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Applied and Environmental Microbiology, October 2004, p. 5715-5731, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.5715-5731.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

MINIREVIEW

Lactic Acid Bacteria as a Potential Source of Enzymes for Use in Vinification

Angela Matthews,1 Antonio Grimaldi,1 Michelle Walker,1 Eveline Bartowsky,2 Paul Grbin,1 and Vladimir Jiranek1*

School of Agriculture and Wine, The University of Adelaide,1 The Australian Wine Research Institute Glen Osmond, Australia2


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INTRODUCTION
 
Two key groups of organisms are involved in the production of red, white, and sparkling wine. The yeasts, typically strains of Saccharomyces cerevisiae, carry out the primary or alcoholic fermentation, in which sugars are converted to ethanol and CO2. Lactic acid bacteria (LAB), especially Oenococcus oeni (formerly Leuconostoc oenos [51]), conduct the secondary or malolactic fermentation (MLF) of wine by decarboxylating L-malic acid to L-lactic acid and CO2 (292). Apart from these two crucial reactions in grape vinification, a myriad of other changes occur to complete the transformation of grape juice to wine. Compounds that stimulate our visual, olfactory, gustatory, and tactile senses are either released from the various ingredients or are synthesized, degraded, or modified during vinification. Many of these processes involve the action of enzymes. Such enzymes can be free or cell associated and originate from sources that include enzyme addition, the grapes themselves, the grape microflora (fungi, yeast, or bacteria), the inoculated microbes, or microbes associated with winery equipment and storage vessels to which the wine is exposed during production.

Current viticultural practices and vinification processes are essentially protocols for favoring the activities of certain enzymes while discouraging the activities of others. Thus, winemakers can broadly achieve desirable outcomes during fermentation by using a selected wine yeast strain characterized by desirable physiological and hence enzymatic properties (149, 225). Conversely, adverse reactions, such as the browning associated with polyphenoloxidases, can be minimized by excluding oxygen from the grape juice or through addition of sulfur dioxide (SO2) to inhibit enzyme activity (25).

A more recent strategy in the history of winemaking is the addition to juice or wine of a microbial culture or enzyme preparation that confers a specific or select group of enzymatic activities. These activities can either amplify the effect of indigenous enzymes or be novel (40, 285). Initially, such additives addressed issues of juice-processing efficiency and wine recovery. Thus, the gelling seen in many fruit juices as a result of pectins has for many decades been reduced or eliminated with pectinase enzymes, most often derived from Aspergillus fungi (283), which increase juice extraction or minimize filter blockage. Enzyme-based solutions that provide a broader range of benefits, such as flavor enhancement or manipulation of color, have now become available.

In the development of new enzyme treatments, efforts have often been centered on desirable activities identified in the microorganisms used or encountered during vinification, especially the yeast (35, 83, 267, 274). In part, this approach has been taken because of legal restrictions on the nature of additives that can be added to wine. It is unlikely that the use in winemaking of wine yeast with a novel enzymatic capability would require regulatory approval, whereas the addition of an enzyme extract or purified enzyme preparation may require such approval. Despite the appeal of this approach, extensive efforts have yielded only a small number of technologically important enzymes, and even fewer of these enzymes perform satisfactorily under winemaking conditions, which include a high sugar (glucose and fructose) content, a low pH (pH 3.0 to 4.0), low temperatures (<15°C), and the presence of ethanol (up to 15% [vol/vol] or more) or SO2.

