Biochimie 70 (1988) 375-379 © Soci6t6 de Chimie biologique/Elsevier, Paris
Role of malolactic fermentation in lactic acid bacteria Pierre RENAULT*, Claude GAILLARDIN and Henri HESLOT Laboratoire de GOnOtique, INA P-G/CBAL 78850 ThivernaI-Grignon, France (Received 24-7-1987, accepted after revision 3-11-1987)
Summary -- Although decarboxylation of malate to lactate by malolactic enzyme does not liberate biologically available energy (e.g., ATP, NADH), the growth rate of many malolactic bacteria is greatly enhanced by malolactic fermentation. The deacidification of the medium due to malate dissipation cannot fully account for this situation. The chemiosmotie theory postulates that another form of energy could generated by translocation of protons through the membrane coupled to end-product effiux. Konings et al. showed that this theory is indeed applicable to lactate efflux in Streptococcus cremori's at pH 7.0. A similar mechanism could account for the observed increzsed activity in malolactic bacteria. The study in wild type and mutant strains of Streptococcus 'actis unable to carry out malolactie fermentation led us to the following conclusions: (1) under giucose non-limiting conditions, malolactic fermentation helps to maintain pH of the medium at a certain level; (2) during glucose limited growth, malolactic fermentation could be coupled with an energetic process independent from that mentioned above. Streptococcus lactis I malolactic fermentation / pH maintenance / chemiosmotic theory I energy-coupled reaction
introduction Malic acid is an organic acid found in large amounts in most vegetable matter such as fruits and vegetables (1-14 g/kg, generally 2-3 g/kg). Different hypotheses regarding malate utilization have been proposed: first, malate could be used as carbon source and/or energy by Streptococcusfaecalis  and Escherichia coli  by a malic enzyme-catalysed decarboxylation ofmalate to pyruvate and concomitant production of NADH; secondly, malate dehydrogenase could also lead to malate assimilation producing oxalacetate and generating NADH; other bacteria also have a second decarboxylating enzyme called malolactic enzyme (MLE) [3, 4, 5]. This last enzyme transforms L-malate to L-lactate directly withtmt production of NADH or other energetic compounds. Lactate is then rejected in the med-
*Author to whom correspondence shouM be addressed.
ium. MLE occurrence in lactic acid bacteria has been well documented for Lactobacillus plantarum , Leuconostoc mesenteroides , Leuconostoc oenos  and Streptococcus iactis ; this enzyme may be found in most lactic acid bacteria in plants and even in milk products. In L. plantarum, this enzyme is induced in the presence of malate and glucose, whereas only malic enzyme is produced if glucose is lacking . Authors postulate that MLE could have an ecological function, allowing better growth of bacteria by increasing extracellular pH, which would otherwise drop rapidly due to lactic fermentation of sugar . When glucose is lacking, however, malate is then assimilated via pyruvate by malic enzyme. In L. oenos as in Streptococcus lactis, no malic enzyme is produced. Nevertheless, malolactic fermentation (MLF) was shown to stimulate growth of L. oenos, even if no pH change was
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found during growth . The significance of high MLE activity in this bacteria is thus unclear since the reaction does not 3,ield any metabolizable end products, or reduction equivalent, or energy equivalent; and, as demonstrated previously , M L F is also not exergonic enough to allow production of ATP. In S. cremoris, Brink et al.  have shown that in addition to metabolic energy generated by substrate level phosphorylation, end product efflux such as lactate combined with protons may supply a significant a m o u n t of additional energy that could be as high as 50% per glucose c o n s u m e d . This m e c h a n i s m involves the ATPase of S. lactis that can catalyse ATP formation with concomitant transtocation of 2 protons. This proton motive force which can be generated by lactate efflux is maintained as long as the energy source is available. Malate decarboxylation by M L E also produces a lactate effiux and M L F may t h e n help the cell to keep the proton motive force required for maintaining its intracellular metabolite pool. Such a meciianism could explain the stimulation of growth observed during MLF. In this paper we report on the comparative effects of M L F on growth under different conditions of wild type S. lactis and a previously obtained mutant. T h e different hypotheses mentioned above on the role of M L F in lactic acid bacteria will be discussed.
