[advances in applied microbiology] advances in applied microbiology volume 9 volume 9 || malo-lactic...

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Ma lo-lactic Fermentation

I. 11. 111. IV.

V. VI.

VII.

VIII.

RALPH E. KUNKEE Department of Viticulture and Enology

University of California, Davis, California

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Early Observations ..... Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malo-lactic Bacteria .., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . ..... ....

A. Taxonomy. ............................................

C. Habitat ..................... ..........

F. End Products ..........................

A. Mechanism ................ B. Enzyme Induction ............................................... C. Energetics .......................................................... D. Secondary Effects of Deacidification

Control of Malo-lactic Fermentation . . . . .. . .. . .... . .. . . . A. Desirability of Control ....................... €3. Stimulation .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ... . . . . .. C. Inhibition ........................................................... Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References

235 236 239 241 24 1 243 246 247 253 257 259 260 260 262 263 269 270 270 271 272 2 73 274

1. Introduction

Acidity and acid taste are pronounced and desirable chemical and sensory characteristics of wine. During storage of new wine a decrease in acidity often occurs. One of the causes of this loss was discovered around the turn of the century when it was found that after the alcoholic fermentation by yeast, certain lactic acid bacteria may carry out a secondary fermentation of wine. During this secondary fermentation, malic acid (a dicarboxylic acid which may constitute as much as half of the acid of grapes) is converted to lactic acid, a mono- carboxylic acid. This conversion, now referred to as malo-lactic fermentation, results in a deacidification. This may be desirable in wines of high acidity but is detrimental to wines with acidities that are already low. In addition to a change in acidity brought about by the

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bacteria, flavor changes, either favorable or unfavorable, may take place. One can see that control (either stimulation or inhibition) of malo-lactic fermentation is of importance to the wine maker. This re- view includes information on the biochemistry and metabolism of bacteria causing malo-lactic fermentation, and discusses how this knowledge may be applied to control malo-lactic fermentation.

The concentration of malic acid is high in apples’ as well as grapes, and malo-lactic fermentation also occurs in cider. Some of the information about the bacteria reviewed here has been obtained from research on microbiology of cider. However, the discussion of the application of malo-lactic fermentation will be restricted to wine making.

Other discussions of malo-lactic fermentation may be found in enologic texts and reference works such as Amerine et al. (1967), Amerine and Singleton (1965), and Ribereau-Gayon and Peynaud (1961), and in articles by Fell (1961), Fornachon (1963a), Lambion and Meskhi (1957), Peynaud and Domercq (1961a), Radler (1962a, 1963, 1966), Rankine (1963a), Schanderl (1959), Suverkrop and Tchelistcheff (1949), Vaughn (1955), and Vaughn and Tchelistcheff (1957).

II. Early Observations

Gross changes in wine can be noticed during malo-lactic fermentation. There is often an increase in cloudiness because of the growth of bacteria, and a deposition of tartrate because of the change in pH (see Section V1,D). Off-odors, which may later disappear, frequently accompany the fermentation. Also noticeable is the effervescence caused by the evolution of carbon dioxide. In the past the loss of acidity was the most mentioned aspecte2 Of course some loss of acid is common in all wine after alcoholic fermentation. The presence of alcohol decreases the solubility of potassium acid tartrate, causing it to precipitate, as does the cool temperature at which wines are stored. The earliest observations of malo-lactic fermentation may have con- fused this precipitation of tartrate with microbial decomposition of

’ Indeed, “malic” is derived from the Latin word for apple, malum. In English, malic acid is sometimes called “apple acid,” and the translation of this term is used in many foreign languages.

‘Even today the most common German translation for “malo-lactic fermentation” is biologischer Saureabbau or Saureabnahme. Cognates of “malo-lactic” are used in other languages. The German term now appears even more ambiguous as it may refer to alcoholic fermentation by some Schizosaccharomyces yeasts which can also ferment malic acid (to alcohol).

MALO-LACTIC FERMENTATION 237

tartrate.3 The first observations of a loss of total acidity that was greater than that caused by loss of tartrate has been attributed to Berthelot and de Fleurieu (1864). In one wine analyzed by them, the total fixed acidity dropped from 10.0 gm./liter to 5.8, but the tartaric acid loss was only 2.5 gm./liter. However, it is not certain that this was a malo-lactic fermentation since the loss of acidity occurred during the alcoholic fermentation. Boussingault (1868) also found a loss of about half the acid in cider, and this may have been a malo-lactic fermentation. In 1891, Ordonneau reported the loss of acid in some aged wine to be the loss of malic acid, and he suggested that malic acid was transformed into another acid.

Pasteur (1858) of course proved that lactic acid was produced by bacteria (or “new yeast” as he called them). Although he did not give much special attention to the loss of acidity in wine, characteristically he correctly related the loss to microbial action. In his studies on wine spoilage, he quoted a letter from de Vergnette-Lamotte (Pasteur, 1866, pp. 66-68). The letter described a wine “disease” which, among other things, brought about a flat (fade) taste. Pasteur (1866, pp. 36-37) described the release of carbon dioxide during the pousse or gassy stage of tourne spoilage. Some of this undoubtedly was caused by malo-lactic fermentation. [It must be pointed out here that although loss of acidity in wine may sometimes be considered spoilage, it does not follow that all bacteria that cause malo-lactic fermentation are necessarily spoilage organisms. One should not identify “malo-” of “malo-lactic” with “bad” instead of with “malic.” Balard (1861) very early recognized the formation of lactic acid in sound wine by microorganisms. Many of the “grand wines” apparently require bacterial activity for their quality, and malo-lactic fermentation is a necessity in the wines of high acidity from the coldest viniculture areas.] Pasteur (1873, pp. 270-277) prevented a small loss of acidity in wine stored for nearly a decade by heat treatment before storage. The untreated wine lost about 0.5 gm. acid per liter of wine. How- ever, this loss seems too small to be a malo-lactic fermentation;, Pasteur claimed the loss was tartaric acid.

Kulisch (1889) was probably the first to prove the biological nature of what is now called malo-lactic fermentation. He pasteurized cider (heated to 60°C.) which then lost very little acid, but in 6 months the

31n the early literature one finds reports of decomposition of tartaric acid in wine by bacteria which seem to have been Lactobacillus spp. These reports may be erroneous; only recently have workers been able to isolate bacteria from wine that are capable of metabolizing tartaric acid. See Berry and Vaughn (1952), Vaughn (1955), Krumperman (1964), and Krumperman and Vaughn (1966).

238 RALPH E. KUNKEE

total fixed acidity of the untreated cider dropped from 0.8% to 0.45%. Kulisch (1889) also claimed he was able to induce the loss of acidity by addition of what he considered a pure yeast culture of “Saccharo- myces ellipsoideus.” Consequently he concluded the acid reduction was caused by yeast. About the same time, Muller-Thurgau (1891a) reported several experiments, probably the first ones, on the control of acid reduction. From his observations he was convinced that bacteria, not yeast, were responsible for the change in acidity. He always found bacteria in wines which had undergone the change, and in “hundreds” of examples h e was never able to induce acid reduction in wine by addition of pure yeast. Unfortunately his attempts (at that time) to induce the acid change by inoculation with the bacteria he had isolated from wine were unsuccessful. The contention that yeasts were causative agents was supported by many other workers: e.g., Amthor (1889), Wortmann (1894), Schukow (1896), and Mestrezat (1907).

Alfred Koch (1900) was the first to isolate malo-lactic bacteria and to induce malo-lactic fermentation by inoculation with these organisms. Furthermore, with the use of salicylic acid he discredited claims that yeast were responsible. Koch (1898) showed that salicylic acid strongly inhibited bacterial growth in wine but had little effect on yeast. Wine with added salicylic acid did not undergo acid reduction, whereas the control wine did (Koch, 1900). H e thus proved Muller-Thurgau’s hypothesis that bacteria were causative agents. Koch also recognized that the loss of acidity involved mainly the loss of malic acid, and he seemed to understand the fastidious nutritional nature of the bacteria and that important bacterial growth factors might arise in commercial winemaking from autolysis of yeast.

Seifert (1901, 1903) also isolated bacteria which would induce loss of acidity. Both Seifert and Koch isolated their bacteria from wine sediment. Seifert gave both a description and a microscopic illustra- tion of paired cocci 1 p in diameter. He showed that the loss of acidity involved the conversion of malic acid to lactic acid and he named the isolate Micrococcus malolacticus. Moslinger (1901) is credited with first presentation of the overall malo-lactic equation (malic acid to lactic acid and carbon dioxide), although Seifert (1901) also published the equation the same year. Moslinger (1901) realized that lactic acid arose both from decomposition of malic acid (malo-lactic fermentation) and by fermentation of other [carbohydrate] materials (lactic acid fermentation).

During the next several years, four mare strains of bacteria capable of malo-lactic fermentation were isolated by Muller-Thurgau and by


him and Osterwalder. A detailed account of their work gave a description of these bacteria (Muller-Thurgau and Ostenvalder, 1913). This report included a taxonomic key for classification of all bacteria (except acetic acid bacteria and bacteria from “ropy” wines) which at that time had been isolated from wine or other fermented beverages.

Koch (1900) suggested that the claims of loss of acidity by yeast were the result of aerobic metabolism, and that wine stored anaero- bically did not lose acidity (except from bacterial activity). It is tempting today to attribute the controversy (see also Muller-Thurgau, 1891b; Kulisch, 1891) over yeast as the cause of malo-lactic fermentation to primitive microbiological techniques. Some of the so-called “pure yeast cultures” or the “sterilized” musts and wines might have been contaminated with aerobic spoilage yeast or perhaps with fermentative yeasts which decompose malic acid, such as some Schizosaccharomyces. Mestrezat (1908) pointed out that most of the claims of loss of malic acid by yeasts were based on loss of total acidity rather than on determinations of malic acid itself. Neverthe- less, the early literature is amazing because of the perspicacity of these researchers. The principles put forward by them are still generally valid.

111. Occurrence

Discoveries of malo-lactic fermentation in wines of various regions of the world spread with the application of technology to the practice of winemaking. Following the first proofs of the microbiological nature of the fermentation in German and Austrian wines (see Section 11) came the reports of malo-lactic fermentation in wines of many areas of western Europe. Moreau (1906) noted the deacidification (by “yeasts”) in wines of Anjou. Rosenstiehl (1908) was of the opinion that wines of Gironde were improved by a slow malo-lactic fermentation, and he also mentioned observing a vigorous secondary fermentation in Alsace. Mestrezat (1908) noted loss of malic acid in wines of southern France (Midi). Although he recognized a difference between a rapid loss of the acid apparently caused by yeast at about the time of alcoholic fermentation and a slower disappearance during aging, he objected to the term “malo-lactic fermentation.” Astruc (1925) also examined wines of southern France and found malo-lactic fermentation. Ferre (1922) and Rousseaux and Ferre (1926) reported malo- lactic fermentation in both white and red wines of Burgundy. Malo- lactic fermentation had already been detected in Bordeaux wines (Rosenstiehl, 1908), and it was further studied by Ribereau-Gayon and Peynaud (cf. 1961, p. 434).