Interestingly, the LAB that grow and thrive in grape juice or wine under conditions that interfere with the production and activity of desirable enzymes in yeast or fungi have been poorly studied as a source of enzymes with potential usefulness in vinification. Young wine can be a nutritionally deficient environment that could be expected to lead to the elaboration by LAB of numerous enzymatic activities for nutrient scavenging. Emerging findings detailed throughout this review are confirming this notion. In this review we examine the potential of LAB as a source of enzymes that could improve wine quality and complexity. Also discussed are the malolactic enzyme, proteases and peptidases, glycosidases, polysaccharide-degrading enzymes, esterases, ureases, phenoloxidases, and lipases. We emphasize findings from investigations in which wine LAB were used or which were performed under wine-like conditions. Where there are no oenological data, we refer to studies of LAB from other processes, such dairy processes, but the analysis is limited to those species also found in wine (based on a consensus derived from references 25, 62, 95, and 231). The activities of greatest interest are those conferred by a single enzyme, ideally one with an extracellular localization. Such enzymes are most amenable to separation from the cell biomass or preparation as an enzyme-enriched extract, which may be desirable when the originating organism is difficult to grow or is not wanted in grape juice or wine. Some topics that fall outside the scope of this review are covered elsewhere, including in broader reviews of the role of enzymes in winemaking (283) or of the implications of the growth and metabolic activity of LAB in grape juice and wine (158).


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THE MALOLACTIC ENZYME
 
The LAB most commonly associated with wine belong to O. oeni and select Lactobacillus and Pediococcus spp. The major function of LAB is the conversion of L-malic acid to L-lactic acid during the MLF. This conversion may be achieved by one of three pathways (reviewed in references 124 and 284). Most wine-borne LAB decarboxylate L-malic acid to L-lactic acid and carbon dioxide in a reaction catalyzed by the malolactic enzyme without the release of intermediates. One exception to this is observed in Lactobacillus casei and Lactobacillus faecalis, which use a malic enzyme (malate dehydrogenase) to metabolize L-malic acid to pyruvate. L-Lactate dehydrogenase then acts on pyruvate to produce L-lactic acid. A second exception is evident in Lactobacillus fermentum, in which metabolism of L-malic acid yields D-lactic acid, L-lactic acid, acetate, succinate, and carbon dioxide.

Despite the importance of the MLF, its occurrence is both highly unpredictable and difficult to control or manipulate (125). Consequently, techniques that facilitate the efficient and complete conversion of L-malic acid to L-lactic acid in grape juice and wine have been sought. Such techniques aim to separate this central enzyme-driven conversion from the often problematic growth of the source LAB in the wine. Examples from the beverage and food industries include bioreactor systems comprising LAB cells immobilized alone (44, 45, 54, 182, 200, 256, 262), LAB cells coimmobilized with yeast (201), or free O. oeni cells (99) or enzymes and cofactors (91, 182, 282). The ability of the malolactic enzyme, as a single enzyme, to conduct the conversion of L-malic acid to L-lactic acid has made it the activity of choice for such bioreactor systems, as well as heterologous expression studies. A bioreactor containing NAD, manganese ions, and the malolactic enzyme from L. oenos strain 84.06 achieved a 62 to 75% conversion rate for L-malic acid to L-lactic acid in wine (91). Incomplete conversion was attributed to enzyme inactivation and instability of the cofactor NAD at the wine pH (40, 102). The expression of the malolactic enzyme encoded by the mleS gene from Lactococcus lactis in an S. cerevisiae wine yeast enabled it to effect the MLF and alcoholic fermentation simultaneously (289). Whether achieved via such recombinant methods or via bioconversions with cells or enzyme preparations, the potential benefits of enhanced application of malolactic enzyme warrant further research. Identification of a malolactic enzyme that is more resilient under wine conditions and improved delivery systems is of foremost interest.


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PROTEOLYTIC AND PEPTIDOLYTIC ENZYMES
 
Grape juice nitrogen compounds include compounds that are variously essential or detrimental to successful fermentation and wine quality. The bulk of the nitrogenous fraction is comprised of the alpha amino acids and ammonium (25, 126), which along with peptides containing up to five amino acid residues (16, 199) represent the assimilable nitrogen that is vital for yeast growth and fermentative activity (30, 139, 191, 238) and suppression of hydrogen sulfide (100, 112, 138, 263). Conversely, the proteins of grapes are considered a nuisance as they become unstable in the finished wine and can precipitate to produce a haze (15, 133, 290).

