Transformation of Citric Acid to Acetic Acid,Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.

Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg²⁺ and Ag²⁺ . The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl₂, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malo-lactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.     

Characterization of a citrate-negative mutant ofLeuconostoc mesenteroides subsp.mesenteroides: metabolic and plasmidic properties

Summary Comparison of the parental strain of the Leuconostoc mesenteroides subsp. mesenteroides (19D) and its citrate-negative mutant, which has lost a 22-kb plasmid, has confirmed the energetic role of citrate. Fermentation balance analysis showed that citrate led to a change in heterolactic fermentation from glucose. High levels of enzyme activity in both mutant and parental strains were found for NADH oxidase, lactate dehydrogenase, acetate kinase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, although NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase were partly repressed by citrate. All these enzymes studied were not plasmid linked. In the parental strain, citrate lyase was induced by citrate. No citrate lyase activity was found in the citrate-negative mutant grown in presence of citrate, but this does not provide evidence that citrate lyase is linked to the 22-kb plasmid. Offprint requests to: C. Diviès     

Expression of citrate permease gene of plasmid pCM1 isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7 in Lactobacillus casei L-49-4

Recombinant vector pJLECit (8,232 bp) was constructed using citrate permease gene contained in the 3,919-bp fragment of plasmid pCM1 (8,280 bp) isolated from Lactococcus lactis subsp. lactis biovar diacetylactis NIAI N-7, repA and ori from pLU1, and pMB1 ori and the erythromycin resistance gene from pJIR418. Lactobacillus casei L-49-4 (plasmid-free mutant of strain L-49) harboring the constructed pJLECit converted citrate into diacetyl/acetoin. Citrate uptake rate of resting cells was the highest at pH 5.5 and 10 mM citrate concentration. Diacetyl formation activity by the cell-free extracts of Lb. casei L-49-4 (pJLECit) grown in de Man–Rogosa–Sharpe (MRS) broth was higher than that of cells grown in MRS broth without citrate. On the other hand, diacetyl reductase activity of cells grown in MRS broth was lower than that of cells grown in MRS broth without citrate.     

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.     

Effect of varying citrate levels on C4 compound formation and on enzyme levels in Leuconostoc mesenteroides subsp. cremoris grown in continuous culture

Summary The effects of citrate on diacetyl, acetoin and 2,3-butylene glycol (2,3-BG) production by Leuconostoc mesenteroides subsp. cremoris grown in continuous culture at pH 5.2 were studied. In glucose alone end-product production agreed with the theoretical stoichiometry. In the presence of citrate, lactate and acetate production was higher than the theoretical stoichiometry from glucose. Lactate production was constant when the initial citrate concentration was increased whereas ethanol production strongly decreased. In the absence of citrate, citrate lyase (CL) exhibited weak activity. Diacetyl reductase (DR) and acetoin reductase (AR) exhibited basal activity. When varying citrate concentrations ranging from 10 to 75 mm were added to glucose broth, DR, AR, lactate dehydrogenase, NADH oxidase and alcohol dehydrogenase decreased as the initial citrate concentration increased suggesting that they were partly repressed by citrate. In contrast, CL increased and the specific citrate utilization rate also increased in the same way, indicating no saturation of the first step of citrate metabolism. Acetate kinase (AK) was slightly higher in the presence of citrate and increased when the initial citrate concentration increased. This result was correlated with an increase of acetate from the acetyl phosphate pathway. More ATP was produced in the presence of citrate, which could explain the increase in biomass formation. Citrate bioconversion into diacetyl, acetoin and 2,3-BG increased as the initial citrate increased. Correspondence to: C. Diviès     

Influence of temperature on flavour compound production from citrate by Lactobacillus rhamnosus ATCC 7469

The citrate utilization by Lactobacillus rhamnosus ATCC 7469 was found to be temperature-dependent. The maximum citrate utilization and incorporation of [1,5-14C]citrate rate were observed at 37 degreesC. At this temperature, maximum citrate lyase activity and specific diacetyl and acetoin production (Y(DA%)) were observed. The high levels of alpha-acetolactate synthase and low levels of diacetyl reductase, acetoin reductase and L-lactate dehydrogenase found at 37 degreesC led to an accumulation of diacetyl and acetoin. Optimum lactic acid production was observed at 45 degreesC, according to the high lactate dehydrogenase activity. The NADH oxidase activity increased with increasing culture temperature from 22 degreesC to 37 degreesC. Thus there are greater quantities of pyruvate available for the production of alpha-acetolactate, diacetyl and aceotin, and less diacetyl and acetoin are reduced.     