240 RALPH E. KUNKEE

After World War 11, paper chromatography became a routine tool for research biologists, and its use became more and more wide- spread in enology laboratories. By replacing the involved chemical procedures for the determination of organic acids with chromato- graphic methods, winemakers throughout the world found it easier to detect and follow the malo-lactic fermentation. In many wine laboratories this determination is now routine. Today malo-lactic fermentation has been reported practically everywhere wine is made. Besides the geographical areas mentioned above, the following countries, at least, can be listed: In Europe: Switzerland (Tonduz, 1920), Spain (Feduchy Marino, 1964), Portugal (Marques Gomes et al., 1956), Italy (Tarantola, 1959), Yugoslavia (Milisavljevic, 1964), and the Soviet Union (Saenko et al., 1965); Africa: Algeria (Bremond, 1937), South Africa (du Plessis, 1964); Australia (Fornachon, 1957); Japan (Nono- mura et a,?., 1963); South America: Argentina (Arena, 1936), Uruguay (Poittevin et al., 1963), Chile (Hernandez and Ortega Tello, 1964); and North America: Canada [Adams (1964) reported high concentrations of lactic acid-presumably from malo-lactic fermentation -in Ontario wines]; and the United States: New York (Rihe, 1965) and California (Suverkrop and Tchelistcheff, 1949; Ingraham and Cooke, 1960; Kunkee et al., 1965).

Sometimes such sophisticated techniques as paper chromatography are not required for detection of malo-lactic fermentation. Some wines of northern Portugal (and certain other parts of Europe) which, because of viticultural practices are very high in acid, require malo- lactic fermentation for palatability (cf. Amerine et at., 1967, pp. 37, 457). The fermentation often occurs after bottling, and the carbon dioxide is retained. This results in a vinho frisante or slightly gassy wine which has become a characteristic type.

It is generally true that malo-lactic fermentation is more common in red wine than white wine. Part of the reason for this is undoubtedly the inhibitory effect on the bacteria of the greater acidity and the higher concentration of sulfur dioxide usually found in white wire. The presence of material extracted from the skins may also stimulate the fermentation of red wine (cf. Garino-Canina, 1943; Kunkee, 1966). Nevertheless, malo-lactic fermentation is an important occurrence in some white wines of high acidity such as those of Switzerland (cf. Ribereau-Gayon and Peynaud, 1960, pp. 438-439).

Malo-lactic fermentation is also inhibited by high alcohol concentration. It has been found only in table wines or wines with alcohol concentration not much higher than 14%.4 In California, sherries and

*All alcohol concentrations are given as ethanol, percent by volume.

MALO-LACTIC FERMENTATION 24 1

other appetizer and dessert wines must be at least 19.5% alcohol. Except for some bacilli (Gini and Vaughn, 1962), Lactobacillus trichodes is the onIy bacterium ever found in these high-alcohol wines, and this species does not attack malic acid (Fornachon et al., 1949).

IV. Malo-lactic Bacteria

A. TAXONOMY By definition, malo-lactic bacteria are lactic acid bacteria which

can ferment malic acid. They are members of the family Lacto- bacillaceae, but the ability to ferment malic acid cuts across generic and*possibly specific lines; and these bacteria are not placed in a taxonomic group of their own (Breed et al., 1957). This sometimes has led to difficulties in classification of these organisms. In the first sys- tematic classification which included malo-lactic bacteria, Muller- Thurgau and Osterwalder (1913) used the ability to ferment malic acid as part of the dichotomous key. They listed four malo-lactic bacteria: Seifert’s Micrococcus malolacticus, their own M . acidovorax and M . varicoccus, and Muller-Thurgau’s Bacterium gracile. By today’s classification, the above micrococci should be renamed because they produce lactic acid and are undoubtedly catalase nega- tive (cf. Arena, 1936). The taxonomy of these organisms has been discussed in detail by Vaughn (1955) who placed them either in the genus Pediococcus or Streptococcus. Since they were isolated from nonanimal sources and do not form chains (cf. Muller-Thurgau and Osterwalder, 1913; Arena, 1936), they should most likely be placed in the genus Ped ioc~ccus ;~ optical activity of the lactic acid produced is required for definite classification (Breed et al., 1957). B . gracile, also, is not an accepted name today. Carr (1952) suggested this organism ought to be renamed Leuconostoc mesenteroides, although the photographs of it (Miiller-Thurgau and Osterwalder, 1918) give the impression of a definite rod rather than an elongated coccus.

Ribereau-Gayon and Peynaud (1961, pp. 465-479) gave a resume of names and classifications used for the most important malo-lactic bacteria isolated from wine since the time of Muller-Thurgau and Osterwalder. Using modern classification (Breed et al., 1957), we

It is ironic that the first characterized malo-lactic organism does not fit modern classification. Miiller-Thurgau and Osterwalder (1913) described Seifert’s M . malolacticus as producing no lactic acid from glucose and not fermenting fructose. We have to consider these observations as errors if we place the organisms in the family Lactobacillaceae. Bidan (1956) isolated an orgamism similar to Pediococcus cereoisiae which he called identical to M . malolacticus and M . varicoccus.

242 RALPH E. KUNKEE

find many members of the Lactobacillaceae are malo-lactic bacteria. Aadler (1962a) listed the following lactic acid bacteria isolated from wine which decomposed malic acid: Lactobacillus buchneri, L. casei, L. delbrueckii, L. fermenti, L. hilgardii, L. pastorianus, and L. plantarum; Leuconostoc citrovorum, L. dextranicum, and L. mesenteroides; and Pediococcus cereuisiae. To this list should be added Lacto- bacillus brevis (Vaughn and Tchelistcheff, 1957) and Lactobacillus leichmannii (du Plessis, 1964). With the exception of L. hilgardii (Vaughn et al., 1949),-each of the above species is described in “Bergey’s Manual” (Breed et ul., 1957). Several species of Strepto- coccus which are malo-lactic have reportedly been isolated from spoiled wines: S. mucilaginosus var. vini, S. malolacticus, and an organism similar to S . damnosus (Carr, 1962; cf. Vaughn, 1.955). There is a question as to the correct classification of some of these strepto- cocci (Vaughn, 1955).

The high concentration of alcohol and acid in wine, plus the presence of sulfur dioxide and the lack of nutrients make wine a fairly hostile environment for microorganisms. Only a few kinds of organisms are found in wine, and part of the classification of malo-lactic bacteria is relatively easy. The family Lactobacillaceae is part of the order Eubacteriales. Members of the family are Gram-positive, asporogenous, nonmotile, anaerobic or microaerophilic, lactic-acid producing bacteria. The absence of catalase6 and a minimum of surface growth in a stab culture separate lactic acid bacteria from other bacteria sometimes found in wine, i.e., from the acetic acid or vinegar bacteria in table wine, and from bacilli in fortified wine.

The Lactobacillaceae family is divided on basis of cell morphology into tribes of cocci and rods (Breed et al., 1957). Of the cocci, only the facultative anaerobes are important as malo-lactic bacteria, and of these only the two genera Pediococcus (homofermentative) and Leuconostoc (heterofermentative) are important. Of the rods, again, only the facultative anaerobes are important; and these include only the genus Lactobacillus. Further subdivision of the three genera is dependent on optimal temperature of growth, optical activity of lactic acid produced, and kinds of sugars fermented. The heterofermentative cocci sometimes are hard to classify because they are especially nutritionally fastidious, which makes it difficult to obtain a defined medium for determination of sugar fermentability (cf. Ingraham et al.,

Some pediococci and lactobacilli have been reported to be weakly catalase positive in presence of low concentrations of energy source (Felton et at., 1953; Dacre and Sharpe, 1956; Whittenbury, 1964).


1960). For example, the separation of L. citrovorum from L. mesenteroides and L. dextranicum is based on the capability to ferment sucrose (Breed et al., 1957). The response of a leuconostoc isolated by Ingraham et al. (1960) and designated “ML 34” (Webb and In- graham, 1960) was poor on all synthetic media. In a complex medium with glucose as the only known carbon source, growth was obtained, but none was found when sucrose was the only known carbon source. The organism was classified L. citrovorum (Pilone et al., 1966). Fornachon (1964) found difficulty in classification of a heterofermentative coccus The organism he described did ferment sucrose and was hence classified as L. mesenteroides, but it did not form dextran when sucrose was the carbon source. Bidan (1956) and Radler (1958a) also found heterofermentative cocci which were similar but not identical to L. citrovorum. Classification difficulties have been found with some homofermentative rods which are similar to Lactobacillus plantarum (Bidan, 1956; Radler, 1962a). Lambion and Meskhi (1957) suggested the homofermentative malo-lactic rods be named Lacto- bacillus plantarum var. gracile and the heterofermentative malo- lactic cocci, Leuconostoc mesenteroides var. gracile.

B. ISOLATION, CULTIVATION, AND IDENTIFICATION Koch (1900) and Seifert (1901) first isolated malo-lactic bacteria

from sediments of wines which had undergone malolactic fermentation. Wine sediment can be a good source of the bacteria; however, one can also use wine itself. Two major difficulties in the isolation of the bacteria are (a) the fastidious nature of the organisms and (b) the presence of yeast in the sample. The first difficulty is solved by use of a culture medium of great complexity. Synthetic media have been described for malo-lactic bacteria (e.g., Peynaud et al., 1965), but these have not proved suitable for the most fastidious malo-lactic bacteria (Kunkee, unpublished results). Media which contains tomato juice has often proved to be excellent for culturing the bacteria (Rogosa et al., 1953). Ingraham (1963) made some modifications of the Rogosa medium to give the following formula: 2% tryptone, 0.5% yeast extract, 0.5% peptone, 0.3% glucose, 0.2% lactose, 0.1% liver extract, and 0.05% Tween 80 in 4.2-fold dilution of tomato juice (canned without preservative) filtered through filter aid. The broth was adjusted to pH 5.5 with hydrochloric acid and 2% agar added for solid medium. In this laboratory, either tryptone or tryptose was found to be satisfactory in this formula, but the shelf age of the tryptone can be important. We have had good results with Difco’s

244 RALPH E. KUNKEE

Rogosa SL broth prepared in diluted, filtered tomato juice. We have also used fresh grape (Vitis uin$era) juice to which 0.05% yeast extract was added, the mixture being adjusted with sodium hydroxide to pH 4.5. Because of the low pH of the medium, extreme sterilization procedures are often not required (Rogosa and Sharpe, 1959). However, little or no deleterious effects from standard autoclave treatment have been detected. Whiting and Coggins (1963) reported improvement in some media after being autoclaved.

The second difficulty, the presence of yeast, has been avoided by use of the fungicide cycloheximide (Acti-dione, Upjohn Co.) at a final concentration from 20 p g h l . (Peynaud and Domercq, 19614 to 100 pg./ml. (Webb and Ingraham, 1960). Sorbic acid has also been used to inhibit yeast (Fornachon, 19641, but Ingraham et al. (1960) found it somewhat inhibitory to malo-lactic bacteria. Peynaud and Domercq (1961a) suggested differential centrifugation of the wine as a preliminary step in removal of the yeast cells. This is undoubtedly also a good practice with old wine in which the proportion of viable bacteria may be small.