Bentonite fining remains the most common and effective method for removal of haze-forming proteins from wine despite the unwanted effects of removing some assimilable nitrogen, modifying the flavor, and changing the kinetics of fermentation (9, 32, 121, 203, 211, 265). Proteases have been sought from a variety of sources, and they have been evaluated as an alternative to bentonite treatment to remove unwanted proteins while possibly also liberating assimilable nitrogen for exploitation by yeast (9, 188, 297). Commercial proteolytic preparations, such as trypsin and pepsin, do not function optimally at the low temperatures and pH used during winemaking (40). Proteases from Aspergillus niger have similarly been unsuccessful under winemaking conditions (9, 121). Modra et al. (188) studied five commercial peptidase preparations in wine, but none was found to significantly reduce the bentonite concentration required to achieve heat stability. Researchers have investigated wine and beer yeasts as alternate sources of such enzymes, reasoning that these organisms would be more suited to the conditions of the corresponding fermentations, but generally the results have been disappointing (21, 55, 148, 202, 236, 267). Alternate enzymes or alternate sources are clearly called for, and thus the proteolytic and peptidolytic activities of LAB are receiving greater attention.

LAB are fastidious in their amino acid requirements (95, 101), and there is clear evidence that some LAB produce the activities needed to procure peptides and amino acids to meet these requirements (81, 190). Comprehensive reviews covering proteolytic and peptidolytic activities across all genera of LAB are available elsewhere (38, 111, 154, 155). Most work in this field has been performed by workers in the dairy industry, in which these enzymes are directly involved in flavor and texture development (65, 287, 294) and are indirectly involved in the maximization of microbial cell growth by provision of essential amino acids (111). Here, only findings from studies of wine-related species of LAB are summarized. From Table 1 it is evident that activities designated proteinases and several types of peptidases are widely distributed across the three genera and 15 wine-related species included. The potential importance of these activities for winemaking is in part linked to the nature of the enzyme, its cellular location, and how it is applied to the wine.


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  		TABLE 1. Proteolytic and peptidolytic activities of LABa

Activities that are lost to the culture supernatant or are associated with whole cells have been reported for most species (Table 1). As a result, such activities could be evident in intact cells of a LAB when it is grown in grape juice or wine. Organisms whose growth mirrors that of O. oeni and is most apparent after or toward the end of the primary fermentation are unlikely to have an impact on yeast growth. At this time yeast needs minimal assimilable nitrogen; therefore, any proteolytic or peptidolytic activity of LAB is beneficial mainly for haze reduction. Conversely, the sensitivity of Lactobacillus and Pediococcus to ethanol (95, 284, 292) relegates their growth in mixed cultures with yeast to the early stages of the primary fermentation. Degradation of proteins and peptides at this early stage might not only affect protein haze formation in the finished wine but also release assimilable nitrogen to benefit yeast growth.

The application of a cell-free enzyme extract is one way to dissociate a desired enzymatic activity from the need to grow a particular LAB in grape juice or wine. This approach also introduces the possibility of exploiting the considerable cohort of intracellular enzymes identified to date, but it might be necessary to consider the stability of these enzymes under wine conditions. In considering the importance of individual enzyme types, proline-specific peptidases might be less important in providing assimilable nitrogen since the liberation of proline has little nutritional value to yeast cells because of their inability to exploit this amino acid under oenological conditions (135, 239).

