Some roles of malic acid in the malolactic fermentation in wine making

Some roles of malic acid in the malolactic fermentation in wine making

FEMSMicrobiologyReviews88 (1991)55-72 @1991Federationof EuropeanMicrobiologicalSocieties0168-6445/91/$03.50 Publishedby Elsevier ADONIS 01686445910005...

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FEMSMicrobiologyReviews88 (1991)55-72 @1991Federationof EuropeanMicrobiologicalSocieties0168-6445/91/$03.50 Publishedby Elsevier ADONIS 016864459100055P FEMSRE00217

Some roles of malic acid in the malolactic fermentation in wine making * Ralph E. Kunkee Department of Viticulture and Enoiogy, Universilyof California, Dat,is, California, U.S.4.

Received19December1990 Accepted9 April1991 Key words: Lactic acid bacteria; Leuconostoe; Energetics; ATP; Deacidification; Malolactic enzyme; NAD

1. SUMMARY The control of the malolactic bacteria, the lactic acid bacteria that carry out the malolactic fermentation, is an important part of the technology of modern commercial wine production. The operative reaction of these bacteria, the decarboxylation of malic acid to lactic acid, can be of small or of large importance, depending upon the climatic environment for the growth of the grapes used as the starting material for the wine and upon the style of wine to be made. In this review, recent information is surveyed on the application of malolactic bacteria in wine production, on the control of these organisms, and on the intermediary metabolism involved in the seemingly simplistic decarboxylation of malic acid to lactic acid of the malolactic fermentation.

Correspondence to: R.E. Kunkee,Department of Viticulture

and lgnology,Universityof California,Davis,CA95616,U.S.A. * Presentedat the ThirdS~anlmsiumon LacticAcidBacteria, Genetics, Metabolismand Applications,September17-21, 1990,Wageningen,The Netherlands.

Central to this survey is the awareness of the special importance of malic acid in the microbiology of the malolactic bacteria and in the production of wine undergoing the malolactic fermentation. In some of the newer winegrowing districts of the world, great strides have been made in the acceleration of this so-called secondary fermentation by use of bacterial starter cultures, often added to the grape must at the same time as the yeast starter cultures are added for the alcoholic fermentation. The early completion of the mainlactic fermentation allows early application of the cellar operations for storage and aging of the wine, which is needed for the protection against further microbial attack. This new technology of addition of bacterial starter is the result of, and in turn has brought about, a surge in the commercial availability of malolactic bacteria in various dried, frozen or liquid forms. There has been a corresponding increase in research interest in these bacteria. Characterizations of the purified malolactic enzymes from several strains of bacteria from several re:eer:h laboratories are presented here as well as infop

mation concerning genetic control of the malolactic activity, including some information about plasmids. Also presented is the new awareness of the discovery of bacteriophages in wine, which has led to research on the susceptibility of the malolaetic bacteria to virus attack and to the associated commercially potential dangers. The continued research interest on some of the environmental influences (pH, nutritional additives, time of removal of the gross yeast lees and temperature) on the malolactic fermentation is discussed, as is the isolation of various new strains of bacteria. From a teleological point of view, it is becoming clear what advantage the malolactic capability has for these bacteria, even though the thermodynamics of the conversion of a molecule of malic acid to lactic acid and carbon dioxide provide for hardly any free energy. This is discussed in terms of the decarborylation reaction, which has been shown to be not absolutely complete. Instead, the reaction seems to provide for a small net produc. tion of hydrogen accepters. This is evidenced by a decrease in the lag period at the beginning of the fermentations in presence of malic acid; that is, a stimulation of the initial growth rate of the bacteria, which under the harsh conditions of practical wine production is sometimes very welcome to the winemaker. In addition to this, the deacidification itself, by increasing the intracellular and extracellular pH, through the conversion of malic acid to lactic acid, is recognized to be advantageous to the cell. Results are also presented, which, in addition to the formation of extra hydrogen accepters, show capture of small amounts of free energy of the reaction for the net formation of high energy storage compounds, again to the cell's advantage.

2. INTRODUCTION The 'malolactic fermentation' is the conversion of malic acid to lactic acid by certain lactic acid bacteria [1-4]. It is most generally thought of in connection with wine production, but it can also be an important aspect of other food fermentations [2]. The control of the fermentation under

commercial wine making conditions has been a wide-spread subject of study. In cold wine regions, the fermentation by these organisms is deemed essential in order to decrease the excess acidity resulting from the high content of malic acid in grapes grown there and in warm regions, the fermentation is considered an essential nuisance for conferring bacteriological stability upon the wine [3,4]. In addition to the review articles cited above, other more recent reviews on the research on the malolactic fermentation in theory and in practice can be consulted [5-10]. The following areas of practical and research interest along with a relevant citation for the reader with special interest have not been addressed in this article: the numerical taxonomy of the malolactic bacteria [11]; karyogamic typing of lactic acid bacterial strains [12]; use of immobilized enzymes and embedded cells for the malelactic conversion and for malolactic fermentation [13]; and the development of special fermentation vessels designed for more efficient malolactic fermentation [14]. Throughout this discussion, for convenience, we will refer to the organic 'acids' as the undisassociated molecules; although it is recognized that at wine and intracellular pHs, substantial portions of the acids are in the dissociated anionic form.