Materials and methods Bacterial strains The bacterial strains used were Streptococcus iactis IL1441  and its derivative M35, selected for its MLF-defective phenotype. M35 mutant was obtained by insertion of the transposon Tn916, as described earlier . Crude extracts of M3S have no detectable MLE activity, and M35 appeared to be a mutant in the structural gene of MLE (Renault, in preparation). Media and culture conditions S. lactis strains were grown in E modified medium  without tomato juice. Glucose and L-malate concentrations were adjusted as mentioned in the text. Concentrated stock media were prepared and supplemented with malate or water for control experiment. Inocula (2%) were cells grown overnight in M17 medium . Batch cultures were performed in 1-1 flasks. The temperature was maintained at 30°C
and the culture controlled by automatic addition of 2N NaOH or 2N HCI (pH meter 28, titrator 11, magnetic valve MNV1, Radiometer, Copenhagen, Denmark). Continuous fermentations were performed in 250-ml flasks. Medium was pumped into the fermentor at the appropriate dilution rate, D (D, proportion of culture replaced per h) and the culture was allowed to reach steady-state (i.e., grown for at least 6 mean generation time) before being sampled. Culture purity was checked by phage sensitivity for each sample. Growth-rate measurement Optical density at 600 nm was measured every 15 min with a LKB 4049 spectrophotometer. Samples were diluted in the same media to obtain OD<0.5. Growth-rate was then calculated both on semi-logarithmic paper and with a computer program. Biomass was expressed as weight of wet cells per I. Known volumes of the culture (250 ml-1 1) were centrifuged at 4°C, washed once and the pellet weighed in a preweighed tube after draining the water. Sampling procedure and measurements Samples (5 ml) were immediately filtered through a 0.22/~m-Millipore filter. Malate determination was performed as described previously by enzymatic method  or by paper chromatography . Malate decarboxylation was determined by measuring CO2 released from malate with a CO2 electrode (Radiometer, Copenhagen, Denmark)  and by following the pH change in resting cell experiments. A computer program was set up to calculate the rate of MLF from change in pH, taking into account the : pK~MALATE , pK2MALATE , pK LACTATE , concentration of malate, pH and buffer capacity of the medium.
Results M a l o l a c t i c f e r m e n t a t i o n by S. lactis G r o w t h ofS. iactis in m e d i a containing 5-10 g/l glucose was followed by rapid acidification of the media down to pH 4.5 after 3 1/2 h, so that cells stopped growing due to this low pH. W h e n L-malate was added (10-20 g/l), cells were still growing after 6-8 h and cell density reached levels 4-6-fold higher than those observed in the absence of malate, pH values remained stable at pH 6.2 until glucose or malate had been completely dissipated. As previously described , S. lactis is able to carry out M L F in the presence of a fermentative sugar such as glucose. This e n h a n c e d growth can be mainly accounted for by the strong deacidification due to malate decarbo-
Role of malolactic fermentation in lactic acid bacte, ia xylation resulting in a buffering effect, balancing glucose metabolism. The pH 6.2 value has been mentioned as the optimum pH for growth of streptococci . A comprehensive study of the role of MLF in S. iactis thus required that the pH be set at different values, in order to dissociate the beneficial effect of MLF (due to maintenance of pH level) from another possible MLF role in the metabolism.
Malolactic fermentation at different pHs A set of experiments was carried out to determine the generation time ofS. lactis IL1441 in a chemostat in which pH was maintained at a constant value. Growth media contained 5 g/l glucose and 20 g/l L-malate when mentioned. As shown in Figure 1, S. lactis IL1441 wild type (A) and M35 (B) had an o~fimum growth rate at pH 6, with a doubling time of 45 min in malate-free media. Addition of L-malate modified the generation time in the wild type (A) but had no effect on the mutant strain M35 (B). At high pH values (> 6.2-6.5) the growth-rate of the wild type strain was enhanced, whereas at lower pH it was reduced. The study of the M35 mutant without the key enzyme for MLF showed that malate had no effect on its growth at any pH; it follows that MLF may be responsible for the change in growth properties. As shown in Figure 1 (C), MLF rate depend,,,4 pH ~ d---' :I t. . W. .a 3. . found that "'-me- maxi~ u Oil " t l l" ~ llU mum MLF rate was obtained at pH ~ 5. Malate decarboxylation decreased rapidly if the pH values were not maintained at this level. Nevertheless, cells grown in the presence ofmalate at pH 5-7 presented the same malate decarboxylating activity (C). Thus, differences observed in the MLF rate probably reflected uptake of malate and not the induction level of the enzymatic system in the cell. This was confirmed by shift-up or shift-down experiments" cultures grown at pH 5, 6 and 7 and shifted rapidly to another pH presented in vivo activity identical to cells grown at the corresponding final pH. At the usual optimum growth of pH 6.2, the generation time orS. lactis IL1441 increased by 10% in the presence of 2% malate, and by 30% lower malate concentration (1%). Glucose concentration (0.5-1%) had little effect on growthrate at any malate concentration (not shown).
Effect o f glucose and nitrogen starvation At pH 5.0, in the presence of excess glucose
g 50l m
Fig. 1. Malolactic fermentation in E modified medium. (a) S. lactis IL 1441; (b) M35. Symbols : (O), 2% malate; (A),
without malate. (c): (l=l)Malolactic fermentation rate (mM malate decarboxylated per h and per g wet cell). (ll): MLF specific activity (#mol CO2 per min and per mg protein in crude extract).
and nitrogen, MLF was shown to have an inhibitory effect on growth rate of S. lactis, so that its value was 16% higher in mala:e free media. Reduced glucose level (0.5 g/l) had little effect on growth rate if malate was omitted from the medium (generation time 49 min). In the presence of malate at pH 5, growth was activated during the first 2 h of growth (generation time 38 min) and then reduced (generation time 60 min). Final cell density was also significantly higher. Continuous fermentation at pH 5.0, D=0.3 and with 0.5 g/l glucose confirmed growth stimulation by malate under glucose limitation.