In the absence of extensive studies of wine LAB, the nature and frequency of proteolytic and peptidolytic activities identified by dairy researchers strongly suggest that similar activities also exist in wine LAB. What data have been reported for wine show promise. It is recognized that the levels of individual peptides and amino acids can increase or decrease during LAB growth in wine, and the only general point of agreement is that the arginine concentration decreases while the ornithine concentration increases during MLF (124, 292). More detailed information comes from a series of studies conducted by Manca de Nadra and coworkers. These workers described two enzymes, proteases I and II, which are produced by several strains of O. oeni during the early and final phases of growth, respectively (233). Protease I displayed optimal activity at pH 4.0 and 30°C, while the optimal protease II activity occurred at pH 5.5 and 40°C (232). Both proteases were apparently repressed by ammonium, tryptone, and casein hydrolysate, were induced by nutrient starvation, and were able to liberate detectable concentrations of amino acids from protein and polypeptide extracts from red and white wines (81, 174, 175, 234). When applied to sterile grape juice, a concentrated, purified exoprotease is thought to degrade proteins at a high rate (80). As encouraging as these findings are, there are some questions that remain to be answered. For example, it is not known whether the observed degradation of grape proteins releases peptides and amino acids in amounts that provide a nutritional benefit to the yeast or bacteria involved in the winemaking process and whether these activities are able to reduce the potential for haze formation in wine in which protein is unstable.


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GLYCOSIDASES
 
The sensory properties of wine are the result of a multitude of individual compounds. Four groups of these compounds, the monoterpenes, C13-norisoprenoids, benzene derivatives, and aliphatic compounds, all can occur linked to sugars to form glycosides (250, 296). Monoterpenes and some benzene derivatives and C13-norisoprenoids play an important role in determining wine aroma, particularly for varieties such as Muscat, Gewürztraminer, and Riesling. Aliphatic compounds, which include the aliphatic alcohols, carboxylic acids, lactones, and ethyl esters, are more related to the flavor of a wine. The remaining benzene derivatives include the anthocyanins, which contribute to wine color. Importantly, the characteristics of the glycosides differ from those of the corresponding aglycones. Generally, the glycosides are water soluble and less reactive and volatile than the aglycones, possibly explaining why plants store a great number of compounds in the glycosidic form (130). In wine, volatile, aromatic compounds that are otherwise detectable by human senses are nonvolatile and undetectable when they are in the glycosidic form. Accordingly, because as much as ~95% or more of such aromatic compounds is present in the glycosidic form, most of the aromatic potential of these compounds is not realized (116). Conversely, monoglucoside anthocyanins represent the principal form in which the anthocyanins that contribute to color in red wines are found (230). When these color compounds are deglycosylated, the corresponding anthocyanidin is less stable and is readily converted to a brown or colorless compound (23, 134). While this outcome may be undesirable in a red wine, these enzymes have been proposed as a means to reduce the color intensity in white or rose wines produced from red grapes (241).

The glycosidase enzymes that cleave the sugar moiety from glycosides can therefore have a major impact on the sensory profile of a wine. The occurrence of many types of such enzymes is a reflection of the complexity of their glycoside substrates, which can contain either mono- or disaccharides. The terminal sugar can be either ß-D-glucopyranoside, {alpha}-L-rhamnopyranoside, {alpha}-L-arabinofuranoside, ß-D-apiofuranoside, or ß-D-xylopyranoside, and the additional central sugar in disaccharides is always ß-D-glucopyranoside (296). Removal of these sugars requires a glycosidase specific for the terminal sugar, followed by, in the case of a disaccharide, a ß-D-glucopyranosidase (116). The latter enzyme is essential for liberation of aglycones from all diglycosides and ß-D-glucopyranosides; hence, research efforts are concentrated on this enzyme to the almost complete exclusion of other enzymes.