Initial inoculations have been made both in liquid and on solid media. Inoculated agar plates are often incubated in sealed glass jars in which the oxygen has been exhausted with alkaline pyrogallol (Peynaud and Domercq, 1961a; Meynell and Meynell, 1965) to inhibit aerobic organisms. An external supply of carbon dioxide has been shown to stimulate growth of lactic acid bacteria under some conditions (cf. Snell, 1952). Rogosa and Sharpe (1959) suggested incubation of lactobacilli in an atmosphere of 5% carbon dioxide. The oxygen concentration in the atmosphere of incubation jars can also be lowered and the carbon dioxide increased by use of a lighted candle (Ingraham et al., 1960). Colony formation on solid media inoculated with 0.1 ml. wine sometimes takes as long as 2 weeks. Because of this relatively long incubation period, mold contamination sometimes de- velops. Beech and Carr (1955) suggested use of 100 pg./ml. diphenyl to inhibit mold. As with any microbial isolation, one assumes that the predominant kinds of colonies are the important organisms. Micro- scopic examination should be made of the isolated organisms to be assured that they are bacteria. After purification of the bacteria by restreaking, they can be stored as stab cultures. In this laboratory, cultures are kept in the cold and transferred every 3 months. Fell (1961) successfully stored malo-lactic bacteria by lyophilization of a mixed culture of the bacteria and yeast. I t is well to point out that often lactic acid bacteria die off quickly and relatively large inocula should be used in making transfers (Rogosa and Sharpe, 1959; Davis, 1960).


For classifications, standard methods for lactic acid bacteria are used, but because of the limited numbers of genera found in wine, the procedure can be somewhat abbreviated. The cultures are first checked for absence of catalase and for minimal surface growth in stab cultures. These positive tests for anaerobes and positive Gram strains of young cultures are presumptive evidence for Lactobacil- laceae. The production of lactic acid can be determined by paper chromatography of the culture medium (Section V) to confirm the presumption. Cell morphology is used at the next stage of classification. Some Leuconostoc become elongated when grown in fruit juice medium (Vaughn, 1955; Breed et al., 1957). Very short rods ought to be “keyed-out” both as cocci and rods before final classification. Heterolactic fermentability is determined by gas production from glucose (Hayward, 1957; Pilone et al., 1966) and by mannitol formation from fructose. The latter is determined by the formation of rosette macrocrystals in evaporated medium (cf. Ribereau-Gayon and Pey- naud, 1958, pp. 256-258). The distinction between homo- and heterofermentative types is sometimes vague (Davis, 1963). From these tests one is able to identify the organism as Pediococcus, Leuconostoc, or Lactobacillus. For further identification, optimal temperature of growth can be determined from an Arrhenius plot of logarithms of specific growth rates (determined by increase in turbidity with time) versus reciprocal absolute temperatures. Optical activity of lactic acid is most easily determined by use of an enzyme assay for L-lactic acid (Hohorst, 1963; Pfleiderer and Dose, 1955) and a chemical method for total lactic acid (Bruno and Moore, 1962; Amerine, 1965, pp. 64-66); the D-lactic acid is then obtained by difference (Cato and Moore, 1965). Determination of fermentable sugars must be made on media with no energy source other than the test sugar. We have used the modified Rogosa medium listed above but lacking glucose, lactose, and tomato juice. A suitable synthetic medium would be pre- ferred. Malic acid fermentability can be determined by growth of the bacteria in broth which contains 0.3% L-malic acid. Paper chromatography (Section V) of the medium will show whether malic acid was utilized. In the same manner, utilization of citric acid can also be determined.

A symposium on isolation, cultivation, and classification of malo- lactic bacteria was recently held by the Commission 11: Oenologie of the Ofice International de la Vigne et du Vin (Husfeld, 1964; Ulbrich, 1964; Milisavljevic, 1964; Peynaud and Dupuy, 1964; Florenzano and Verona, 1964; Fell, 1964; Feduchy Marino, 1964).

It is comforting for the wine lover to learn that no Leuconostoc

246 RALPH E. KUNKEE

or LactobaciZZi are known to be pathogenic (Niven, 1952), and that no pathogenic bacteria have been found in spoiled or sound wine (Vaughn, 1955).

C. HABITAT

It is natural to wonder about the source of malo-lactic bacteria in wine. On the one hand, the most obvious source would seem-to be the grapes themselves. Peynaud and Domercq (1961b) and Kunkee et al. (1965) got spontaneous malo-lactic fermentation in wines in which care had been taken to obtain the musts aseptically. Japanese workers have obtained malo-lactic fermentation in grapes stored at 40°C. (Otsuka et u Z . , 1964). [We have been unable to confirm this with tests on a dozen varieties of grapes (Kunkee, unpublished experiments).] Radler (1958a) detected malo-lactic bacteria on grape leaves. Garino-Canina (1943) reported an old experiment of Nessler which indicated the presence of bacteria on grape skins. On the other hand, the sources of the bacteria might be the winery. Webb and Ingraham (1960) obtained spontaneous malo-lactic fermentation in grapes vinified in one winery but not when the same grapes were used in another winery. Radler (1958a, 1966) was unable to isolate malo-lactic bacteria from fresh must, but he was able to obtain bacteria from it 2 weeks after the beginning of alcoholic fermentation. In the results of Peynaud and Domercq (1961b) mentioned above, the spontaneous fermentations occurred only in a few instances. Both sources of the bacteria, in fact, may be important. We might suppose that the bacteria arise originally from a small population on the grapes. Under favorable conditions, especially after alcoholic fermentation has brought about added nutrients from yeast autolysis, the bacteria would multiply to an easily detectable level. Peynaud and Domercu (1959) pointed out that malo-lactic fermentation more likely occurs in large vessels where the chance for significant levels of viable bacteria is higher than in smaller vessels. Perhaps the presence of an occasional grape leaf also contributes to the original natural inoculum. It would seem reasonable that fermentation and storage containers, especially those which are difficult to clean, would retain malo-lactic bacteria after the secondary fermentation. Ribereau-Gayon and Peynaud (1961, pp. 440,468) reported malo-lactic organisms on walls or floors of wine cellars and on wooden barrels. Bacteria also might be associated with pomace. Wineries often dispose of pomace by spreqd- ing it in nearby vineyards. Thus, winery operations might lead to a perpetuation of the bacteria in and around the winery from year to


year until characteristic malo-lactic microflora were built up in much the same way certain yeast strains become established in vineyard areas (Amerine and Singleton, 1965, p. 54).

D. GROWTH CONDITIONS

Descriptions of isolated malo-lactic bacteria usually include some information about the growth requirements for the organisms. Even in the very first reports, Koch (1900) indicated a need for special growth factors for the bacteria he isolated, and Seifert (1903) showed that M. malolacticus was inhibited by high alcohol and high acidity. The requirements for growth of malo-lactic bacteria are generally those of lactic acid bacteria. However, as a class malo-lactic bacteria are more tolerant to alcohol and to acidity than many other lactic acid bacteria.

Even though one determines the best conditions for growth of a particular organism, the knowledge of the requirements may not be directly helpful to the enologist, since the information obtained under laboratory conditions is not necessarily applicable to winery operations. The winemaker usually does not have control over the kinds of organisms with which he must be concerned. Furthermore, the most favorable conditions for bacterial growth may not be the same as those for most rapid conversion of malic acid to lactic acid. More information on the latter subject is especially needed.

Experiments on malo-lactic fermentation itself can tell us something about growth and metabolism of malo-lactic bacteria, but careful inter- pretation is required before these kinds of results can be generally applied.

In this report, detailed descriptions will not be given of specific bacteria. Rather, a generalized discussion of malo-lactic bacteria as a group will be presented.

1 . Nutrition

Lactic acid bacteria require an extensive complement of amino acids, purines, pyrimidines, and vitamins, besides minerals and carbon and nitrogen sources. These bacteria are often used in bioassays for growth factors. Several synthetic media for malo-lactic bacteria have been published (e.g., Luthi and Vetsch, 1959; Peynaud et al . , 1965). du Plessis (1963) and Radler (1966) have published lists of amino acids and other growth factors required for strains of malo- lactic bacteria studied by them. We have found that complex medium containing all of the suggested growth factors gave only minimal

248 RALPH E. KUNKEE

growth for some bacteria. As mentioned above, L. citrovorurn ML 34 is especially fastidious. It grew poorly in a medium containing, in addition to the components of Difco’s C F [Citrovorum Factor Assay Medium (itself a very complex mixture)]: folinic acid, lipoic acid, cyanocobalamine, inositol, L-asparagine, y-aminobutyric acid, thymidine, deoxycytodine, Tween 80, sodium pyruvate, .xylose, arabinose, galactose, and sucrose (Kunkee, unpublished results). The medium was adjusted to a final pH of 4.5 and sterilized by filtration. It also contained 0.5 pg./ml. pantothenic acid, which is sometimes needed by lactic acid bacteria at a relatively large concentration (Snell, 1952). Addition of L-malic acid, citric acid, acetic acid, or ethanol to the medium did not give a better growth response. y-Aminobutyric acid was added because it was found to be an unex- pectedly large component of the amino acids of grapes (Nassar and Kliewer, 1966); Radler (1966) found it ineffective in his tests of it as a nutrient. Not all malo-lactic bacteria are as fastidious as this, of course, and some will grow well on simpler media.

All nonsynthetic media for malo-lactic bacteria have yeast extract or yeast autolyzate added. It was discovered very early that the presence of yeast can supply the nutrients for the fastidious bacteria (Koch, 1900). Storage of wine on gross lees for several weeks stimulates malo-lactic fermentation (Fornachon, 1957). Liithi and Vetsch (1959) commented on the ancient practice of adding one wine to another to “start” malo-lactic fermentation. The practice was effective because it supplied bacteria as inoculum and probably because the addition of resting yeast cells supplied nutrients for them. The nutritionally active components of yeast extract have been isolated (Luthi and Vetsch, 1959), and seemed to be mostly simple nitrogen compounds.

Discovery of bacterial contamination of tomato ketchup led to the use of tomato juice in nutrient media for bacteria (Mickle, 1924). For all the malo-lactic bacteria we have tested, the addition of tomato juice, or grape juice, was helpful. Stamer et al. (1964) found the ash of tomato juice to be the active fraction. By testing various minerals, they found manganous sulfate addition resulted in bacterial growth equal to that obtained on medium supplemented with tomato juice. Other inorganic components are also important. Zickler (1964) showed that potassium, sodium, magnesium, and manganese ions were required for growth of malo-lactic B . gracile. Malic acid decarboxylation rate was dependent on potassium ion. Bocker (1964) tested demineralized wine. He also found that the four inorganic ions mentioned above plus calcium had to be added back to the medium


for malo-lactic fermentation. The growth of the bacteria was influenced by magnesium. Radler (1966) found that 2 mM potassium ion was required for maximal growth in synthetic medium. Fornachon (1964) added a mixture of inorganic salts (Wright and Skeggs, 1944) to a complex medium used for growth of Leuconostoc.

There probably is no universal medium for all malo-lactic bacteria. A major problem in development of a synthetic medium is the an- tagonism between certain growth factors. For example, small amounts of folic acid, copper, or purine deoxyribotides can inhibit growth of some lactobacilli (Rogosa and Sharpe, 1959; Morris and Williams, 1965). Even with nutritionally balanced media, the concentration of certain components can be surprisingly influential on the metabolism of the organisms. For example, Nossal (1952) found changes in pyruvate metabolism in the presence of glucose in a lactobacillus. To add to the difficulties, two or more strains growing together may show nutritional symbiosis by growth in medium in which neither could grow alone. Doctor and Couch (1954) showed that both Leuconostoc citrovorum, which required citrovorum factor and produced vitamin B12, and Lactobacillus leichmannii, which required vitamin B12

and produced citrovomm factor, could grow in medium lacking both of these biochemicals.