Citrate utilization by free and immobilized Streptococcus lactis subsp. diacetylactis in continuous culture

Summary The effects of pH, temperature and concentration of citrate were investigated to achieve an optimal production of diacetyl, acetoin and C₂ compounds such as acetaldehyde, acetate and ethanol for free and immobilized cells. The critical conditions of culture, 22°C, pH 4.8, increased the production of C₄ compounds (diacetyl, acetoin, 2, 3 butylene glycol), C₂ compounds (acetaldehyde, ethanol, acetate) and formate. A higher yield of C₂ and C₄ compounds was observed for the immobilized cells than for the free cells in continuous culture. At 75 mMol/l of citrate, the citrate bioconversion yield was 42.8% and 80% for free and immobilized cells, respectively. This paper discusses citrate and lactose utilization and NADH₂ part on diacetyl reduction.     

Diacetyl, Acetoin, and Acetaldehyde Production by Mixed-Species Lactic Starter Cultures

Citrate utilization and acetoin, diacetyl, acetaldehyde, and lactic acid production in milk at 21 C by five different mixed-strain starters, containing Streptococcus diacetilactis (D type), Leuconostoc (B type), and S. diacetilactis and Leuconostoc (BD type), were measured. BD and D cultures utilized citrate more rapidly and produced more diacetyl, acetoin, and acetaldehyde than B types. All cultures produced much more acetoin than diacetyl, with the BD and D cultures producing four to five times larger amounts of acetoin than the B cultures. Reduction of diacetyl and acetoin toward the end of the normal incubation period was characteristic of BD and D cultures, whereas a similar reduction of acetaldehyde was characteristic of BD and especially of B cultures. Continued incubation of B cultures beyond 17 h also resulted in reduction of diacetyl and acetoin. Addition of citrate to the milk retarded diacetyl and acetoin reduction. Mn²⁺ had no effect on diacetyl production by a BD culture but increased citrate utilization and, as a consequence, caused greater diacetyl destruction in one of the B cultures.     

Localization of Lactococcus lactis ssp lactis bv diacetylactis in alginate gel beads affects biomass density and synthesis of several enzymes involved in lactose and citrate metabolism

Summary Lactococcus lactis ssp lactis bv diacetylactis, immobilized in calcium alginate beads, was grown in synthetic medium in a continuous flow reactor. Cell distribution inside the gel, as well as the activity of various enzymes, was measured after 30 h of operation. The included biomass tended to concentrate at the periphery of the bead along a section of radius about 100 m long. ATPase activity was maximal in this zone. The activity of NADH oxidase, alcohol dehydrogenase, diacetyl reductase and acetoin reductase, which are repressed in the presence of citrate, were higher in the deeper zones than at the surface of the beads. This result shows that only the peripheral zone of the bead is responsible for the bioconversion of citrate into flavour compounds (diacetyl and acetoin).     

Diacetyl and Acetoin Production by Lactobacillus casei

Agitation of broth cultures of Lactobacillus casei retarded cellular dry weight accumulation but enhanced production of both diacetyl and acetoin. Addition of pyruvate overcame this retardation, but addition of sulfhydryl-protecting reagents did not. Both pyruvate and citrate enhanced accumulated dry weight of L. casei incubated without agitation, but only pyruvate increased diacetyl accumulation. Both actively dividing cells and cells suspended in buffer converted pyruvate to diacetyl and acetoin. Maximum production of diacetyl and acetoin occurred during the late logarithmic or early stationary phases. Cells isolated from pyruvate- or citrate-containing cultures showed the greatest ability to convert pyruvate to diacetyl and acetoin. The optimum pH for diacetyl and acetoin formation by whole cells was in the range of 4.5 to 5.5. The presence of citrate or acetate enhanced diacetyl and acetoin formation by L. casei cells in buffer suspension.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu2+

Citr⁺Lactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu²⁺. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu²⁺, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu²⁺. On the other hand, the Y_Glu value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu²⁺ enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu²⁺ stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP_i. In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Glucose/Citrate Cometabolism in Lactococcus lactis subsp. lactis biovar diacetylactis with Impaired α-Acetolactate Decarboxylase

The pyruvate metabolism of a Lactococcus lactis subsp. lactis biovar diacetylactis mutant deficient in α-acetolactate decarboxylase and its wild-type strain was studied during batch cultivations. A chemically defined medium was used containing glucose as carbon- and energy-source. The α-acetolactate decarboxylase deficiency had no effect on the specific growth rate. Addition of citrate was found to increase the specific growth rate of both strains under aerobic and anaerobic conditions. The product formation was monitored throughout the cultivations. The carbon- and redox-balances were within the accuracy of the experimental data. When citrate was added, α-acetolactate, diacetyl, and acetoin were formed, and aeration was shown to have a positive effect on the formation of these metabolites. By omitting lipoic acid (required for a functional pyruvate dehydrogenase complex) from the growth medium, a similar stimulatory effect on α-acetolactate, diacetyl, and acetoin formation was observed under aerobic conditions. The strain with impaired α-acetolactate decarboxylase activity accumulated α-acetolactate which resulted in an increased diacetyl formation compared to the wild-type strain, under aerobic and anaerobic conditions.     