With the aim of increasing the aromaticity of wines, glycosidases have been widely studied in several organisms, including both wine-related and non-wine-related organisms. Grapevines produce glycosidases, although these enzymes have little activity against wine glycosides (6). Given its importance in winemaking, much attention has been paid to S. cerevisiae, but this yeast shows very limited production of glycosidases, much of which is intracellular (49, 179). Studies of other wine yeasts, including the apiculates and the spoilage yeasts, have yielded wide-ranging levels of activities, primarily ß-D-glucosidase (ß-D-glucopyranosidase) activities (35, 176, 237). Sanchez-Torres and coworkers (241) have also heterologously expressed a ß-D-glucosidase from Candida molischiana in an S. cerevisiae wine strain and have demonstrated readily observable anthocyanase (decolorizing) activity in microvinification experiments. Several grapevine fungal pathogens, such as Aspergillus and Botrytis, produce large amounts of glycosidase activities that also have high levels of specificity for purified wine glycosides (177). Accordingly, Aspergillus is a common source of commercial enzyme preparations that have glycosidic activities; however, these preparations are often impure, requiring resolution before characterization in the laboratory (258, 259, 261), and they have undesirable effects on the wine (1, 115, 296). More importantly, the enzymes of fungi are frequently ineffective in wine (6, 43, 114). The same is true for many of the glycosidic activities from the various source organisms examined to date, which can be limited by sensitivity to one or more of the following key wine parameters: low pH (pH 3.0 to 4.0), ethanol content (9 to 16%, vol/vol), or residual sugar content (<10 g/liter) (reviewed in reference 296). Interestingly, the LAB, which can thrive under these conditions, have received little attention as a potential source of glycosidic enzymes.

While the glycosidases of some LAB have been studied, wine isolates have only recently been included. Limited data have been reported for O. oeni, and no data are available for wine Lactobacillus and Pediococcus spp. McMahon and coworkers (183) observed no enzymatic activity by O. oeni against arbutin, an artificial glycosidic substrate. In another study (24), changes in the glycoside content of Tannat wines during MLF indirectly supported the existence of such activities in the commercial O. oeni strains used. More specific data have come from examinations of commercial wine O. oeni isolates (11, 110, 176), which were shown to have the potential for high glycosidase activity against nitrophenyl glycosides. ß-D-Glucosidase was the predominant activity, and some ß-D-xylopyranosidase and {alpha}-L-arabinopyranosidase activities were also detected (110). Notably, these activities were only partially inhibited under wine-like conditions. At pH 3.5 and in the presence of glucose (20 g/liter) and ethanol (12%, vol/vol), one isolate retained ~50% of the activity seen under optimized conditions (110). Mansfield and coworkers (176) could not detect activity against glycosides extracted from the Viognier variety; however, more recent work demonstrated that some O. oeni strains are able to act on glycosides extracted from the highly aromatic Muscat variety (275) or the nonaromatic Chardonnay variety (53). In agreement with results obtained with synthetic substrates, the pattern of hydrolysis of selected glycosides from the Chardonnay variety showed that strain EQ 54 had little activity other than a ß-D-glucosidase activity, and greater hydrolysis of the mixture occurred only after addition of commercial {alpha}-L-rhamnopyranosidase and {alpha}-L-arabinofuranosidase preparations (53).

While the use of enzymes and/or selected cultures to liberate aroma compounds from natural grape aroma glycosides is still in the early stages of development, the findings to date for LAB and synthetic or natural glycosides are very encouraging and justify further investigation. LAB appear to possess the full array of glycosidases needed to hydrolyze many of the glycosides found in grapes and wine, although some enzymes have limited activity. Determining the precise sensory significance of glycosidic activities, as well as the longevity of any positive effects, is an important objective of future studies.


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POLYSACCHARIDE-DEGRADING ENZYMES
 
The polysaccharides of higher plant cell walls and middle lamellae that affect wine production include cellulose (primarily ß-glucans), hemicellulose (primarily xylans), and pectic substances (291). Such compounds are present in grape juice as a result of berry disruption or release through the action of degradative enzymes from the grapes. In fruit infected with the mold Botrytis cinerea, ß-glucans are excreted by this pathogen directly into the berry (32, 59), and fungal enzymes release grape polysaccharides, particularly type II arabinogalactan and rhamnogalacturonan II (92). While fungal enzymes appeared not to enhance the release of polysaccharides (mannoproteins) from yeast (92), these compounds can be released during yeast cell growth, through exposure to shear (e.g., during pumping and centrifugation) (157, 251), and particularly upon autolysis (56, 90).