3. MALOLACTIC BACTERIA IN COMMERCIAL WINE PRODUCTION 3.1. Widespread usage of bacterial starter cultures in newer wine growing regions One of the hallmarks of the technical advances being utilized by the California wine industry, and somewhat in other newer wine growing regions also, is the increasing use of bacterial starter cultures to initiate the malolactic fermentation, especially in red wine. When the malolactic fermentation is desired, the addition of bacteria is a general practice there and completion of the malolactic fermentation is expected within a few weeks of harvest date. For white wine production, the controlled stimulation of the malolactic fer-

mentation in some Chardonnay wines is also emerging as a distinct style. In this case, bacterial starters are also employed; but the difference in the methodology as compared to red wine production is that generally only a portion of the white wine is encooraged to undergo the fermentation. With the increased use of malolactic bacteria inoculations, or because of it, there has been a corresponding increase in the commercial avail. ability of starter cultures. At least five preparations are currently on the market. These starter materials are usually received as lyophilized ('freeze-dried') cultures, as compared to wine yeast starters, which are generally sold in the 'active dry' form. (However, promising results for production of'active dry' malolactic bacteria, with the use of fluidized-bed drying, has been reported [15]). In at least one case, as is common in the marketing of cultures for the dairy industry, the bacteria culture is shipped frozen, rather than frozen-dried. With two exceptions, all of the successful commercial cultures of which we are aware are strains of Leuconostoc oenos. For one of the exceptions, the nomenclature of the distributed strain is given as L. oenos, whereas in our hands, it is Pediococcus damnosus. The other exception is a special ease of a strain of Lactobaciilus plantarum and will be discussed in section (3.3.3.). The origin of the commercial cultures is not always of public knowledge; we are aware that various of the commercial strains were originally isolated from wines from California, Pennsylvania, France and Switzerland. The commercial cultures are usually 'expanded' by the trained winery personnel to obtain the high populations of bacteria needed for the desired speedy rate of malolactic fermentation. The expansion involves standard microbiological laboratory techniques, taking care to employ materials that are legally allowable as additions to wine. For example, grape juice may be slightly diluted with water; the pH may be raised with CaCO 3, and limited amounts of yeast extract may be added [16]. In some cases, the inocula for the expanded starters may come from 'in house' cultures, maintained as 'stabs' by the winery, or

as specially ordered cultures from various commercial wine laboratories. Expansion of cultures on a rather large scale has been proposed where aseptic inoculation is bypassed by addition of a very low inoculum of wine yeast to gra~ juice at the same time as a rather large inoculum of bacteria is added [16]. This method allows growth of a high population of bacteria, which is followed by an alcoholic fermentation to protect the starter from further microbial attack, assuming the expanded culture is used expeditiously. The time-honored methodology, where the winemaker relies on the bacteria resident in used barrels in which a desirable malolactic fermentation has previously occurred, is still in evidence, especially in the older wine production regions and where t?~e time of bottling of the wine is not demanding. For this, the wine is stored in a protected condition, i.e. cool and with added sulfur dioxide, which may itself delay the eventual malolactic fermentation for months or possibly a year. One supposes that the infecting organisms are identical strains, from barrel to barrel, from year to year. 3.2. Characte~tics of desirable strains of mat.olactic bacteria for wine production The successful use of bacterial starter cultures for the rapid completions of the malolactic fermentation has led to newer definitions of the acceptable criteria for desirable stains. The most important criterion is that the strain of bacteria will grow reasonably fast in wine under wine making conditions. At present, this selection must be obtained empirically. Only limited new information has appeared (see section 4.1.) giving the definitive micronutrient requirements of the current popular strains, even supposing the winemaker armed with better ~nformation could command the micronutrient composition of the grape must to be fermented. Just as important is the criterion that no offflavors or off-odors are produced by the bacteria during the commercial production of wine. We have previously shown that sensory quality of the resulting wine by 'good' strains of bacteria is affected, but only to a very limited or subtle extent, by that strain [17]. More recent work, with

Chardonnay and with red French-American hybrid grapes from the Eastern United States [18,19], has also shown a slight dependence of wine flavor upon the strain of malolactic organism used [20]. However, we have also observed that some strains can produce obvious off flavors and should be labeled as spoilage organisms; at least one of the strains in our collection can be easily identified by its production of a characteristic off-odor (R.E. Kunkee, unpublished results). A third equally important criterion, not so well recognized, has to do with the commercial production of the cultures. These bacteria are nutritionally fastidious; in the economics of their production it is necessary to determine whether very complex, and probably expensive, growth medium is required. In addition, the fully grown cultures must be able to sustain the rigors of being concentrated, frozen or lyophilized with a minimal loss in viability. All of the currently commercial available malolactic strains meet these criteria. It is interesting to note, however, that L. oenos ML34, the first strain of malolactic bacteria to be used extensively in commercial wine production, some twenty to twenty-five years ago in California, is still not available as a single strain in a concentrated stabilized form because of difficulties in its production. ML34 is of more than historical importance; its effect on the resulting flavor of the wine seems to be as neutral as any strain available [17]. A special word should be said concerning one of the most evident of the end products of most malolactic fermentations: diacetyl. The amounts produced by the acceptable malolactic bacteria in red wine are generally low enough to be barely detected organoleptically. These amounts (1-5 rag/l) are often thought to increase favorably the complexity of the wine flavor [2,20]. However, for white wine production, even the low amounts of diacetyl produced may be excessive, since the white wines are generally more delicate in flavor, Nevertheless, the emerging style of some California Chardonnay's, alluded to above, may depend on detectable amounts of the 'buttery notes' associated with diacetyl. This puts special demands on the winemaker to employ the malolactic fermentation with finesse, and to be especially care-

ful in selection of bacterial stain in order to arrive at wine with just the desired concentrations of these flavor tones. Recent work in Portugal [21], on the content of diacetyl in red and white wines undergoing malolactic fermentation, has confirmed earlier work [20,22] upon which the above discussion is based. Winemakers seem generally satisfied with the spectrum of the bacterial strains available for inducing the malolactic fermentation. Nevertheless hope springs eternal that some 'super-bug' might be isolated, especially from untapped wine regions [23,24]. Studies on 166 isolates from Australian wines revealed an interesting heterogeneity among the wine lactic acid bacteria, but no exceptional strains for commercial use were necessarily identified [8]. Henick-Kling et al. [25] examined several new isolates from a cool viticulture area (Oregon state), and found two strains promising to be more tolerant to low pH as compared with other well-known strains. Some researchers (R.B. Beelman and T. Henick-Kling, personal communications and this author) have noticed that the vigor of recently isolated malelactic strains seems to diminish over the first year or so of its domestication in a culture collection.

