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A Tentative Model to Evaluate the Kinetics of Malolactic Fermentation

A Tentative Model to Evaluate the Kinetics of Malolactic Fermentation
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  A Tentative Model to Evaluate the Kinetics of Malolactic Fermentation G. ANDRICH? s. CASELLA: R. FIORENTINI,~ AND P. SPETTOLI~ bIstituto di Industrie Agrarie 'Istituto di Microbiologia Agraria Universith di Pisa Pisa. Italy dScuola Superiore Studi Universitari e Perfezionamento S. Anna Pisa, Italy INTRODUCTION Malolactic fermentation in wine-making greatly contributes to wine quality because it lowers the overtly high acidity and improves the sensory characteristics and microbiological stability of the wine. The conversion of L-malate to lactate, carried out by heterolactic bacteria working in batch or in immobilized form, has been studied widely,'-' but knowledge of the kinetic steps involved would allow for the control of this fermentation process in order to obtain more efficiently the desired bioconversion. This discussion reports some of the preliminary results concerning the kinetics of the malolactic fermentation induced by Leuconostoc oenos ML 34. MATERIALS AND METHODS In order to evaluate L-malate fermentation induced by Leuconostoc oenos ML 34, a suitable experimental apparatus was set up. The apparatus mainly consisted of a flask connected to a sampler that was able to collect the desired amount of liquid from the reactor bulk under sterile conditions; it used a pressure of sterile nitrogen as the drawing force. Also, in order to ensure an anaerobic and sterile environment during the whole experimental run, he apparatus previously autoclaved was kept under a flow of sterile nitrogen. The different microorganisms, if present, were removed from the reaction solution by filtration (0.2 Fm), whereas the desired pH was ensured either by an acetate buffer or through a partial titration of the employed L-malic acid with KOH. Leuconostoc oenos ML 34 was grown in a tomato juice broth as described in a previous paper.' The components of the growth medium were removed from the Leuconostoc oenos ML 34 bacteria by filtration (0.2 Fm) and then the cells, carefully washed with a sterile NaCl solution (l ), were resuspended in the reaction medium 'This work was supported by the Italian Research Council (CNR), Rome, Italy. 356  ANDRICH r al.: MALOLAmIC FERMENTATION 357 (T 25 C) by utilizing an inverse flow of sterile water. The time corresponding to the bacteria inoculum was assumed to be the start of the kinetic run, and the concentra- tions of involved compounds, as well as the cells employed, were analyzed as a function of the run time. The chemical determinations were carried out by enzymatic assays, whereas the bacteria concentration was determined by a total plate count on tomato juice agar.' RESULTS AND DISCUSSION Because the count of viable cells remained nearly constant (about 10 microorgan- isms/L) in the adopted reaction conditions, it was possible to determine the kinetic TIME ~~103) FIGURE 1. Time evolution of concentrations of L-malate (A), L-lactate B), D-hCtate (C). and viable cells (D). order of the decrease of L-malate. A first-order kinetic equation was able to fit the experimental points (FIGURE with good accuracy r2 - 0.9): -d[~-mal] t - )/dt kl [~-mal] t t), (1) [~-maI] t - ) [~-maI] t - 0 e- .'. Then, after linearization of the integrated form, the kl could be evaluated: n {[~-mal] t t)/[~-mal] t - O)} -kI t. In addition, by knowing the mean number of viable cells N), he kinetic constant, klb, measured for active microorganisms could be calculated: klb - kl/R 2.35 x lo- (L/s . n'viablecells) (SEM - 0.32 x 10-17).  358 ANNALS NEW YORK ACADEMY OF SCIENCES By fermentation of 1 mole of L-malate in the adopted reaction conditions, 0.85 moles of L-lactate was obtained, together with 0.15 moles of the D-isomer. Only the two enantiomeric forms of lactic acid and the residual amount of L-malate were detected in the reaction bulk, and the sum of the concentrations of these three compounds was nearly constant and close to the initial value of L-malate FIGURE ). Furthermore, because the lactic fermentation of glucose induced by Leuconostoc oenos ML 34 produced only the D-lactate i~omer,~ t could be hypothesized that pyruvate is an intermediate in the production of D-lactate, whereas the L-isomer is synthesized directly from L-malate. Consequently, the following two alternative, concurrent pathways could be hypothesized: (a) direct decarboxylation of L-malate to L-lactate; (b) production of D-lactate through a pathway involving pyruvate as an interme- diate compound. 40 1 I I I I 0 1 2 3 4 5 TIME ~~10~) FIGURE 2. Calculated time evolution of concentrations of L-malate (A), L-lactate B), and D-lactate (C). A hypothetical isomerization of a previously synthesized L-lactate to give D-laCtate did not seem to occur, probably because of a lack of L-lactic-dehydrogenase in Leuconostoc oenos ML 34. In fact, no L-lactate decrease was detected when these bacteria were incubated with this substrate. Therefore, the following equations concerning the formation rates of the two enantiomeric forms of lactic acid can be formulated: d[~-lac] t t)/dt kl [~-mal] t - ), d[D-laC] (t - )/dt - k2 [L-mall (t - ). (2) 3) Because the L(D)-lactate/L-malate ratio at a random time, t t, is equal to the product of the time, t, and the kinetic constant, kl (k2), the kinetic constant involved can be  ANDRICH et a/.: MALOLACI'IC FERMENTATION 359 evaluated as the slope of the straight line obtained: kl klb N; k2 k2b N; klb 1.75 x k2b 0.35 x (L/s n'viablecells); (L/s . o viable cells). Moreover, because the forms of the equations concerning the rate of the L-malate decrease and the L/D-lactate increase are the same (compare equation 1 with equation 2 or 3) and also because only L- or D-laCtate is detected in the reaction bulk during L-malate fermentation, the sum of the two kinetic constants due to D and L formation (2.1 x lo-'') must be equal to the value of the constant of the L-malate decrease On the basis of the kinetic model hypothesized and of the two resulting equations (equations 2 and 3), it is possible to evaluate the time evolution of the L-malate and L(or D)-hCtate in the reaction conditions adopted (FIGURE ). Furthermore, these equations, together with those concerning the time evolution of the bacteria concentra- tion, would allow for the calculation of the development of the species involved in malolactic fermentation induced by Leuconostoc o nos ML 34. (2.35 x 10-1 1. REFERENCES 1. SPEITOLI, P., A. BOTTACIN, . NUTI A. ZAMORANI. 982. Immobilization of Leuconostoc oenos ML 34 in calcium alginate gels and its application to wine technology. Am. J. Enol. Vitic. 33 1-5. 2. SPETTOLI, ., M. NUTI, A. CRAPISI A. ZAMORANI. 987. Technological improvement of malolactic fermentation in wine by immobilized microbial cells in a continuous flow reactor. Ann. N.Y. Acad. Sci. 501: 386-389. KUNKEE, . 1974. Malo-lactic fermentation and winemaking. In Chemistry of Winemaking. Advances in Chemistry, series 137. A. Webb, Ed.: 151-170. Amer. Chem. SOC. Washing- ton, District of Columbia. 3.
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