We have been surprised to discover that some of the most fastidious organisms grow better in winery operations, or at least carry out malo-lactic fermentation faster, than do some of the least fastidious ones (Pilone et al., 1966). Perhaps the presence of certain natural components of the grape or wine overcome the nutritional handicap of the fastidious bacteria and then other factors become more influential.

Red wines are more susceptible to malo-lactic fermentation than white wines. This has been attributed to the greater acidity and concentration of sulfur dioxide in white wines. Malo-lactic fermentation was found to be stimulated by the presence of skins during the alcoholic fermentation (Garino-Canina, 1943). This kind of result was also reported by Fornachon (1957) and by Kunkee (1966). Possibly some growth factors extracted from the grape skins are beneficial to malo-lactic bacteria and stimulate the fermentation in red wine. High glucose concentration had no effect on malo-lactic fermentation (Radler, 1958b).

2. p H Wine is highly acidic and bacteria isolated from it tolerate low pH.

Malo-lactic fermentation readily occurs in wine at pH 3.4 and often

250 RALPH E. KUNKEE

at lower pH. It has been reported in wines with low alcohol at pH 3.0 (Rankine, 1963a). Optimal pH for the bacteria is much higher than the pH of wine, but it is generally lower than the optimal pH for other lactic acid bacteria (Ingraham et al., 1960). The pH range for growth varies with each organism, of course. We found it difficult to obtain growth of strains of P . cerevisiae in complex medium at pH below 4, even though the bacteria had been isolated from wine with pH lower than that (Pilone et al., 1966; Kunkee, unpublished results). The acidity of the medium influences malic acid fermentation as well as growth of the bacteria. Ribereau-Gayon and Peynaud (1961, pp. 478-479) found different pH thresholds for fermentation of malic acid than for the fermentation of sugars and the formation of volatile acidity. They suggested that the best organisms for malo-lactic fermentation in wine would be those which had the greatest differences between these pH thresholds, so that rnalo-lactic fermentation could be obtained with a minimum of other kinds of metabolism.

The pH of the medium also greatly influences the activity of sulfur dioxide present (see below).

3. Alcohol

The antiseptic quality of ethanol is well known, and its presence in wine, depending on the concentration, has an inhibitory effect on malo-lactic bacteria. The legal limit in California table wine is 10 to 14% alcohol, and malo-lactic fermentation is delayed, but not necessarily prevented, in medium with this much alcohol. At lower concentrations the inhibition is less but sometimes is still detectable to 6%. The tolerance to alcohol varies with bacterial strain and, again, generalizations are difficult to make.

In California the lower alcohol limit in fortified wines is 19.5%, thus one is not concerned about malo-lactic fermentation in these wines. The only reports we have seen of malo-lactic bacteria which tolerate this amount of alcohol are a strain of Lactobacillus fermenti (Bidan, 1956) and another Lactobacillus sp. (Vaughn et al., 1949). Neither of these organisms was isolated from high-alcohol wine.

4 . Temperature

Optimal growth temperatures for Leuconostoc spp. range from 20" to 25"C., for P . cerevisiae, 25" to 32"C., and for Lactobacillus spp., from 28" to above 37°C. (Breed et al., 1957). Malo-lactic fermentation is inhibited at 5" to 10°C. (Garino-Canina, 1943); but the fermentation


occurs, more or less readily, at cellar temperatures of 10” to 15°C. We must be cautious in assuming that the lower temperature brings about merely a change in rates of growth and of malo-lactic fermentation. Temperature has been shown to have a profound effect on the regulation and activity of anabolic systems of prototrophic bacteria (Ng et al., 1962). Temperature could influence metabolic pathways of the fastidious lactic acid bacteria also.

Lactic acid bacteria do not form spores and are relatively sensitive to heat. Two exceptions, with optimal temperatures about 45”C., are Lactobacillus bulgaricus and L. thermophilus, but these have not been reported as malo-lactic bacteria. Carr (1952) reported survival of cider Leuconostoc sp. at 60°C. for 10 minutes and Lactobacillus sp. at this temperature for 2.5 minutes. For bacteria in wine, less drastic heat treatment is required. Wine can be pasteurized by flash heat for 1 minute at 82°C. or by bottling hot at 54°C. (Ough and Amerine, 1966).

5. Oxygen and Redox Potential

Malo-lactic bacteria are not obligate anaerobes, and they will de- velop in presence of dissolved oxygen (cf. Ribereau-Gayon and Pey- naud, 1961, p. 490). Usually they will form colonies on surfaces of solid media. Oxygen seems to have a beneficial effect on their growth. Charpentie (1954) found malo-lactic fermentation started most rapidly when the redox potential was about t0.4 V; Bocker (1964) got malo- lactic fermentation in wine starting with redox potential between f0.1 and +0.2 V. (Obligate anaerobes usually require redox poten- tials less than -0.1 V.) However, Ribereau-Gayon and Peynaud (1961, p. 490) found saturation of new wine with air stimulated malo- lactic fermentation, but saturation with oxygen inhibited it. Thus microaerophilic (“love of small amounts of air”) is an apt description for malo-lactic bacteria.

6. Sulfur Dioxide

Sulfur dioxide is universally used for production of wine of all qualities. The antiseptic is strongly inhibitory to malo-lactic bacteria, if not lethal (Fell, 1961). Reports of malo-lactic bacteria as being sensitive to certain levels of sulfur dioxide are ambiguous because of the variety of forms of the compound in solution. A large fraction is “bound” (combined with aldehydes and pigments). The “free” form exists as sulfurous acid, bisulfite, or sulfite ion. Of the free form only sulfurous acid is directly active on metabolic systems of the bacteria-

252 RALPH E. KUNKEE

probably by oxidation of disulfide group of proteins (Wyss, 1948). The bisulfite ion is indirectly inhibitory by its combination with acetaldehyde needed by the cell in alcoholic fermentation for reoxidation of reduced coenzymes. Thus the amount of active sulfur dioxide is dependent on the concentration of aldehydes and on the pH, both of which are variable. Instability of the bisulfite-acetaldehyde addition compound also adds to the difficulty. Fornachon (1963b) showed that lactic acid bacteria metabolized part of the addition compound and thus “freed” sulfur dioxide and increased the inhibitory effect. Sulfur dioxide is also relatively easily removed from solution by oxidation to sulfate or reduction to elemental sulfur. [Most lactic bacteria do not produce free acetaldehyde as do yeast (Wilkinson and Rose, 1963). Thus in laboratory cultures of bacteria, the inhibitory effect of sulfur dioxide ought to be greater than in mixed cultures with yeast.] Thus, the concentration of total, or even free, sulfur dioxide does not give a precise estimation of inhibitory activity. Nevertheless, practical concentrations for control can be arrived at empirically. Fornachon (1957) gave a table listing the proportion of wines which did not undergo malo-lactic fermentation at a variety of total sulfur dioxide concentrations and pH values.

7. Tannin

We have already pointed out the inhibitory activity of wine against pathogenic organisms. Acid and alcohol are only partly responsible for the antiseptic qualities of wine. Masquelier (1959) showed that charcoal treatment of wine removed much of the antibiotic activity against salmonella, staphylococcus, and coliform bacteria. The tannins (phenolic compounds) were shown to be responsible. Lactic acid bacteria, however, are apparently resistant to grape tannins (Mas- quelier, 1959). Fornachon (1943) pointed out that if tannins were inhibitory to naturally occurring organisms of wine, bacterial activity should occur more readily in white wine than in red, but the opposite is true. Tannins from other plants are sometimes added to wine to facilitate fining and to improve flavor and protect color. Definite retardation of bacterial spoilage can be found with tannin addition of 0.5 gm./liter, but the usual amount added is less than this. It would seem the effect of tannin is of minor influence on malo-lactic fermentation.


E. METABOLISM 1. Sugar Fermentation

Two distinct types of sugar fermentation are recognized in the lactic acid bacteria. One group produces mostly one product (lactic acid) from glucose and thus is called homofermentative. The other group, heterofermentative bacteria, convert about half of the sugar to lactic acid, one-third to ethanol, and one-sixth to carbon dioxide. (Heterofermentation is characterized by gas production from glucose.) A brief account of these two kinds of metabolic activity is necessary here. More thorough discussions can be found elsewhere (see especially Wood, 1961).

The route of catabolism of glucose by the homofermentative bacteria is via the familiar glycolytic pathway (Embden-Meyerhof scheme). In glycolysis, hexose diphosphate is fragmented into two %carbon units which are oxidized to pyruvic acid and reduced to lactic acid. There is no net change in redox state of coenzymes, but there is a theoretical net formation of two high-energy phosphate bonds (in the form of adenosine triphosphate, ATP) per molecule of hexose. In contrast, the heterofermentative bacteria lack the enzyme aldolase which catalyzes the splitting of hexose diphosphate in two (Buyze et al., 1957). Thus complete glycolysis is blocked in heterofermentative bacteria, and glucose is fermented via the first part of the hexose monophosphate shunt (Warburg-Dickens-Horecker scheme). The phosphorylated glucose is oxidized and decarboxylated to give phosphorylated pentose. The latter is again phosphorylated and split into a %carbon and a 2-carbon compound (Heath et al., 1958). The 3-carbon fragment is converted to lactic acid in the way just described for the homofermentative bacteria. The 2-carbon unit, acetyl phosphate, is reduced to ethanol and inorganic phosphate. Again, there is no net change in the redox state of the coenzymes (two molecules of which are reduced in the formation of pentose and are reoxidized in the formation of ethanol). [Glucose oxidation not involving phosphorylation has been reported in Pediococcus spp. (Lee and Dobrogosz, 1965). There was no indication that this was a significant part of the energy metabolism of the organism.]

The above pathways represent only the major routes of catabolism of hexoses. Other pathways are sometimes important. For example, in heterofermentative bacteria, the reduction of fructose to mannitol (Peterson and Fred, 1920) is important and is used as a diagnostic test for this kind of organism. Three molecules of fructose give

254 RALPH E. KUNKEE

approximately two molecules of mannitol and one of lactic acid, acetic acid (or ethanol), and carbon dioxide. This pathway apparently com- petes with the hexose monophosphate pathway, and various amounts of end products can result (cf. Wood, 1961).

In the fermentation of pentoses, no reduction of coenzyme in the initial steps occurs; it is not necessary to use acetyl phosphate as a hydrogen acceptor. The high-energy phosphate of acetyl phosphate can be conserved by transfer to adenosine diphosphate (ADP) (Lip- mann, 1944). This results in a greater yield of high-energy phosphate and in formation of acetic acid rather than ethanol as an end product. This fermentation of pentose occurs in both homo- and heterofermentative bacteria, It would seem that malo-lactic bacteria could use pentoses found in wine with greater efficiency per carbon atom than hexoses.

Other sugars which should be considered here are those found in wine. Besides hexoses and pentoses (Melamed, 1962), other sugars have been detected (Esau and Amerine, 1964): altroheptulose, man- noheptulose, and glyceromannooctulose. Unfortunately, little is known about their metabolism by lactic acid bacteria. Sedoheptulose is involved in the pentose phosphate cycle of the hexose monophosphate shunt. However, studies with radioactive glucose show that the pentose phosphate cycle is not operative in lactic acid bacteria (Rappoport et al., 1951; Bernstein et al., 1955).