3.3. Timing of the malolactic fermentation 3.3.1. Simultaneous alcoholic and malolactic fermentations. We have mentioned the tendency in the newer wine growing regions to hasten the malolactic fermentation. In fact, it is now common in California, with the ready availability of bacterial starters as well as yeast starters, to encourage the two fermentations to occur simultaneously. Blackburn [7] has pointed out that some French enologists are apprehensive about the application of the malolactic fermentation before the completion of the alcoholic fermentat:on. Bordeaux researchers especially have noted an unacceptable formation of acetic acid, said to be formed by the malolactic bacteria in the presence of sugar when the malic acid has been depleted or when the bacteria are no longer in the exponential growth phase [26,27]. Alternatively, it has been suggested that excess formation of acetic acid, when it occurs, may not come from the

bacteria, but rather from the yeast under the stressed conditions resulting from competition between the yeast and the bacteria for nutrients [28]. We have not been able to mimic conditions to bring about formation of acetic acid, other than the small amount normally associated with the malolactic fermentation [29], aor have we been able to substantiate any reports of an increased acetic acid formation from this practice in the California industry. An Australian report [30] supports the use of simultaneous fermentations where sensory evaluation revealed such wines to be free from undesirable odors. Further research is required to establish the importance of the individual strain of yeast or bacteria employed, and of the composition of the grape must used, before one can recommend with complete confidence the use of simultaneous alcoholic and malolactic fermentations. Some anecdotal information is of interest here. Even without the problem of increased acetic acid production, some California winemakers retain the opinion that the sensory quality of the wine suffers somewhat unless the bacterial fermentation follows the alcoholic fermentation. Others suggest that any objectionable diacetyl formation (see above), which could be formed by the bacteria in the presence of high concentrations of sugar and be especially noticeable in white wines, seems to be removed by the yeast during the alcoholic fermentation. For more information on the metabolic interactions between wine yeast and malolactic bacteria, the reader is directed to research by King and Beelman [31] and a general review article by Bisson and Kunkee [100].

3.3.2. Malolactic fermentation preceding the alcoholic fermentation. It has been earlier suggested [2] that technology, which would allow the malolactic fermentation to occur before the onset of the inhibitory formation of ethanol by the yeast, might be the method of choice in the control of the malolactic fermentation. Required for this is either a procedure to inhibit the indigenous yeast, perhaps by application of carbon dioxide pressure; or to encourage a rapid malolactic fermentation before the yeast inoculation is made (or before the indigenous yeast express their activity).

Two methods for encouraging the early catabolism of malic acid have recently appeared and are briefly discussed here.

3.3.3. induction of malolactic fermentation by 'Viniflora' strain. 'Viniflora' is a strain of Lactobacillus plantarum, and as such would not be expected to grow or to carry out the malolactic fermentation in grape juice or wine, because of the low pH of these media. Thus the 'expansion' of such bacterial cultures by the winemaker, as outlined above, would not be applicable. However, Prahl et al. [32] suggest the employment of high concentrations of the organism, to be added to the grape juice before the onset of the alcoholic fermentation by the yeast. With a large enough initial concentration of cells, acting as a stationary culture, the need for growth of the bacteria (expansion) is avoided. The bacteria are capable of making the malolactic conversion, in spite of the low pH, as long as the yeast have not begun their inhibitory production of ethanol. The resulting wines are said to be free from any adverse sensory characteristics. One drawback to this procedure would seem to be the expense, since the winemaker must purchase, rather than grow, the large amount of bacteria needed; however, the apparently relatively easy production (growth and lyophilization) of the culture by the producer, as compared to the production of more fastidious strains, say, of L. oenos, seems to allow the production of it to be economic enough to override this objection. Another drawback is the precarious condition in which the grape juice needs to be placed while waiting for the malolactic conversion to be completed before the onset of the alcoholic fermentation. Reports we have heard from the field indicate that this has not been a serious concern.

3.3.4. Malic acid catabolism by Schizosaccharomyces. The possibility of decreasing the acidity of wine by use of the yeast Schizosaccharomyces, which can ferment high concentrations of sugars to ethanol, and which also degrades malie acid, has intrigued winemakers from very cold wine regions over the years [33,34]. The use of this yeast for deacidification has never been reduced to practicality because the fermentation has commonly been accompanied by noticeable off-odors.

A new and promising means of using this genus has appeared, and although the reaction is not a 'malolactic' reaction, a brief mention is given for the sake of completion. Rodriguez and Thornton [35] have obtained a mutant of Schizosaccharomyces malidervorans, which requires malic acid for growth and which ferments glucose poorly. This strain can be ~sed to remove the malic acid before the inoculation with the normal wine yeast. The latter then carry out the bulk of the fermen. tation of sugar with the end result being a wine without malic acid and without any of the bad flavor characteristics associated with Schizosaccharomyces. It needs to be emphasized that this practice leaves tartaric acid as the only acid in the wine. Tartaric acid, itself, is subject to loss (as potassium bitartrate, which is relatively insoluble). The resulting wine, which may have initially been unacceptably acid, may now be in need of acidulation.

