lactose, lactulose, or arabinosyl-beta-D-galactoside) were induced for a galactose-phosphoenolpyruvate-dependent phosphotransferase system (gal-PTS). This high-affinity system (Km for galactose, 11 microM) was inducible in eight strains examined, which were representative of all five subspecies of L. casei. The gal-PTS was also induced in strains defective in glucose- and lactose-phosphoenolpyruvate-dependent phosphotransferase systems during growth on galactose. Galactose 6-phosphate appeared to be the intracellular inducer of the gal-PTS. The gal-PTS was quite specific for D-galactose, and neither glucose, lactose, nor a variety of structural analogs of galactose caused significant inhibition of phosphotransferase system-mediated galactose transport in intact cells. The phosphoenolpyruvate-dependent phosphorylation of galactose in vitro required specific membrane and cytoplasmic components (including enzyme IIIgal), which were induced only by growth of the cells on galactose or beta-galactosides. Extracts prepared from such cells also contained an ATP-dependent galactokinase which converted galactose to galactose 1-phosphate. Our results demonstrate the separate identities of the gal-PTS and the lactose-phosphoenol-pyruvate-dependent phosphotransferase system in L. casei.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

Citrate metabolism by Enterococcus faecalis FAIR-E 229.

Citrate metabolism by Enterococcus faecalis FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used, E. faecalis FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of E. faecalis FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as well. Finally, when increasing initial concentrations of citrate (10, 40, 70, and 100 mM) were used as the sole carbon sources in MRS broth without glucose, the main end products were acetate and formate. Small amounts of lactate, ethanol, and acetoin were also detected. This work strongly supports the suggestion that enterococcal strains have the metabolic potential to metabolize citrate and therefore to actively contribute to the flavor development of fermented dairy products.     

Cometabolism of Citrate and Glucose by Enterococcus faecium FAIR-E 198 in the Absence of Cellular Growth

Citrate metabolism by Enterococcus faecium FAIR-E 198, an isolate from Greek Feta cheese, was studied in modified MRS (mMRS) medium under different pH conditions and glucose and citrate concentrations. In the absence of glucose, this strain was able to metabolize citrate in a pH range from constant pH 5.0 to 7.0. At a constant pH 8.0, no citrate was metabolized, although growth took place. The main end products of citrate metabolism were acetate, formate, acetoin, and carbon dioxide, whereas ethanol and diacetyl were present in smaller amounts. In the presence of glucose, citrate was cometabolized, but it did not contribute to growth. Also, more acetate and less acetoin were formed compared to growth in mMRS medium and in the absence of glucose. Most of the citrate was consumed during the stationary phase, indicating that energy generated by citrate metabolism was used for maintenance. Experiments with cell-free fermented mMRS medium indicated that E. faecium FAIR-E 198 was able to metabolize another energy source present in the medium.