It has been pointed out that malo-lactic bacteria are microaerophilic. If the reduced coenzyme formed during the oxidation of glucose in the heterofermentative bacteria could be reoxidized by oxygen as in aerobic organisms, then high-energy phosphate of acetyl phosphate produced could be conserved as in the fermentation of pentoses. There are hints of oxidative phosphorylation or other reactions with oxygen in lactic acid bacteria and other anaerobes (Davis, 1960; Wood, 1961; Gallin and VanDemark, 1964). Johnson and McCleskey (1957) tested a strain of Leuconostoc mesenteroides in which they obtained definite oxygen uptake- 1 mole oxygen per mole of glucose. This extra source of energy would be of great benefit to malo-lactic bacteria in wine which contains very low levels of energy sources.

Many of the residual sugars, such as pentoses, which are found only in trace amounts in wine may not be available as energy sources for all malo-lactic bacteria. Often the machinery for metabolism of sugars (at least some hexoses) must be induced in the bacteria, and the induction may require concentrations as high as 2%, many times higher than that in wine (Rogosa and Sharpe, 1959).


The above pathways of sugar fermentation are the major pathways with the major end products of lactic acid, ethanol, and carbon dioxide. The extent of utilization of these pathways by the organisms depends not only on strain, but also on pH, substrate, redox potential, stage of growth of the organism, etc. (Gunsalus and Campbell, 1944; DeMoss et al., 1951). Even in homofermentative bacteria, as much as two-fifths of the glucose can go, under special conditions, to products other than lactic acid (cf. Wood, 1961). Davis (1963) pointed out that in practice, the distinction between homo- and heterofermentative bacteria often is indefinite.

2. Pyruvic Acid

Pyruvic acid is present in wine in relatively small amounts, but it is implicated in malo-lactic fermentation; the concentration is lower in wine which has undergone the deacidification (Rankine, 1965). Pyruvic acid is of course extremely reactive biologically, and some of its reaction products are important flavor components.

Already mentioned was the reduction of pyruvic acid to lactic acid. Here pyruvic acid acts as a hydrogen acceptor in the reoxidation of coenzymes. Pyruvic acid can a1 so be oxidatively decarboxylated with an uptake of phosphate to give acetyl phosphate. In one case, another molecule of pyruvic acid acts as hydrogen acceptor, the hydrogen being transported via a flavoprotein carrier (Hager et al., 1954). In another case, oxygen is reduced and the resulting hydrogen peroxide oxydizes a second molecule of pyruvic acid to give acetic acid and carbon dioxide (Hager et al., 1954). In either case, high-energy phosphate, which can be transferred to ADP, is formed from two molecules of pyruvic acid. Acetyl phosphate itself can be a hydrogen acceptor in its reduction to ethanol and inorganic phosphate. Free acetaldehyde is not formed in this reaction as it is in yeast alcoholic fermentation (Wilkinson and Rose, 1963).

Pyruvic acid is a precursor to three related 4-carbon compounds: diacetyl (2,Sbutandione), acetoin (3-hydroxy-2-butanone), and butylene glycol (2,3-butandiol). All three are found in wine. The pathway of their formation is not completely understood. For acetoin, two molecules of pyruvic acid react to give a-acetolactic acid and carbon dioxide (Juni, 1952). a-Acetolactic acid is then decarboxylated to acetoin. Rowatt (1951) and Moat and Lichstein (1953) showed quantitative conversion of pyruvic acid to acetoin and carbon dioxide at low pH, in presence of glucose. Using radioactive substrates, Shira- kawa et aZ. (1964) explained the need for glucose by what they called

256 RALPH E. KUNKEE

“entangled” fermentation in homofermentative lactobacilli. In this scheme, acetoin is formed from a molecule of free pyruvic acid and a molecule of pyruvic acid from glucose fermentation. The other molecule of pyruvic acid from glucose goes to lactic acid. Another molecule of free pymvic acid is reduced to lactic acid to balance the coenzyme redox state.

Acetoin can be reduced to butylene glycol (DeMoss et al., 1951; Charpentie et al., 1951). The relative amounts of acetoin and butylene glycol depend on the amount of available hydrogen. Diacetyl is an oxidation product of acetoin. However, research with cell-free ex- tracts indicated that the pathway of formation of diacetyl in lactic acid bacteria is not by way of acetoin as was supposed. It apparently arises, as acetoin does, from pyruvic acid via acetaldehyde-thiamine pyrophosphate. The latter forms an addition product with acetyl coenzyme A which rearranges to give diacetyl and to regenerate thiaminepyrophosphate carbanion (Speckman and Collins, 1966).

Another product of pyruvic acid metabolism, at least in some bacteria, is formic acid which comes from the splitting of pyruvic acid. High-energy phosphate is produced in this reaction in the form of the other product, acetyl phosphate (cf. Utter et al., 1944). Fornachon (cf. Rankine, 1965) obtained faster malo-lactic fermentation when pyruvic acid was added.

3. Citric Acid

Citric acid is normally found in grapes at much lower levels than tartaric or malic acids, but it is sometimes added to acidify wines artificially. Not all lactic acid bacteria will attack citric acid. du Plessis (1964). found that malo-lactic strains of P. cerevisiae and Lactobacillus buchneri did not metabolize it. In addition to these two species, the same was found to be true with malo-lactic Lactobacillus delbrueckii (Pilone et al., 1966). Bacteria that can metabolize citric acid can use it as an energy source (Deffner, 1938; Campbell and Gunsalus, 1944; Charpentie et al., 1951). In the first step, citric acid is split to oxaloacetic acid and acetic acid. Decarboxylation of oxaloacetic acid gives pyruvic acid which is metabolized in the ways outlined above. Oxalo- acetic acid formed from citric acid can also act as a hydrogen acceptor, if need be, and be reduced to succinic acid. Bacteria which do not metabolize citric acid probably lack citrate permease (Collins and Harvey. 1962). In Streptococcus diacetilactis, citric acid is metabolized but is not used as an energy source; apparently citric acid metabolism provides some sort of detoxification (Harvey and Collins, 1963).


Resting cell fermentation of citric acid by several malo-lactic lactobacilli have been studied by du Plessis (1964). The major end products were lactic acid, acetic acid, and carbon dioxide. Also found were formic acid, ethanol, succinic acid, and acetoin. Charpentie et al. (1951) found butylene glycol also an end product of citric acid fermentation by malo-lactic bacteria.

4 . Malic Acid

It is generally believed that malic acid is stoichiometrically converted to lactic acid and carbon dioxide by malo-lactic bacteria. du Plessis (1964) obtained 97% recovery of the carbon of malic acid as lactic acid and carbon dioxide in resting cultures of Lactobacillus buchneri. With other malo-lactic bacteria the recovery was less, as low as 85%. Ethanol was also formed. With the homofermentative bacteria some acetoin and diacetyl were found. The stoichiometry of the malo-lactic reaction is important in the consideration of the energetics of malo-lactic fermentation. This will be discussed further in Section V1,C.

L-Malic acid is the acid found naturally in grapes. D-Mak acid is sometimes added to wine (in the form of the DL-malic acid mixture) to increase the acidity. Malo-lactic bacteria do not attack the D-isomer. Further, Radler (1966) reported the inhibition of the induction of “malic” enzyme by D-malic acid (see below, Section V1,B). However, malo-lactic fermentation was readily obtained in musts in which DL- malic acid had been added to supplement or replace the natural acid (Ough and Kunkee, 1967). D-Malic acid has little or no effect on the activity of malic dehydrogenase used in enzymic assay of L-malic acid (Kunkee and Combs, unpublished results).

F. END PRODUCTS During malo-lactic fermentation, lactic acid is the product formed

most abundantly; it is formed from sugars, malic acid, and citric acid. Lactic acid exists in two optically active forms. The types formed- from glucose - depend on the organism. Of characterized malo-lactic bacteria, the types producing D-lactic acid or a mixture of D- and L-isomers predominate: P . cerevisiae form DL-lactic acid; Leuconostoc spp. form D-; and of the malo-lactic lactobacilli only L. delbrueckii form just the L-isomer (Breed et al., 1957). There is some confusion about the isomers: L-lactic acid (“sarcolactic”) has the same con- figuration as L-glyceric acid. Solid L-lactic acid is levorotatory, but in solution the formation of a strongly dextrorotatory ethylene oxide bridge causes the acid to rotate polarized light to the right (Lockwood

258 RALPH E. KUNKEE

et al., 1965). Thus the isomer is designated L(+)-lactic acid. The opposite is true with D(-)-lactic acid (“paralactic”).

Some of the lactic acid in wine is produced by yeast during alcoholic fermentation and is of the D type. In wines that have undergone malo-lactic fermentation most of the lactic acid is produced by the bacteria from malic acid. It is surprising that examination of many malo-lactic wines revealed L(+)-lactic acid as the predominant form present (Peynaud et al., 1966a,b; Brechot et al., 1966). We have seen no information ab,out kinds of lactic acid produced by bacteria from malic acid as the substrate.

Increase in volatile acidity (mainly acetic acid) was very early shown to accompany malo-lactic fermentation (Seifert, 1901). Riber- eau-Gayon and Peynaud (1961, p. 437) suggested that most of the acetic acid comes from citric acid. Wines with no fermentation of citric acid had little increase in acetic acid. Addition of more malic acid after malo-lactic fermentation brought about a second malo-lactic fermentation with no additional formation of acetic acid. However, du Plessis (1964) found acetic acid in resting cell fermentations of malic acid. Lactic acid is also slightly volatile with steam distilla- tion. Small amounts, depending on pH and type of equipment used, are distilled during the usual volatile acid determinations (Amerine, 1965; Pilone, 1965). Some of the increase in volatile acidity found after malo-lactic fermentation may be contributed by lactic acid, but there seems to be no doubt that an increase in acetic acid also occurs. There is one report of no increase in volatile acidity after malo-lactic fermentation (Bremond, 1937, p. 29).

Acetoin and related compounds are usually produced in very small amounts, but their organoleptic effects may be large. Butylene glycol has a slightly sweet taste similar to glycerin (Amerine, 1965). Acetoin is rather odorless and tasteless, but its general sensory effect can be pleasant (Niven, 1952). Acetoin and butylene glycol have optical isomeric forms, but there is no information about organoleptic differences between them. Diacetyl has a potent odor often associated with dairy products. Vaughn and Tchelistcheff (1957) reported that addition of diacetyl and acetoin to wine gave a flavor and odor similar to sauerkraut. Diacetyl in very small amounts may improve wine (Fornachon, 1963a). Citric acid generally increases diacetyl production, especially under nonoptimal conditions (Davis, 1963). Homo- fermentative bacteria usually produce higher concentrations of diacetyl than heterofermentative types (Fornachon, 1963a; Davis, 1963; du Plessis, 1964). The concentrations of acetoin and diacetyl are usually determined as the total of both. A higher concentration


of these products is found after malo-lactic fermentation (Radler, 1962b; Fornachon, 1963a; Dittrich and Kerner, 1964; Fornachon and Lloyd, 1965), but Kunkee et al. (1965) found no statistical significance between concentration and malo-lactic fermentation.

Short-lived off-odors often accompany malo-lactic fermentation in wine. Undoubtedly part of this is hydrogen sulfide (Suverkrop and Tchelistcheff, 1949) produced from the sulfur dioxide added to wine but not to laboratory media.