4. CONTROL OF MALOLACTIC BACTERIAL GROWTH AND END PRODUCT FOR. MATION 4.1. Growth conditions and nutritional require. merits As a group, the malolactic bacteria are unusual in their ability to tolerate the hostile envi. ronment of wine with its low pH, high concentration of ethanol and low concentrat" ~n of nutrients, cool temperature, and presence of sulfur dioxide. Earlier reviewers [1-4] have outlined the general growth parameters of these bacteria. Later studies have been confirmatory; but with the isolation of new strains, the earlier limits tend to have become somewhat less restrictive. For example, malolactic fermentations occurring with the initial pH of 3.0 is not now so incredible as once thought [8,25]. (Although, as we have already mentioned, some of the 'native resistance' of the newly isolated strains may diminish with time in laboratory captivity). The present trend in use of lower and lower additions of sulfur dioxide, and sometimes even none at the time of crushing the grapes, has allowed easier opportunities for the malolactic fermentation [36], and

incidentally, easier opportunities for infections from spoilage bacteria and yeast! Also, some winemakers are employing selective production operations, which tend to encourage the malolactic fermentation, such as, in white wine, increased skin contact time at the beginning of the alcoholic fermentation, and increased gross yeast contact time at the end. Both of these operations would tend to increase the availability of the micronutrients, that is, tend to encourage the bacterial growth. Early researchers, and earlier review articles, have provided ample information on the effectiveness of various growth media for the malolac. tic bacteria, both for optimal culture in the laboratory and for wine production. What is missing is more definitive information on the micronutrient requirements of the various strains. In fact, a few studies were made several years ago in attempt to profile the requirements of strains extant at that time [37,38]. More recently, Tracey and Britz [23] have examined nine additional malolactic strains (of L. oenos and P. damnosus) for their amino acid requirements. It was interesting that the substitution of casein hydrolysate was the least effective means of those tried for supplying the necessary amino acid complement. More of this kind of research is needed to include the current familiar maloiaetic strains. The role of oxygen, if any, in malolactic bacteria has had little definitive study. Rib~reau.Gayon and Peynaud [39] found that saturation of new wine with air stimulated malolactic fermentation, but that saturation with oxygen inhibited it. This led one reviewer [2] to remark that mi. croaerophilic ('loving of a small amount of air') would seem to be an apt description for malolactic bacteria. Kelly et al. [40] have provided new, and better replicated, infom~ation on the relative importance of aerobic or anaerobic conditions, with some 20 strains of L. oenos. Their work showed an important stimulatory effect in absence of oxygen. (The stimulation was not caused by an effect of carbon dioxide, often used to displace the oxygen in these kinds of studies; the stimulation also occurred when the air was replaced by nitrogen). They found formation of colonies as large as 1 mm in diameter in two or

three days under strictly anaerobic conditions, compared with five to ten days under air. It is not clear how important this information will be when applied to commercial wine production operations, but it certainly can be of great practical importance for the wine microbiologist endeavoring to cultivate these bacteria directly from wine or from grape juice. The apparent inability of the malolaetic bacteria to use even limited amounts of oxygen as a hydrogen acceptor adds weight to the suggestion (section 5.3) that the role of malic acid in its stimulation of the mak,lactic fermentation is to provide additional hydrogen acceptors. The use of yeast hulls ('yeast ghosts') as a nutritional supplement for yeast to enhance the alcoholic fermentation has had some recent appeal. The mechanism of action of these agents, which are derived from the 'spent lees' from vinifications is not clear at this writing [41]. The hulls seem to be a mix of fragments of cell walls and cell membranes and thus could provide nitrogenous materials, or 'survival factors' (unsaturated long chain fatty acids and sterols), or surfaces for absorption of inhibitory medium chain fatty acids (for example, decanoic acid), or all three. It is to be expected that these agents would be tested for their effect on the malolactic fermentation also. So far, experience seems to be limited. We are aware of reports of both stimulatory [42] and inhibitory effects (G. Wann and R.B. Beelman, personal communications) of yeast hulls on the malolactic fermentation.

4.2. End products With regard to end products of the malolactic fermentation, besides those chemicals contributing to the sensory quality of the wine (section 3.2), we are forced to examine the possibility of the formation of compounds reported to be health hazards. Two major candidates, at least from the popular literature, are histamine and ethyl carbamate (urethane). Recent research supports the notion that there is no important formation of either of these compounds in connection with the malolactic fermentation. In the first case, Ough et al. [43] and Delfini [44] showed essentially no production of his-

tamine, in presence or absence of histidine, by several malolactic strains. This validates previous work by Weiller and Radler [45], which showed that most wine lactic acid bacteria do not contain the enzyme histidine decarboxTlase. Buteau et al. [46] also found no production of histamine, or of other biogenic amines (ethano!amine, tyraraine, cadaverine, agmatine, putrescine) coming from malolactic fermentation. In the second case, Tegmo-Larsson et al. [47] found no detectable amounts of ethyl carbamate in wines fermented by several malolactic strains. Upon heat treatment of the wines, detectable levels were found, but the amounts were the same as those in the control wines without malolactic fermentation.

4.3. Inhibitory conditions and compounds in order to discourage the malolactic fermentation where it is not wanted, California winemakers have already been advised to remove the wine from the gross lees and to make fining and filtration operations as early as possible, to store the wine at low temperatures in non-infected (stainless steel) cooperage, and to maintain judicious concentrations of sulfur dioxide [48]. However, physical or chemical stabilization is required to insure complete inhibition of subsequent malolactic fermentation. For physical stabilization, sterile filtration (with the employment at the final stage of filtration of a 0.45 pm membrane filter for security), followed by sterile bottling, is now common practice, For chemical stabilization against malolactic bacteria, fumaric acid is allowed in California [49], although its low solubility has made its use difficult for treatment of very large volumes of wine. Sulfur dioxide, itself, of course is not an absolute deterrent, except if used in unrealistic and unaIlowed concentrations. However, a new chemical, dimethyl dicarbonate ('Velcorin'), is now legal in the United States. This chemical was foreseen for use especially in wine yeast stabilization of semidry wine; but it has also been found to be an effective inhibitor of malolactic bacteria, providing that the pH is reasonably low and that there has been nominal use of sulfur dioxide [50].