Collectively, polysaccharides reduce juice extraction and are primarily responsible for fouling of filters during clarification steps. Wine quality also can be affected through changes in clarity (60), while an effect on viscosity may influence mouth feel (240) and the perception of tastes and aromas (61, 119, 120, 151, 173). Enzymes capable of degrading polysaccharides therefore have the potential to improve juice yields (18, 118, 208, 210) and wine processability through the removal of problem colloids, to increase wine quality via breakdown of grape cell walls to yield better extraction of color and aroma precursors (10, 98, 215, 229, 293), and to alter the perception of wine components (61, 151). These complex macromolecules are hydrolyzed by a number of distinct enzymes, including pectinases (protopectinase, pectin methylesterase, polygalacturonase, and pectin and pectate lyase activities), cellulases (endoglucanase, exoglucanase, and cellobiase activities), and hemicellulases (ß-D-galactanase, ß-D-mannase, and ß-D-xylanase activities) (reviewed in reference 283). There have been few reports of attempts to specifically identify polysaccharide-degrading enzymes in LAB, despite the importance of these enzymes to winemaking.

The pectinolytic activities of LAB have largely been addressed in studies of fermentation processes other than winemaking, in which their significance remains unclear. For example, early work on silage microflora suggested that cellulases and hemicellulases are produced (195), whereas more recent work indicated that combinations of Lactobacillus plantarum and Pediococcus cerevisiae (Pediococcus damnosus) had negligible ability to degrade plant cell walls (147). Pectin methylesterase and polygalacturonate lyase activities have been detected in the spontaneous fermentation of cassava roots, but the study neither confirmed nor discounted the involvement of the LAB present in the fermentation (28). At the very least, L. plantarum is able to liberate reducing sugars from polymeric carbohydrates during corn straw ensiling (299).

An extracellular glucanase that is produced early in the stationary phase of cell growth has been demonstrated in O. oeni (113). This enzyme was determined to be ß-1,3 in nature and to be capable of hydrolyzing yeast cell wall macromolecules; thus, it was proposed that the enzyme plays a role in yeast cell autolysis following alcoholic fermentation. Further work is required to confirm the significance of this activity along with its efficacy at temperatures below 10°C, at which currently available glucanases are insufficiently active (32, 286). Similarly, the absence of additional polysaccharide-degrading enzymes cannot be assumed until a comprehensive and specific search for such activities, such as the search conducted for wine yeasts and fungi, has been completed for LAB.


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ESTERASES
 
Esters are a large group of volatile compounds that are usually present in wine at concentrations above the sensory threshold. Most wine esters are produced by yeast as secondary products of sugar metabolism during alcoholic fermentation (149, 300). Esters can also be derived from the grape (226) and from the chemical esterification of alcohols and acids during wine aging (75). The importance of esters in winemaking lies in their prominent role in determining the aroma and, by extension, the quality of wine. Esters are responsible for the desirable, fruity aroma of young wines (149, 178), although they can also have a detrimental effect on wine aroma when they are present at excessive concentrations (264).

Quantitatively, the most important wine esters are mainly yeast derived and include (i) ethyl esters of organic acids, (ii) ethyl esters of fatty acids, and (iii) acetate esters (75). Ethyl acetate is usually the predominant ester in wine, and with a low sensory threshold, it is often an important contributor to wine aroma (75). At low concentrations, ethyl acetate aroma is desirable and described as fruity, but at higher concentrations it imparts an undesirable nail polish remover character to wine (13). Other important wine esters and their aromas include isoamyl acetate (banana), ethyl hexanoate (fruity, violets), ethyl octanoate (pineapple, pear), and ethyl decanoate (floral) (149, 178). Esterolytic activity during wine production could result in either an increase or a decrease in wine quality, depending on the ester involved (47). In addition, the compounds liberated by the esterases (for example fatty acids and higher alcohols) could contribute to wine aroma (75, 149).