We should also mentioned the esterification of acids by ethanol and higher alcohols. Yeasts are probably the causative agents (Nord- strom, 1966), but acid products of malo-lactic bacteria are involved. The tastes of the various nonvolatile organic acids are nearly indis- tinguishable (Pangborn, 1963), but the esters are more volatile and may play an important role in flavor and odor of wine. We have noted that lactic acid exists in two forms which probably have very similar sensory properties but the various esters of each of the acids may have a distinctive sensory character. There may, of course, be other products arising from the presence of lactic acid.

In the laboratory the substrates, organisms, and conditions can be controlled. In practical operations many of these factors are variable, and they can influence each other profoundly. du Plessis (1964) speculated that there was little difference between malo-lactic bacteria and spoilage bacteria; he believed the environmental conditions were of more importance than the bacterial strain. Luthi (1957) reported he found no case where an organism from wine with some special characteristic produced that same characteristic when used as inoculum in sterile wine. He attributed this to symbiotic relation- ships existing between the several organisms in the natural, mixed culture in wine.

V. Detection of Malo-lactic Fermentation

Changes in acidity and pH are not reliable indices of malo-lactic fermentation because they can be influenced by many other factors. The qualitative determination of malic acid by paper chromatography is simple and has proved to be invaluable for routine analysis. Several procedures using butanol-acid solvents have been described (Lugg and Overell, 1948; Dolle, 1958; Ribereau-Gayon and Peynaud, 1958). Acid spots can be detected by spraying the paper with acid-base indicator or by addition of the indicator directly to the solvent. Absence of the malic acid spot is satisfactory proof of malo-lactic fermentation (Ingraham and Cooke, 1960). The presence of a spot at the lactic posi-

260 RALPH E. KUNKEE

tion is not confirmatory proof, because in these systems succinic acid and lactic acid are not separated. Solvents for separation of succinic acid and lactic acid were reported by Hartley and Lawson (1962) and Blundstone (1963). These acids can also be separated by two-dimen- sional thin-layer chromatography (Higgins and von Brand, 1966).

Quantitative measurement of malic acid can be made enzymically. In one method, suggested by Korkes and Ochoa (1948), manometric determination is made of carbon dioxide formed by the decarboxylating activity of “malic” enzyme on malic acid (Kolar, 1962). In the other, spectrophotometric measurement is made of reduced coenzyme brought about by the action of malic dehydrogenase on malic acid (Mayer and Busch, 1963). D-Malic acid is not active in these assays.

We have already discussed (Section IV,B) the determination of total and L-lactic acid, and of D-lactic acid by difference. A simple and reliable method of quantitative determination of D-lactic acid would be very useful.

VI. Deacidification

A. MECHANISM

Ochoa and co-workers obtained an enzyme preparation from malic acid-adapted Lactobacillus urubinosus (plantarum) that decarboxylated malic acid to lactic acid (Korkes and Ochoa, 1948). This enzyme was similar to the carbon dioxide-fixing enzyme, “malic” enzyme, which had been obtained from pigeon liver (Ochoa et al., 1948). (We will use the name “malic” enzyme for the bacterial enzyme, but it is now properly called L-malate: nicotinamide adenine dinucleotide oxidoreductase (decarboxylating) E. C. 1.1.1.38.) Nicotinamide adenine dinucleotide (NAD) was required, and the reaction was assumed to involve a redox change. In analogy to the pigeon liver enzyme, a two-step reaction involving pyruvic acid was suggested:

malic acid + NAD+ - pynivic acid + COn + N A D H + H + (1)

Pyruvic acid as an intermediate is a convenient explanation for the coenzyme requirement. However, free pyruvic acid has not been found in the reaction. In exchange reactions with radioactive malic acid, no label was found in pyruvic acid (Kaufman et at., 1951).


Apparently there is a very close association between “malic” enzyme and lactate dehydrogenase, the enzyme for reaction (2), and pyruvic acid. The latter is thus reduced to lactic acid before being released from the enzyme surfaces. Lactate dehydrogenase activity has not been separated from “malic” enzyme, although the former is a constitutive enzyme of the bacteria, and the latter is inducible. With highest purification of “malic” enzyme, L-lactate dehydrogenase activity is present, while in the more impure preparations both D- and L-lactate dehydrogenase activities were found (Kaufman et d., 1951).

The reactions are undoubtedly reversible, as shown, but this is difficult to prove. The equilibrium of the second reaction is far to the right (Burton and Wilson, 1953), but its reversibility can be demon- strated (Hohorst, 1963). In the first reaction direct fixation cannot be shown with the bacterial enzyme because the substrates, pyruvic acid and NADH are rapidly removed from the solution by the lactate dehydrogenase activity. However, in exchange experiments, uptake of radioactive carbon dioxide was found in malic acid (Korkes et aZ., 1950).

“Malic” enzyme decarboxylates oxaloacetic acid, but oxaloacetic acid is apparently not an intermediate in the decarboxylation of malic acid. If it were there would be no situation where malic acid would be decarboxylated faster than oxaloacetic acid, but this did occur at pH 6 (Korkes et al., 1950). Decarboxylation of oxaloacetic acid required only manganous ions as cofactor. Malic acid decarboxylation required NAD, specifically, as coenzyme, and for an unexplained reason phosphate was required for maximal activity (Ochoa, 1951).

Recent studies with Bacterium “L” (a strain of LactobaciZZus plantarum) show different enzymes for decarboxylation of malic acid and oxaloacetic acid (Flesch and Holbach, 1965). “Malic” enzyme activity was inhibited by p-chloromercuribenzoate, but oxaloacetic decarboxylase was not. Other evidence was presented against the oxidation of malic acid to oxaloacetic acid before decarboxylation. This oxidation would be catalyzed by malic dehydrogenase which was shown to have optimal activity at pH 10. If the intracellular pH was anywhere near that of wine, it would seem malic dehydrogenase would be nonoperative under malo-lactic fermentation conditions.

A malic-lactic transhydrogenase in Micrococcus lactilyticus has been described (Allen and Galivan, 1965; Dolin et al., 1965). This enzyme reversibly converted malic and pyruvic acids to oxaloacetic and lactic acids. It had no dehydrogenase or decarboxylase activities and required no exogenous cofactors.

262 RALPH E. KUNKEE

B. ENZYME INDUCTION

The original studies on bacterial “malic” enzyme showed that malic acid decarboxylating ability of the bacteria was inducible. The enzyme developed in bacteria grown on malic acid and even in noninultiplying cells incubated in presence of malic acid (Blanchard et al., 1950). Biotin and an energy source were required for “malic” enzyme induction. Flesch and Holbach (1965) showed “malic” enzyme synthesis was inhibited by avidin, an antibiotin factor.

Nathan (1961) used enzvme induction to separate malic acid and oxaloacetic acid decarboxylating activities in three malo-lactic bacteria. She found L-malic acid induced both these activities, but oxaloacetic acid induced only oxaloacetic acid decarboxylase activity. Oxaloacetic acid permease was induced only by oxaloacetic acid. Isolation of the enzymes is required before one can say whether malic acid was an inducer for one enzyme with two activities or for two separate enzymes, one of which can also be induced by oxaloacetic acid.

Whiting and Coggins (1963) studied “malic” enzyme induction in several malo-lactic bacteria isolated from cider. Some of the bacteria were found to have inducible “ma1ic”- enzyme, but in others it was constitutive. In the bacteria studied, citric acid fermentation occurred only in those strains in which “malic” enzyme was inducible.

In the first studies on induction of “malic” enzyme, relatively high concentrations of inducer were used [150 mM DL-mahc acid (Blan- chard et d., 1950)l. Other workers have used concentrations of inducer approximating that found in grape juice. Nathan (1961) and Flesch and Holbach (1965) used 30 mM L-malic acid. Results showing the dependency of “malic” enzyme induction on concentration of malic acid would be of special interest to enologists. Strains of malo- lactic bacteria that required high concentrations of malic acid for induction of “malic” enzyme would not carry out malo-lactic fermentation in wines with low malic acid. These strains should not be used for inoculation of wine for malo-lactic fermentation. The presence of these bacteria would explain situations where wines become biologically stable after incomplete malo-lactic fermentation. If the bacteria began to multiply again during the secondary fermentation, say because the wine was racked, then the newly formed bacteria might not be able to make the induced enzyme because of the lower level of inducer (malic acid) present.


C. ENERGETICS

In his excellent monograph Schanderl (1959, p. 164) described the malo-lactic reaction as one which yields no energy. This statement, which has been widely quoted, was based on a consideration of the difference between the molar enthalpies (AH) of the substrate, malic acid, and the products, lactic acid and carbon dioxide. [AH, determined as the heat of combustion, for malic acid is usually given as -320.1 kcal./mole and for lactic acid as -326 (Kharasch, 1929). (The heat of combustion of carbon dioxide is, of course, 0.) More recent evaluation gives the heat of combustion for L-malic acid as -318.0 kcal./mole (Wilhoit and Shiao, 1964) and for L-lactic acid as -321.2 (Saville and Gundry, 1959). Calorimetry experiments with malic acid and the enzymes and cofactors required for conversion to lactic acid resulted in a slight rise in temperature (Schmidt, 1959).] Thus the conversion, in the solid state, of malic acid to lactic acid and carbon dioxide is endothermic and requires an enthalpy input of 3.2 kca1.l mole. However, whether energy is required for the reaction depends not only on the enthalpy difference but also on the change in entropy at the temperature of the reaction; a large increase in entropy would overcome the increase in enthalpy to make the reaction actually exergonic. The change in free energy (AG) (Lewis and Randall, 1961; Lehninger, 1965) tells us whether the reaction will proceed and if it is a potential source of energy for the ba~ te r i a .~

“Standard”8 free energy changes (AG’) for these reactions have been calculated by Burton and Krebs (1953):

(4) A G ‘

malate-2 + NADP+ = pyruvate-* + NADPH + COP -2.0 kcal.,mo,e

pyruvate-’ + NADH + H+ = lactate-’ + NAD- 6.0 (5)

Under “standard” conditions -at pH 7 -the acids are completely ionized, as written. Note that at this pH, hydrogen ion is not involved in the decarboxylation equation (4).

The free energy changes for oxidation and reduction of NAD and

‘Under conditions of constant temperature heat cannot be converted to work, and any change in enthalpy is not available as energy anyway.

‘“Standard” conditions, as used by Burton and Krebs (1953), are 25”C., pH 7,0.05 atm. carbon dioxide and 0.2 atm. oxygen, and 0.01 M aqueous solutions of other re- actants and products.

264 RALPH E. KUNKEE

NADP (nicotinamide adenine dinucleotide phosphate) are not ex- actly equivalent (Burton and Wilson, 1953):

NADPH + Hr = NADP+ + 2[H] 4.1 (6)

NAD+ + 2[H] = NADH + H+ -4.3 (7)

Summation of equations (4) through (7) gives:

malate-2 + H+ = lactate-’ + GO2 AG’ = -8.2 kcal./mole (8)

Equation (8) shows that the reaction actually is exergonic and is therefore a potential source of energy for the bacteria. However, the above calculations must be modified to consider the reaction in wine rather than at “standard” conditions. Let us examine the reaction at (a) low pH in alcohol, (b) in presence of carbon dioxide, and (c) at cellar temperature.