Acetoin Fermentation by Citrate-Positive Lactococcus lactis subsp. lactis 3022 Grown Aerobically in the Presence of Hemin or Cu

CitrLactococcus lactis subsp. lactis 3022 produced more biomass and converted most of the glucose substrate to diacetyl and acetoin when grown aerobically with hemin and Cu. The activity of diacetyl synthase was greatly stimulated by the addition of hemin or Cu, and the activity of NAD-dependent diacetyl reductase was very high. Hemin did not affect the activities of NADH oxidase and lactate dehydrogenase. These results indicated that the pyruvate formed via glycolysis would be rapidly converted to diacetyl and that the diacetyl would then be converted to acetoin by the NAD-dependent diacetyl reductase to reoxidize NADH when the cells were grown aerobically with hemin or Cu. On the other hand, the Y(Glu) value for the hemincontaining culture was lower than for the culture without hemin, because acetate production was repressed when an excess of glucose was present. However, in the presence of lipoic acid, an essential cofactor of the dihydrolipoamide acetyltransferase part of the pyruvate dehydrogenase complex, hemin or Cu enhanced acetate production and then repressed diacetyl and acetoin production. The activity of diacetyl synthase was lowered by the addition of lipoic acid. These results indicate that hemin or Cu stimulates acetyl coenzyme A (acetyl-CoA) formation from pyruvate and that lipoic acid inhibits the condensation of acetyl-CoA with hydroxyethylthiamine PP(i). In addition, it appears that acetyl-CoA not used for diacetyl synthesis is converted to acetate.     

Formation and stabilization of diacetyl and acetoin concentration in fully grown cultures ofStreptococcus lactis subsp. diacetylactis

Summary The effects of pH and temperature on diacetyl and acetoin concentration and diacetyl:acetoin ratio evolution of non-growing cells ofStreptococcus lactis subsp. diacetylactis CNRZ 124 were studied. A cooling down to 10°C allowed the cells to retain 6 to 10 times as much diacetyl. A large reduction of acetoin was promoted at pH 7 whereas a twice increase was observed at pH 4,8. As a result of these variations, the ratio diacetyl: acetoine showed an opposite evolution according to the pH.     

Effect of lactic acid on diacetyl and acetoin production byLactobacillus casei subsp.rhamnosus ATCC 7469

Lactic acid or its acidity apparently play an important role in the regulation of the biosynthesis of flavor compounds inLactobacillus casei subsp.rhamnosus ATCC 7469. In pyruvate-containing media,L. casei produces lactic acid, acetoin, and diacetyl. A specific pH-dependent system is necessary for both the use of pyruvate and the induction of acetoin and diacetyl production. In cell extracts ofL. casei, lactic acid inhibits the enzymatic activity of acetolactate decarboxylase (ALD) and acetolactate synthetase (ALS); this effect does not occur in whole cells under standard physiological conditions. Lactic acid prevents the use of pyruvate, and the induction of acetoin and diacetyl production. When pyruvate-containing media are used, the pH must be kept close to 6.0 in order to obtain the best production of acetoin and diacetyl.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates

Lactobacillus rhamnosus is a heterolactic acid bacterium, which can be used to produce flavour compounds like diacetyl and acetoin. Various startegies have been applied to improve the growth rate and diacetyl yield. The use of multiple substrates affected growth as well as the yield of diacetyl. Growth on a medium containing glucose demonstrated a diauxic growth profile, with the second phase of growth being on the product, lactic acid. L. rhamnosus also grew on a medium containing citrate. Growth on medium containing glucose+citrate demonstrated simultaneous utilization of carbon sources. L. rhamnosus did not grow in a medium containing acetate and also did not co-metabolize it with glucose. Maximum specific growth rate ( _max) was found to increase in the case of simultaneous utilization of glucose+citrate (0.38 h^–1) as compared to glucose as the sole carbon source (0.28 h^–1). The yields of diacetyl were also found to increase for glucose + pyruvate and glucose + citrate (0.10 and 0.05 g g^–1 of glucose, respectively) as compared to glucose alone (0.01 g g^–1 of glucose). The productivity of diacetyl on medium containing glucose and citrate was double that of a medium containing only citrate, although the yields were comparable.