5. INTERMEDIARY METABOLISM

5.1. Thermodynamicsof the malolactic reaction In early studies, thermodynamic calculations, based on the enthalpies of the substrates and products, revealed that the essential part of the maIolaetic reaction, that is, the decarboxs'lation of L-malic acid to L-lactic acid, was endothermic [51]. These considerations led to the notion that the reaction was probably endergonic as well, and agreed with the observations that energy sources other than malic acid were required to support the fermentation. The observations proved to be accurate, but ft~rther calculations, based on heat of combustion, revealed that the reaction was actually exergonic, if only slightly so (AG= approx. - 2 kcal/mol) [2,52]. From the energetics, it was difficult to assign an operative role for the reaction in the economy and metabolism of the cell. First of all, the low molar amount of energy released is not enough to provide for any of the common high energy biological storage compounds, and if the reaction were strictly stoichiometric, there would be no mechanism for trapping of the small amount of energy produced anyway, Secondly, even though it had earlier been shown that nicotine adenine dinucleotide (HAD +) was a required coenzyme for the reaction [53], if stoichiometry prevailed, the reaction would bring about no change in the coonzyme's redox state, which is an important consideration in the intermediate steps of the fermentation of glucose (section 5.3).

5.2. Stimulation of the initial growthphase of malolactic bacteria by malic acid Nevertheless, the malolactic bacteria do seem to benefit from the presence of malic acid. Pilone and Kunkee [54] showed a stimulation of the initial phases of bacteria growth in the presence of malic acid. Morenzoni [55,56] provided a partial explanation for the effect of malic acid in that the decarboxylation of malic acid to lactic acid under wine making conditions was not precisely stoichiometric, after all. There was a slight 'spilloff' of intermediates: pyruvic acid and reduced NAD + (NADH). The formation of NADH was extremely small; it was necessary to use fluorime-

try, rather than spectrophotoraetry for its detection. Morenzoni's [55,56] explanation had to depend, in part, on the identity of the malolactic enzyme. In the earliest demonstrations of the reaction in a ceil-free system [57], the requirement of NAD + as a eofactor gave the natural implication that reaction involved two enzymes, namely malate dehydrogenase (L-malate:NAD ÷ oxidoreductase) and L.lactate dehydrogenase, with the intermediate formation of pyruvate. Many arguments were put forth in support of a single enzyme (L-malate: NAD + carboxylyase), including Morenzoni's work with inhibitors. The controversy was laid to rest by the purificatiDn studies showing a single protein with the malolactic activity, and demonstrations of a single gene in control of the formation of the enzyme (section 7.2). It was speculated that pyruvic acid (from the 'spill-off from the single enzyme) could be converted to acetyl phosphate, probably by a phosphoroclastic enzyme [58], and acetyl phosphate could serve as a hydrogen accepter to re.oxidize the NADH formed during the spill-off. Furthermore, acetaldehyde, from acetyl phosphate, then can act as an additional hydrogen accepter, in its reduction to ethanol, thus bringing about a net formation of oxidized coenzyme.

5.3. Increased formation of hydrogen accel~,~rs provided by malic acid One of the roles for the malolactic reaction would be to provide, in the presence of ma!ic acid, a slight excess of oxidized NADH (NAD+). The resulting extra NAD + woul~ .'-~ctto 'spark' the initial stages of the fermentati~a; since NAD + is a necessary substrate in the pa,~hways of the fermentation of glucose at the oxic~adon of the phosphorylated.C 3 intermediates in both the heterolactic and homolactic fermentations. Alizade and Simon [59] substantiated this suggested role of malic acid in their studies, which utilized tritium and deuterium-labeled substrates. The)' also showed that in the decarboxylation reaction, the conversion of malic acid to lactic acid was not stoichiometricaUy complete; there was a spill-off of reduced coenzyme from the

malolactic enzyme. (This was also evidenced by the formation of labeled o-lactic acid, showing that the decarboxylation of malic acid gives rise to some o-lactic acid, after all. Pukrushpan's work [60,01] further corroborated this suggested role of maiic acid as providing hydrogen acceptors far oxidation of NADH by showing a striking c.Jnelation between the extent of the 'spill-off' (~,f the intermediates in the malolactic enzyrae ac,ivity) and the extent of the stimulation of the in.tial growth rates of other strains of malol~.ctic Lacte~ia. Stimula~ory results were also found by Lee and Pack [62], who showed that in the presence of malic acid there was an increased synthesis and activity of o-lactate dehydrogenase, bringing about a more rapid conversion of glucose to o-lactate. They suggested that this rapid production of o-lactic acid from glucose could help explain the stimulator)' effect of malic acid on the growth rate [59]. This role, too, involves the provision of increased (oxidized) NAD +. Fructose can serve as a hydrogen acceptor in some of the heterolactic bacteria, for example, in the leuconostoes. This results in the reduction of fructose to mannitol. Fructose also shows a stimulation of initial growth in these organisms, giving support to the role we are assigning to malic acid of providing for increased hydrogen acceptors [631.