Because of the uptake of a proton by the substrate [cf. equation (S)], it would seem the high concentration of hydrogen ion in wine would tend to drive the reaction toward the formation of products. But at low pH one must also consider the change in ionic form of the acids.Y The p&’s for L-malic acid are 3.40 and 5.13 (Jones, 1962). The p& for lactic acid has recently been redetermined and found to be lower than previously given (Lockwood et al., 1965). For either isomer of lactic acid, the plc, is 3.73. The thermochemical equilibrium constants for these acids in alcoholic solution were not found in the literature; but for tartaric acid, in 10% alcohol at 20°C., there is an increase of p&, of 0.05 and of p&, of 0.20 (Berg and Keefer, 1958). With the use of these values as estimates of the alcohol effect on the dissociation of L-malic and DL-lactic acids, the approximate thermodynamic p&’s are: for L-malic, p&, = 3.45 and p&% = 5.33; for lactic acid, p& = 3.78. Then at the pH of wine, say pH 3.4, 52.5% of the malic acid is not ionized, 46.9% is malate-’, and 0.55% is malate-‘; and 70.6% of the lactic acid is not ionized and 29.4% is lactate-’.

At pH 3.4, equation (8) would be represented by the following four reactions:

(0.55%) malate-2 + H+ (pH 3.4) = lactate-’ + COz b)

3Thermodynamically it is probably unimportant which ionic forms of the acids are actually involved in the enzymic reactions as long as the change in protonation is con- sistent, but the change in free energy in equation (8) was obtained from reactions with acids in completely ionized form (pH 7 ) .


(b)

(28.8%) malate-' = lactate-' + COP (4

(d)

The free energy of equation (a) can be obtained from equation (8) by considering the change in pH at 25°C. ( R is the gas constant, T is the absolute temperature):

(18.1%) malate-' + H+ (pH 3.4) = lactic acid + COZ

(52.5%) malic acid = lactic acid + COz

10-7 M H+ AG, = AG' + RT In 10-3,4 K+ = -8.2 - 4.9 = -13.1 kcal./mole

For the other equations the free energy of ionization of the organic acids is required. For 1 molal solutions, AG = 2.3 RT PI( , (see Fruton and Simmonds, 1961). Substitution with the above thermodynamic equilibrium constants gives:I0

malic acid = malate-' + Hf AGM, = 2.3 RT (3.45) = 4.7 kcal./mole

malate-' = malate-2 + H+ AG,wn, = 2.3 RT (5.33) = 7.3

lactic acid = lactate-' + H+ AGl, = 2.3 RT (3.78) = 5.2

(9)

(10)

(11)

Thermodynamic equilibrium constants were used, thus the free energies of ionization in kcal./mole are the same at 0.01 M concentration. Addition of equations (a) and (10) and subtraction of (11) gives equation (b): AGb = AG, + AGW, - AG,, = -13.1 + 7.3 - 5.2 = -11.0 kcal./mole. For equation (c), the pH of equation (10) is changed from pH 2 (0.01 M ) to pH 3.4 (on right side of equation): malate-l= malateP2 + H+ (pH 3.4). The change in free energy

M H+ RT In H+ = 7.3 - 1.9 = 5.4 kcaI./mole

Addition of this last equation to (a) gives equation (c): AGc = AGM~ + AGa = 5.4 - 13.1 = -7.7 kcal./mole. Equation (d) is calculated from equations (c), (11) and (9): AGd = AG, - AGL + AGM, =-7.7 - 5.2 + 4.7 = -8.2 kcal./mole. By combination of the proper proportions of the changes in free energies as represented by equations (a), (b), (c ) and (d), the overall change in free energy at pH 3.4 is:

AG, -13.1 x 0.55% = -0.07 kcal./mole

'OThe activity coefficient for hydrogen ion in water is nearly 1 at these concentrations (Kielland, 1937).

266 RALPH E. KUNKEE

AC& -11.0 X 18.1% =-I .%

-8.2 X 52.5% = -4.30 AGc -7.7 x 28.8% = -2.22 AGd

AG” = -8.6 kcal./mole

The pH effect is not great. Even though free energy changes for reactions involving hydrogen ion, (a) and (b), are larger than (c) and (d), the relative amount of the former is smaller than the latter. If carbon dioxide were released in the form of bicarbonate ion this would not be the case. Hydrogen ion would appear on the right side of equations (c) and (d) and the equilibria would be shifted toward the left. This is a very important consideration. Schmidt (1959) calculated the change in free energy, at pH 0, for the following reaction:

COz + H2O = HCO3- + H+ 11.2 kcal./mole (12)

At pH 3.4, the change in free energy would be 11.2 - 4.6 = 6.6 kca1.l mole. Addition of the latter to equation (8) would give a reaction much less exergonic, -2.0 kcal./mole. Carbonic anhydrase studies on decarboxylation of pyruvic acid by yeast pyruvic carboxylase indicated carbon dioxide, not bicarbonate ion, as the product (Krebs and Roughton, 1948). This has also been assumed to be true for malic acid (Harary et al., 1953). The carbonic anhydrase studies ought to be repeated with “malic” enzyme.

Carbon dioxide concentration in wine is higher than at “standard” conditions. Newly fermented wine has about 1 gm./liter carbon dioxide from alcoholic fermentation (see Amerine, 1958). The head space above the wine in storage tanks is often blanketed with carbon dioxide gas at about atmospheric pressure. The concentration of carbon dioxide in new wine could and probably does reach saturation. The maximum concentration of carbon dioxide in wine in equilibrium with 1 atm. of the gas is about 2 gm./liter (see Amerine, 1958) or about 26.8 times, say 30 times, the concentration under “standard” conditions” used in equation (8). The adjusted free energy change is at least: AG“‘ = AG“ + RT In 30 =-8.6+2.0=-6.6 kcal./mole. Peynaud (1955) observed that at low temperature, where solubility of carbon dioxide is higher, malo-lactic fermentation can occur with no detectable gas production. More often effervescence is a noticeable aspect of malo-lactic fermentation in new wine. Carbon dioxide loss would

“Henry’s Law constant for solubility of carbon dioxide in water at 25°C. is 1.25 X

lo6 mm Hg per molar fraction (Loomis, 1928). At 0.05 atm. carbon dioxide, the concentration of carbon dioxide in water would be 0.0745 gm./liter.


tend to make the reaction more exergonic. The high acidity of wine also decreases the solubility of carbon dioxide; carbonic acid pK1 is 6.4. The decreased solubility because of acidity may be overcome by the increased positive charge on the proteins and pigments of wine at low pH which tend to cause electrostatic attraction of bicarbonate ion (Amerine et al., 1967, p. 214). At any rate, the actual concentration of carbon dioxide at the pH of wine was considered in calculation of AG"'. l2

Finally, cellar temperature is different from "standard" temperature and must be considered. The change in free energy with temperature is related to temperature by the entropy change (AS) of the reaction (Lewis and Randall, 1961):

AG - AH--6.6 - 3.2- (g) =-AS=-- - -0.011 kca1.imole-" aAT T 298

Thus the correction for change in free energy at cellar temperature of 18°C. is 0.08 kcal./mole, and the adjusted free energy change for the reaction at this temperature is -6.5 kcal./mole. (At 18"C., the concentration of carbon dioxide would be higher than at 25"C., but the actual concentration found under cellar conditions was used for calculation of AG"').

Several uncertainties in the calculations were noted above, but an estimate of change of free energy in the malo-lactic reaction in wine under cellar conditions (pH 3.4, 18"C., 1 atm. carbon dioxide) is -6 kcal./mole. From this estimate, the reaction is favorable and the equilibrium is on the side of the products. Whether this potential energy is actually available to the bacteria is another question.

The potential energy is not available to the bacteria whenever malic acid is completely converted to lactic acid. Useful energy for the cell requires the formation of high-energy phosphate. We see there is no direct formation of ATP in the malo-lactic reaction. Reoxidation of reduced coenzymes can lead to formation of high-energy phosphate, but in malo-lactic fermentation there is no net change in redox state of

l2 We might consider malo-lactic fermentation which occurs after bottling. Bottled wine has about 0.5 gm./liter carbon dioxide (see Amerine, 1958), and the concentration is less than in new wine blanketed with carbon dioxide. Even after malo-lactic fennenta- tion, the additional carbon dioxide produced from, say 0.4% malic acid, would make the final concentration of entrapped carbon dioxide only about 2 gm./liter, Iess than the maximum concentration in wine in equilibrium with carbon dioxide at atmospheric pressure. Higher concentrations might be obtained when alcoholic or other fermentations occurred after bottling.

268 RALPH E. KUNKEE

coenzymes. The conversion of malic acid to lactic acid, however, is not necessarily complete. Early work (Ribereau-Gayon and Peynaud, 1938) has been cited to show the reaction to be complete, but the lactic acid found was only 75% of that required for complete conversion of malic acid. Furthermore, substantial amounts of lactic acid found were undoubtedly from sources other than malic acid. du Ples- sis (1964) reported 10 to 15% of malic acid was recovered in products other than lactic acid and carbon dioxide in resting cell siisnensions of malo-lactic bacteria. One might speculate on the conversion of the intermediate, pyruvic acid, to acetyl phosphate. The hydrogen produced in the formation of pyruvic acid and acetyl phosphate could be used for the reduction of acetyl phosphate to ethanol (Section IV,E). The reduced coenzyme might also be reoxidized by reduction of another molecule of malic acid to succinic acid.

In spite of these possibilities, it has not been shown that malic acid can be used as an energy source for malo-lactic bacteria. Two exceptions have been reported. Krasil’nikova (1965) found growth of Lactobacillus delbrueckii on wort was stimulated somewhat by malic acid; Bidan (1966) reported some growth of malo-lactic bacteria on malic acid. Radler (1958~) found no loss of malic acid unless carbohydrate was fermented simultaneously. Melamed (1962) reported a decrease in residual sugars of wine after malo-lactic fermentation. Peynaud (1955) found addition of glwose stimulated malo-lactic fermentation in wine. The best proof of complete malo-lactic conversion was with tracer experiments; Schmidt et al. (1962) got nearly complete recovery of lactic acid and carbon dioxide from 1,4-malic acid-W. Unfortunately it was necessary to use DL-malic acid, and the disappearance of the substrate was greater than could be accounted for by fermentation of L-malic acid only. The average recovery of lactic acid was 97% with a range from 90% to 106%.

It is strange that malic acid is not used for energy in the schemes outlined above. Perhaps pyruvic acid is available only for reduction to lactic acid. We have seen that free pyruvic acid is not formed as an intermediate. It is apparently bound tightly to the enzymes and reduced to lactic acid before being released. For citric acid to be used as an energy source, the oxaloacetic acid formed from citric acid may be decarboxylated to give unbound pyruvic acid. Kaufman et al. (1951) did not test oxaloacetic acid in their exchange experiments with pyruvic acid.

Mutants of malo-lactic bacteria might be obtained in which enzymes were altered such that there was less affinity for pyruvic acid. In these mutants, pymvic acid from malic acid would not be bound as tightly


as in the wild type and would be available for exergonic reactions. This hypothesis could be tested by searching for mutants of malo- lactic bacteria which could grow on malic acid as an energy source.