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At steady-state, biomass (wet cells) was 3 g/l in media containing malate, whereas only 2.4 g/l was obtained without malate. Under these conditions, we checked that the malolactic system was fully induced. The same experiment with M35 mutant showed that addition of malate had a slight negative influence on growth and biomass at steady-state. We thus concluded that MLF was responsible for the stimulation observed in the wild type strain. Preliminary studies on batches with slowly assimilated carbon sources such as starch, mannitol or arginine also showed a higher cell density in the presence of malate. To study the effect of nitrogen starvation, we carried out continuous fermentations at pH 5.0 and pH 7.0 with D=0.3 and D=0.6. The medium used was modified medium E diluted 4fold and adjusted to 40 g/l glucose. Without malate, steady-state was reached at any D, but addition of 20 g/l rnalate induced cell lysis so that the OD of cultures dropped to less than one fifth of the initial value.
Discussion The role of MLF has been questioned. We postulate a dual role for MLF in lactic acid bacteria: (1) under glucose non-limiting conditions, MLF helps maintain the external pH around pH 7.0; (2) under glucose-limiting conditions, MLF may generate energy through a coupling mechanism. Both effects are discussed below in view of our results and those obtained in other lactic acid bacteria. Under conditions where glucose and nitrogen sources are not limiting, it is clear that MLF is not favourable to S. lactis, especially at low pH (
the lactate gradient and the energy produced may offset the metabolic cost involved in carrying out this fe~'mentation at pH 7.0, but not at pH 5.0. Indeed, at low pH (<6) MLF is not favourable to S. lactis. It has been reported that malate uptake in L. plantarum requires energy . If MLF had an energy-dependent step (e.g., for malate uptake), differences observed in growth inhibition at different pHs could be explained by a higher MLF rate at pH 5.0 than at pH 7.0. At pH 5.0, MLF rate and therefore energy consumption is maximal, whereas energy recycling by lactate efflux is minimal. There still remains another question: why is an optimal MLF rate observed at pH 5.0, at which level no energy can be generated by lactate efflux? We conclude that when there is an excess of energy, the only positive effect of MLF on the cell is an increase in pH of the medium due to malate decarboxylation. A positive effect of MLF on cell-yield was clear under glucose limitation even at pH 5.0, where lactate efflux produced by glucose fermentation was not coupled with additional proton translocation through the membrane (see above). This effect only appeared if energy source was the limiting factor and was not detectable in a mutant affected by MLF. Such a result strongly suggests that MLF may supply energy to the cell. As malate decarboxylation by MLF is not very exergonic, and since the free energy of the reaction is not biologically available to the cell , a coupling mechanism must be involved. Previously [9, 24] it was shown that: (i) entrance ofmalate required a transport system; (ii) MLF was inhibited by sodium azide, which inhibits the generation of an electrical potential; (iii) internalized malate was rapidly converted to lactate and reexported. Furthermore, MLE unlike other decarboxylating enzymes, does not release anhydrous carbon dioxide, but carbonic acid, bicarbonate ions or carbonate ions . We thus postulate the existence of a mechanism operating at pH 5.0 which would link malate influx and/or lactate and carbonate efflux to an energy-consuming process in the membrane by a reverse flow of protons and/or positive ch~,rges. This hypothesis could be tested by measuring the effect of MLF on electrical potential, pH gradient and gradients of malate, lactate and carbonate across the membrane. This hypothesis is strengthened by other reports on lactic acid bacteria. Experiments on
Role o f maiolactic f e r m e n t a t i o n in lactic acid bacteria
growth rate and biomass production with a strain of L. oenos  put forward similar arguments: (i) growth rate increased in the presence ofmalate; (ii) optimum pH growth was 5.5 without malate and 5.0 with malate; (iii) dry weight and cell density increased significantly if malate was added. Also, after M L F had been carried out in wine, addition of L-malate to the latter allowed growth to resume but malate was not incorporated into the cell and was not transformed into volatile acidity . In this case growth of L. oenos may occur at the expense of energy produced by M L F as we postulate, and residual sugar, citric acid and nitrogen compounds liberated by yeast autolysis [26, 27] may be assimilated as carbon and nitrogen source [28, 29]. It has also been shown  that catabolism of malic acid increases proton motive force and inter.nal pH ofL. oenos and L. plantarum, which again is in agreement with an energetic role for MLF. Further study of our mutants will thus help us to characterize genes involved in this process (at least 4; manuscript in preparation), but further studies regarding the role of M L F in natural biotopes will require study of wild type strains freshly isolated from plants or fruits, and development of suitable maintenance media. A better knowledge of the role of MLF, which is of considerable importance for the wine industry , is of great interest. Understanding its mechanism and effect on ceil viability could be of some help in the production of an active starter for M L F m wine-making.
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