S.4. Increased formation of high energy phosphate provided by malic acid The p, oposed formation of acetyl phosphate, from the 'spilled off' pyruvi¢ acid, probably by a Dhosphoroclastic enzyme [58], could indicate another role of the malolactic reaction. Depending upon the physiological state of the cell, the acetyl phosphate should also serve as a substrate for the formation of additional adenosine triphosphate (ATP), besides serving as a hydrogen acceptor, as suggested above. The extra ATP ought to be evidenced as an increased formation of biomass. In fact, an increase in biomass was noticed in the original work showing the stimulation of initial growth rate [54] (section 5.2). However, the increase in biomass was explained by the increased

pH, which accompanied the malolaetic fermentation [541. Nevertheless, some other reaction, besides the initial growth rate, seems to be stimulated in the presence of malic acid. For example, Pilone and Kunkee [64] found an unexplained increase in the fermentation end product, D-lactic acid, from glucose by malolactic bacteria in the presence of malic acid. The increase was not simply the result of the accompanying change in pH. The formation of excess ATP would seem to be implicated. The fermentation of glucose in the leuconostoes provides energy to the cell by supplying the formation of one or two molecules of ATP per molecule of glucose [65]. The stimulated initial growth rate by the malic acid should also resum)in a stimulated formation of ATP, which in turn would be used in the stimulation of biomass. The question as to whether there is a net formation from the malic acid in the malolactic reaction, per se, rather than from a fleeting stimulated increase coming from the faster fermentation of glucose is not so easily answered. In the above results, the increases in biomass, representing an increased ATP production, were seen as an indirect effect of increased pH coming from the conversion of malic acid to lactic acid. In some elegant experiments, Cox and Henick-Kling [66] have brought some new answers to this old question. By use of bioluminescence techniques (the photon production from ATP in the presence of luciferin and catalyzed by iuciferase), they were able to detect considerable increased formation of ATP in cells in the presence of malic acid. The cells were either starved or not, but in all cases suspended in buffer without substrate (except malic acid) before lysis of the cells for the bioluminescence measurements. The concentration of ATP in the control cells, without malic acid, was very low, indicating essentially no fermentation. The increased ATP formation can be explained by the same mechanism as the stimulatory effect of malic acid on the rate of fermentation. The fermentation of any residual substrates in the controls is arrested because of the absence of NAD+; however, the latter is provided by the malolactic enzyme activity when malic acid is added.

Cox and Henick-Kling [66] were unable to detect any formation of ATP in a cell-free extract of malolactic bacteria. This is not surprising. The formation of first stage intermediates in the malelactic reaction has been detected in cell-free extracts [55,59,60], but the amounts were exceedingly small. A test for ATP, as above, with a purified malolactic enzyme preparation, plus the addition of a phosphoroclastic enzyme, such as pyruvate formatelyase [58], would be extremely interesting.

5.5. Energy from the efflux of lactate ions The increase in internal cellular pH by the conversion of malic acid to lactic acid, in itself providing for the uptake (disappearance) of one proton per molecule of conversion [2], is of obvi. ous benefit to the cell immersed in a high external concentration of protons. Renault et al. [67] and Cox and Henick.Kling [66] have suggested that the efflux of the extra lactic acid produced by the malolactic fermentation may provide extra relief of some of the energy requirements for the cell, namely that required for maintenance of the electrochemical proton gradient. Their suggestions stem from mechanisms proposed by Michels et al. [68], who calculated that in lactic acid fermentations of glucose (without malic acid), the process of excretion of the electrically charged end products, i.e., lactate ions and protons, could theoretically increase the energy released from the fermentation by 30%. Otto et al. [69] have verified the calculations. They showed that the addition of sodium lactate to the medium diminished the ratio of the internal to external solutes and gave the predicted decreased growth rate. Other anionic solutes were not effective. The results implicated a carriermediated excretion of lactate ion with 'more than one proton', a consideration allowed in the application of the proposal of Michels et al. [68], and which would account for a larger effect in the sparing of energy from the efflux of lactic acid than expected. The above proposal leads to the suggestion that the formation of lactic acid (lactate ion) by the malolactic conversion can also provide an energy saving situation for the cell. Renault et al.

[70] tested the suggestion in wild type Streptococcus lactis and in mutants unable to carry out the malolactic conversion. They concluded that the malolactic conversion either helped maintain the pH of the medium, or when glucose was limiting, it provided energy generated by endproduct elflux. Cox and Henick-Kling [68] also showed that in starved malolactic cells, when membrane ATPase was inhibited by N,N'-dicyclohexTlcarbodiimide, the ATP formation found in the presence of malic acid (see above), and formed in response to efflux of lactate ion, did not occur.

5.6. Effect of the deacidiflcation by the malolactic reaction The above discussions come from a desire to realize that the benefits of the malolactic reaction, in those organisms that possess it, are something more than the 'simple' accompanying deacidification. However, the advantages from the increased pH (~:,~ernally or externally), in the presence of malic acid, as compared to the decreased pH, in its absence, may be far and away the most important practical facet of the phenomenon. We have indications from some continuous culture experiments, with glucose limitation, that there is an increase in the viability of the culture (L. oenos ML34), as a whc,le, in the presence of malic acid. This is undoubtedly a reflection of the increase in pH, as compar~d to the continuous culture without malic acid, resulting from the malolactic fermentation (E. Bordeu and R.E Kunkee, unpublished experiments). 6. PURIFICATIONS AND PROPERTIES OF THE MALOLACTIC ENZYME The malolactic enzyme, properly named Lmalate:NAD+ carboxy lyase, has not as yet been crystallized (and is not to be confused with Lmalate:NAD ÷ oxidoreductase EC 1,1.1.38. How. ever, several research groups have achieved purifications of the malolactic enzyme from various strains of bacteria, and a characterization of the enzyme has emerged. One of the major intentions of the purification in the earlier work was to determine the identity