The ruison d’etre of malo-lactic fermentation is puzzling if the reaction does not provide energy for the organism. From an evolutionary point of view, the increase in pH of weakly buffered solutions provided by malo-lactic fermentation would be advantageous for bacterial growth, but growth of all organisms present in the primitive medium would be favored. Another explanation might be that enzymes involved are used for other reactions and malo-lactic fermentation is an accidental expression of their activity. Additional studies on inducer and substrate specificities of “malic” enzyme are necessary. Metab- olism of malic acid may provide some sort of detoxification as has been shown with citric acid (Harvey and Collins, 1963).

D. SECONDARY EFFECTS OF DEACIDIFICATION

We have seen that malo-lactic fermentation causes loss of acidity and increase in pH in wine. These changes in turn can bring about other changes.

The color of anthocyanin pigments of red wine is dependent on pH and oxidation state. Malo-lactic fermentation can cause up to one-third loss in red color. Part of the color loss is from the change in pH. However, Vetsch and Liithi (1964) found that fermentation of citric acid, rather than malic acid, by malo-lactic bacteria caused a color change by providing hydrogen as reductant. They also claimed loss of color of anthocyanins by NADH2 in absence of bacteria.

If the acidity loss is SO large that an unusually high pH is obtained, not only is there loss in color, but the quality of the color changes from a natural full red to a bluish hue.

With decreased acidity, wine is a more favorable medium for microbial growth and thus more susceptible to spoilage. This results directly from the change in pH and also from the indirect effect on sulfur dioxide (Section IV,D).

The midpoint between p&’s for tartaric acid (Berg and Keefer, 1958) is about 3.6. Thus if wine is saturated with potassium bitartrate and malo-lactic fermentation brings about an increase in pH which approaches pH 3.6, the potassium bitartrate will precipitate. The precipitation causes turbidity and further loss of acidity.

Fornachon (1963a) pointed out that wine fined with an excess of gelatin may become hazy after malo-lactic fermentation because of the increase in pH.

270 RALPH E. KUNKEE

VII. Control of Malo-lactic Fermentation

A. DESIRABILITY OF CONTROL Winemakers from regions where wines of high acidity are pro-

duced have praised malo-lactic fermentation and declared it an essen- tial ingredient for premium quality wine (e.g., Ferre, 1928; Ribereau- Gayon, 1946; Peynaud and Domercq, 1959; Marques Gomes et aZ., 1956). Much of the utility of the fermentation in these wines is, of course, the deacidification, but other benefits may result.

In warmer regions, as in parts of California, the deacification effect is usually not desirable. Nevertheless, malo-lactic fermentation was found to occur most frequently in California wines of highest quality, vintage and varietal wines (Ingraham and Cooke, 1960). It has been suggested that by-products of malo-lactic fermentation make subtle flavor changes to give a distinctiveness and complexity the wine would otherwise not have (e.g., Suverkrop and Tchelistcheff, 1949; Vaughn and Tchelistcheff, 1957; Ingraham and Cooke, 1960). Bac- teria associated with certain wineries (see Section IV,C) may add distinctive characteristics to their wines (cf. Marques Gomes et al., 1956; Webb, 1962). Pilone and Kunkee (1965) made sensory tests of wines especially fermented with different malo-lactic bacteria. Differences in the organoleptic qualities of the wines were found, but the differences were not as striking as might have been expected. In the chemical analyses of the volatile components of these wines, small differences were found in acetoin (plus diacetyl), volatile acidity, and diethyl succinate (Pilone et al., 1966). The authors suggested that use of grapes with higher varietal character than used by them might provide substrates for the bacteria for greater differences in metabolic end products.

Biological stability is another beneficial aspect of malo-lactic fermentation. As long as malic acid is present, nonsterile wine should be considered unstable, although wines over 2 years old rarely undergo malo-lactic fermentation. However, gassy wines of Burgundy and Italy are found on the American market.

Malo-lactic fermentation may not be of benefit to less-than-premium wines (Ough and Amerine, 1963). Some of these wines are ready for consumption about the time of the secondary fermentation. Malo- lactic fermentation would be considered spoilage in these wines. Furthermore, the changes in quality provided by the fermentation would probably be unappreciated in these wines.

Undesirable aspects relating to deacidification caused by malo-


lactic fermentation were discussed in Section VI,D. Other disad- vantages are gassiness, the temporary formation of off-odors, and the increase in some divalent ions. Malic acid, the most potent chelating agent of the organic acids found in wine, complexes copper and iron ions (Rankine, 1960). Loss of malic acid brings about release of the ions which often cause haziness in wine and are oxidation catalysts. This effect is partly compensated by the increase in chelating activity of the other acids because of the increase in pH.

Off-odors such as diacetyl are sometimes removed by refermenta- tion by yeast (see Dittrich and Kerner, 1964).

The decision to encourage or discourage malo-lactic fermentation can be difficult to put into practice. The fermentation has been described as “capricious.” Moreover, wines which need the fermentation most - the high acid wines -are the very ones in which it is most difficult to induce because of the low pH, and vice versa. The usual suggested procedures are helpful, but the best control requires a better knowledge of the strains of bacteria most likely to be involved and a thorough knowledge of the wine components.

B. STIMULATION

Laboratory descriptions of induction of malo-lactic fermentation in wine by bacterial inoculation began with the first studies on malo- lactic fermentation (Koch, 1900) and now are prevalent (see Peynaud and Domercq, 1959; Sudraud and Cassignard, 1959; Domercq et al., 1960; Webb and Ingraham, 1960; Fell, 1961; Fornachon, 1963a; Kunkee et al., 1964). It is difficult to make an estimate of the degree of commercial use of bacterial inoculation. It is apparently used in France, Germany, Switzerland, and Portugal. We know of at least one California winery which has used the procedure consistently and successfully and of others which encourage natural malo-lactic fermentation for production of premium quality wine (Tchelistcheff, 1966).

For stimulation of fermentation by inoculation, careful selection of bacteria is important at this state of the art. We recommend Leu- conostoc citrouorum ML 34 because of its pH and temperature tol- erances and its negligible formation of off-flavors. (The volatile acidity of the wine is increased about 200 mg./liter.) From the references listed, and the author’s observations, the following procedure can be suggested: The selected strain can be propagated in sterile grape juice containing 0.05% yeast extract and titrated to pH 4.5. The presence of malic acid in the propagating medium will assure

272 RALPH E. KUNKEE

adaptation of enzymes for malic acid fermentation, but adaptation probably also will occur during growth of the bacteria in the wine. Grape juice at pH 3.5 with low levels of sulfur dioxide has been used in an attempt to adapt the bacteria to some of the conditions of wine. Inoculations of 0.01% to 1% have been used. Wine which has recently undergone malo-lactic fermentation can also be blended with other wine to induce the fermentation. Here large amounts of “inoculum” are used: 15% to 50%. Some workers suggest the inoculation be made at the beginning of alcoholic fermentation, before the uptake of micronutrients by the yeast; others suggest the middle of alcoholic fermentation when free sulfur dioxide is lowest (because of the high concentration of acetaldehyde formed by the yeast at this time); and still others at the end of alcoholic fermentation, when yeast autolysis begins and when sugars are at low concentrations to mini- mize formation of metabolic end products. With relatively large inocula, the speed of malo-lactic fermentation is independent of time of inoculation (Kunkee et al., 1964). Good data on the effect of size of inoculum and time of inoculation on the quality of the wine are not available.

Natural malo-lactic fermentation can be encouraged by use of low levels of sulfur dioxide (less than 50 mg./liter), delay in addition of acidifying or fining agents, and storage at warmer temperatures. Aeration can stimulate malo-lactic fermentation (see Section IV,D), but Domercq et al. (1960) found it impaired fermentation. These conditions are also those which encourage bacterial spoilage, and the wine should be frequently examined.

It is nearly impossible to obtain malo-lactic fermentation in wines with very high acidity -approaching pH 3.0. In regions producing these wines, it is often legal to increase the pH by addition of chem- icals, by amelioration with water, or by ion exchange. Further deacidification can then be made by malo-lactic fermentation. Increase in pH can also be obtained by blending the wine with another wine which has undergone malo-lactic fermentation and has less acidity. The pH of the blended wine may then allow further malo-lactic fermentation.

After malo-lactic fermentation, wines have less acidity, are turbid, and often have temporary off-odors. Good winery practice suggests addition of extra sulfur dioxide; settling or fining, aeration if hydrogen sulfide is high (Rankine, 1963b), and storage before bottling.

C. INHIBITION If absolute prevention of malo-lactic fermentation is desired,


sterilization of the wine is required. Pasteurization (Ough and Amerine, 1966) has been used, but undesirable changes in flavor from heat do occur. Sterile filtration is gaining acceptance. Removal of lactic acid bacteria by commercially available membrane filters with 0.65 p porosity has been reported (Tchelistcheff et al., 1964). Chemo- sterilants for wine such as sorbic acid and diethyl pyrocarbonate are relatively inactive against lactic acid bacteria (Peynaud, 1963; Mayer and Luthi, 1960) and are not used for control of malo-lactic fermentation.

Secondary fermentation can be discouraged, if not always prevented, by attention to good winery practices. Scrupulous care should be made to maintain clean filling and pumping Iines and storage containers, especially wooden cooperage. Sulfur dioxide is an effective inhibitor if cleanliness and low pH are maintained. Low acid wines should be acidified by chemical addition or by ion exchange -as much as legally permitted and economically feasible. Because of the variability in activity of sulfur dioxide, it is unrealis- tic to make specific recommendations as to the amount of sulfur dioxide to use. Addition of 100 to 200 mg./liter, depending on the condition of the grapes, has been suggested for red wine (Amerine et al., 1967, p. 363). Free sulfur dioxide ought to be determined regularly and maintained at some empirically selected concentration. Removal of gross lees soon after alcoholic fermentation and early fining with high concentrations of fining material also have been suggested, but these procedures may delay malo-lactic fermentation rather than prevent it (Fornachon, 1957; Kunkee, 1966).

Ribereau-Gayon and Peynaud (1961, p. 496) discussed antagonisms between malo-lactic fermentation and certain yeasts used in alcoholic fermentation. Apparently some yeasts can inhibit the secondary fermentation by exhaustion of micronutrients of the medium or by ex- cretion of some unknown inhibitory materials. Practical use could be made of these observations. Radler (1958~) found no symbiotic rela- tionship between the yeast and bacteria as long as adequate amounts of amino acids were present.

VIII. Conclusions

True today as it was 60 years ago is the statement of Alfred Koch (19OO), “. . . acid reduction in wine is a completely normal process [and] therefore the bacteria play a very important role in the normal development of wine.” Malo-lactic fermentation is necessary not only to deacidify some wines and to stabilize others, but also to improve some wines apparently by addition of certain products of metabolism

274 RALPH E. KUNKEE

which make the flavor more complex. Information on the latter is still scant. Metabolism of malo-lactic bacteria has been well studied, but more information about the organisms under conditions of malo-lactic fermentation in wine is needed. Malic acid is not used as an energy source, but the reason for this seems to be biochemical rather than thermodynamic. Malo-lactic fermentation is most likely to occur under conditions which also favor wine spoilage. However, with knowledge of the wine and the bacteria, the fermentation can be controlled - stimulated or inhibited -as desired.

ACKNOWLEDGMENT The author thanks G. J. Pilone and Mrs. Leslie Westergaard for their help with some

of the literature.

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