of the enzyme, and to see whether the activity resulted from a single decarboxylating enzyme, or rather from the amalgamated activity of two enzymes. If the latter, the following scheme had been suggested [71]: a malate oxidative decarboxylase, bringing about the formation of pyrnvic acid (and the reduction of coenzyme NAD+). This activity would be coupled with a second enzyme such as L.lactic dehydrogenase, bringing about the reoxidation of NADH and a reduction of pyruvic acid to L-lactic acid. L-lactic acid was always found as the product of the malolactic reaction [72], and that fact spoke against the duel enzyme scheme, since many species of malolactic bacteria contain v.lactic dehydrogenase as well. Nevertheless, the proof of a single enzyme awaited the purification. The major difficulties in the purification seemed to be in the elution of the enzyme from the various selective adsorption agents tried, and in the instability of the enzyme in its purified form during storage. The first purifications of the enzyme, by Schiitz and Radler in 1974 [73] from Lactobaciilus casei M40, using sephadex gel filtration chromatography; and by Lonvaud et al. in 1977 [74] from L. plantarum 8014, using isoelectric focusing, clearly indicated that the purified enzyme was a single protein. However, there was a reluctance to conclude firmly that the activity was due to a single protein since the possibly contaminating enzyme, L-lactic dehydrogenase, had a similar molecular mass [73] and an isoelectric point [74] similar to the purified enzyme. Nevertheless, all attempts to separate the purified malolactic enzyme into the two contended activities were unsuccessful. Furthermor¢, a multi-enzyme complex or an aggregation of several enzymes seemed highly improbable since the use of sodium dodecyl sulfate denaturing eloctrophoresis [74] yielded only a single band. Further work on purification of the enzyme, outlined below, supported the final conclusion that the native malolactic enzyme was a dimer of two identical sub-units, each carrying the malolactic activity only. Lonvaud-FuneI and Strasser de Saad [75] purified the enzyme from Leuconostoc mesenteroides. Some kinetic studies with this system revealed a

molecular mass of 220000, for the native enzyme. They found a sequential pattern of binding: first NAD and Mn +÷, followed then by malic acid. At pHs substantially away from the optimum pH there was a positive cooperation between malic acid molecules. They also found succinic, citric and tartaric acids to be competitive inhibitors of malic acid. Further purification of the enzyme was made by Caspritz and Radler [76], this time from Lactobacillus plantarum, using isoelectric focusing. They determined the purity by use of sodium dodecyl sulfate polyacrylamide gel electrophoresis. They found a single band corresponding to a molecular mass of 70000. The gradient gel electrophoresis of the native protein exhibited a band corresponding to a molecular mass of approximately 140000. They assumed that the enzyme in the active state consisted of two subunits and that they were most likely identical. They also reported the Michaelis-Menten Km values for the substrates L-malic acid and NAD + and for the cofactor Mn ++ as 9.5 raM, 0.059 mM and 0.012 raM, respectively. Malolactic activity is almost exclusively found in lactic acid bacteria associated with food fermentations. One exception is that of Lactobacillus murinus, a bacterium isolated from rodent intestine. Strasser de Saadet al. [77,78] choose to purify and study the malolactie enzyme from this organism. The results with respect to the molecular mass were similar to those found (above) with Leuconostoc mesenteroides (also not really a wine organism) in having about double the molecular mass found by Radler and Caspritz [76] in Lacto. bacillus plantarum. The lower molecular mass values were also found by McCord [79] in purifications of the enzyme from strains PSU-1 and MLA of Leuconostoc oenos, using ion-exchange with ~phadex resin. The native protein from these two strains had a molecular mass of 138000 and it was concluded that they most likely consisted of two identical subunits of molecular mass of 65500 each. The E m values for L-malic acid were also found to be identical with that found by Radler and Caspritz [76]. Apparently the only attempt at purification of

the malolactic enzyme from ML34 (L. oenos) has been by Spettoli et al. [80], who devised an affin. ity chromatography technigue (using a column consisting of agarose-hexane-NAD +) to separate completely the malolactic activity from the offending lactic dehydrogenase. The purification was not high (five.fold), but the purified enzyme was free from lactic dehydrogenase. The Km for malic acid was lower, 2.8 raM, then for strains PSU-1 and MLA. Lautensach and Subden [81] examined the malolactic enzymes of 18 enological strains of leuconostoc, lactobacilli and pediococci by electrophoretic mobility. The activities in the gels were identified by a complex staining system designed to detect the resulting end product, L-Iactic acid. The mobilities of the malolactic activity were, with the exceptions of those from Lacto. bacillus plantarum, essentially identical. The highest purification so far seems to have been made by Chagnaud et al. [82] with the enzyme from a strain designated as Lactobacillus sp. 89, using several fractionation steps followed by elutions from several absorption columns. The molecular mass of the native protein and its two subunits and the Km for L-malic acid was as above: 120000 and 60000 and 4.1 raM. The competitive inhibition found with L. mesenteroides (above) with succinic, citric and tartaric acids, was also found here. Surprisingly, the optimum temperature of the purified material was 48°C.

7. SOME GENETIC ASPECTS OF THE MAL-

OLACTIC BACTERIA

7.1. Bacteriophage of malolactic bacteria Although the discovery of bacteriophages with specificity for the lactic acid bacteria has not (yet) had the impact on molecular genetics as did the discovery of the T-phages for Escherichia coil some 40 year ago, this section on 'genetics' seems a logical place to discuss the bacteriophages of the malolactic bacteria. While bacteriophages are well known as inhibitors of dairy lactic acid bacteria, their discovery as inhibitors of wine lactic acid bacteria has

not been well publicized. They were first reported in wines of Switzerland as early as 1976 [83] and ten years later in wines of Australia [84,85]. The first reports, which supplied descriptions and photographs of the phages, and of the plaques they produced, raised little concern, since the phages were reportedly inactivated at wine pH and in the presence of sulfur dioxide. There was no compelling reason at first to think that any 'stuck' malolactic fermentations might have resulted from phage infection [83-85]. A later report [86] clearly showed the potential danger a bacteriophage infection could have in a commercial wine situation: the inactivation at wine pH was not necessary true, and a failure of a malolactic bacterial starter culture by bacteriophage infection was demonstrated. As yet there has been no incidence of bacteriophages in the Calf.. fornia wine industry. One means of protection is to employ the same procedure as that used in the dairy industry: have other strains of starter cultures in readiness, since the range of sensitivity of the host for the virus is expected to be narrow. The relationship between bacteriophages and genetic manipulation (ttansduction or plasmid interaction) in the lactic acid bacteria is only beginning to be studied [87].

Z2. Cloning and describing the malolactic gene The first attempt, by Williams et al. [88], to clone the malolactic gene was in its own way successful. This work was reported in 1984 before much understanding of the molecular biology of the malolactic bacteria, even before much notion as to whether plasmids might be involved or not. In any case, the ligation of DNA fragments of a malolactie strain of Lactobacillus delbrueckii into a pB327 plasmid, which was then inserted into E. coil, conferred the malolactic activity to a colony of the latter, selected by brute force. Transfer of the genetic material from this organism by means of the yeast shuttle vector pRC3 into a wine strain of Saccharomyces cerevisiae also transferred the malolactic activity. There was no attempt to incorporate the new genetic material into the host chromosome. For this reason, and probably for others, the experiment was not practically successful: the new activity in the yeast was

only a small fraction corresponding to that in the bacteria, and no attempt had been made to stabi. iize the activity in the yeast. Nevertheless the results demonstrated that the gene inducing the malolactic enzyme formation could be identified in the malolactic bacteria, and it confirmed that the malolaetic enzyme was a single protein. Other workers have also cloned a malic-assimilating gene, not necessarily the malolactic gene, from L. oenos into E. coil [89]. Renault et al. [90] have gone farther and have described the gene in a strain of Lactococcus lactis. The gene, which they have designated mleR, is necessary for induction of malolactic activity in the bacteria. The gene consists of an open reading frame and codes for a protein they calculated to have a molecular mass of 33813. The predicted gene product is homologous to the gene products of several gram-negative bacteria (the lactic acid bacteria are generally Gram-positive). It has long ago been shown that the malolaetic enzyrne, and its permease, are induced by malic acid [91]. All of the good efforts to identify, describe and induce the malolactic gene need to be supplemented with more information on the role, or need, for permease(s) for the transport of malic acid into the cell. 7.3. Screening and enrichment methods for malolactic mutants An important difficulty in genetic manipulations of the malolactic activity has been the lack of methods to detect, and to enrich for, the presence or absence of this expressed genotype. Several methods have been reported, including the use of acid/base indicating dyes with special substrates [92], with varying concentration of substrate [70] or with embedded particulate material [93], and enzyme preparations and redox dyes to detect formation of L-lactic acid from malic acid [891. 7.4. Piasmids in malolactic bacteria The possibilities of application of genetic manipulation techniques so productive for research with Escherichia and Saccharomyces finally have

begun to show promise for the lactic acid bacteria with the discovery, preparation and ligation of plasmids of this bacterial group. A survey of plasmid distribution in the lactic streptococci showed that all 38 strains tested contained from 1 to 12 distinct plasmids [94]. In some cases, the plasmids were responsible for induction of key functions of the cell, such as iactase formation and diacetyl formation from citric acid. A good example of the possible use of the new plasmid information is shown in an improvement in a streptococcal strain used as a starter culture for milk fermentations [95]. The desired new strain was made to be resistant to a certain bacteriophage. The new strain could be selected for after trahsformation of the original strain with a recombinant plasmid. The recombinant plasmid was from another strain that itself had the desired selective marker but had no replicationorigin functional in the initial bacterium. Many of the plasmids display induction for antibiotic resistance, which can also be used to help in selection of altered cells, as was done in the example above. This was also important in one of the first demonstrations of plasmids in the leuconostocs [96], and in an intragenic conjugal transfer between streptococci and leuconostocs [97]. Although plasmids had been demonstrated, and used, in several species of leuconostoc, the plasmid situation in the wine leuconostoc, i.e.L. oenos, does not at first seem to be so promising. Janse et al. [98], using a specially sensitive two dimensional electrophoretic technique, did find plasmids in this species. Eleven plasmids were isolated from eight strains. However, no plasmids were found in the other 34 strains tested. Furthermore, the small size (2-5 kb pairs), lowfrequency and low-copy numbers confirmed the belief [96] that L. oenos strains contain very little genetic information on extraehromosomal elements. Nevertheless, Sgorbati et al. [99] characterized a plasmid (pBI.34) in L. oenos, which induces pesticide resistance in this organism. Continued research on the plasmids and their functions in the wine bacteria is encouraged and awaited.

8, CONCLUDING REMARKS We have tried to provide and discuss information and application concerning the roles of malic acid in the metabolism of the malolactic bacteria, In general the information presented is that from the literature or personal experience of just the last several years. We have left it to the reader to consult the later definitive reviews on this subject for additional background information as desired. In the introduction several items were listed that were not addressed in this article, and which had to do mainly with engineering aspects of the malolactic fermentation. The present practical information for control of the malolactic fermentation in the commercial situation was covered, with special respect to the situation in the newer wine regions, such as California. The conditions for control of the organisms in culture were also examined. The various efforts at purification and characterization of the malolactic enzyme were presented. These topics have to do with the importance of the malolactic fermentation from the winemakers point of view. We also looked at the malolactic reaction from the bacteria's point of view, that is, its intermediary metabolism. This includes the advantages to the cell of the presence of malic acid, which brings about increased availability of hydrogen aeeeptors, increased formation of ATP, increased internal and external pHs, and extra energy coming from the efflux of additional lactate ions. The emerging capabilities of genetic manipulations of the lactic acid bacteria, including the wine malolactic bacteria were scanned, with expectations of things to come. Areas where new research would seem to be the most urgent, and at the same time the most productive, include: better definition of the mi. cronutrient requirements of the industrially important strains; examination of the permeases, or other systems, for transport of malic acid into the cell; better control of all aspects of the malolactic fermentation in commercial white-wine production; better control of the threat from bacteriophage infection; and evaluation of the effect of malic acid on the viability of the culture under continuous culture conditions with controlled pH.

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