While the esterases of yeast have been extensively researched (for example, see references 180, 218, and 235), there has been little work focusing on the esterases of wine LAB. Our current knowledge of LAB esterases is based primarily on work carried out in the dairy industry, in which such enzymes contribute to the characteristic flavors and defects of cheeses (132). Most of this work has focused on the metabolism of esters by LAB, and it is now suspected that these enzymes have the ability to both synthesize and hydrolyze esters (158). Thus, dairy LAB synthesize esters, including ethyl butanoate and ethyl hexanoate, while ester hydrolysis is also supported by abundant experimental evidence (82, 131, 159). A summary of the literature describing hydrolytic esterases from dairy Lactobacillus and Pediococcus species is presented in Table 2. Similar results have been obtained for LAB genera not endogenous to wine, including Streptococcus (160), Leuconostoc (141, 280), Lactococcus (41), and Enterococcus (141). The appearance of esterase activity in association with whole cells or in culture supernatants of some LAB shown in Table 2 implies that growth in grape juice or wine of these species may modify the ester profile of the beverage. Where intracellular esterase activities are reported, cell disruption would presumably be required in order for these activities to have an impact on wine.


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  		TABLE 2. Esterases of LABa

In a screening of the enzymatic activities of wine LAB, Davis et al. (47) found 23 strains that were able to hydrolyze an ester substrate, but no steps were taken to characterize the enzymes further or to determine their ability to synthesize esters. In some wine flavor studies workers have reported changes in the concentration of individual esters during MLF. For example, increases in ethyl acetate (48, 172), isoamyl acetate (152, 172), and ethyl lactate (48, 97) levels have been observed, while Zeeman et al. (301) reported a decrease in the levels of some esters following MLF. These results suggest that like the esterases of dairy isolates, esterases of wine LAB are involved in both the synthesis and hydrolysis of esters. No further investigation has been reported. Therefore, further research into the esterase systems of wine LAB should help determine the precise nature of these enzymes and their effects on the sensory properties of wine.


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UREASES
 
Ethyl carbamate is a known carcinogen and is formed in wine via the spontaneous acid ethanolysis of certain carbamyl precursors, including urea and citrulline (145, 192, 207, 209, 266). Due to the health risks associated with elevated levels of ethyl carbamate in wine, the sources of the precursor compounds have been studied extensively. The pathways by which LAB can contribute to the ethyl carbamate precursor pool in wine have also been investigated (161).

Ureases produced by microorganisms are substrate-specific enzymes that catalyze the hydrolysis of urea (249). Urea-degrading enzymes are a potential tool for reducing the concentration of urea in wine in order to avoid dangerous and illegal concentrations of ethyl carbamate. Most commercial urease products are derived from nonwine sources (e.g., beans) and are unsuitable for use in wine, which has a pH that typically is well below the neutral pH optima (144). Ureases derived from LAB have been investigated as alternate enzymes for the removal of excess urea in wine. The acid ureases produced by L. fermentum (144, 212, 273) and Lactobacillus reuteri (140) were very effective over the pH range from 2.0 to 4.0, which included typical wine pH values. Wine components, such as phenolic compounds (e.g., grape seed tannins), sulfur dioxide, ethanol, and organic acids (e.g., malic acid, lactic acid, and pyruvic acid), did, however, inhibit the activity of the urease from L. fermentum (79, 273). Whether such inhibition limits the usefulness of ureases, at least in wine of a certain type or composition, remains to be determined through further study.


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PHENOLOXIDASES
 
Laccases (p-benzendiol:oxygen oxidoreductase) and tyrosinases (monophenol monooxygenase) are two groups of phenoloxidases which are widely distributed in nature. They have been found in bacteria (39), filamentous fungi (181, 196), insects (7, 253), and higher plants (109, 181). Both groups of enzymes catalyze the transformation of a large number of (poly)phenolic and nonphenolic aromatic compounds and thus have potential uses in bioremediation processes in the paper and pulp, tanning (63, 196), and food industries (olive mill and brewery wastewater) (187). One of the main applications of laccases in the food industry is product stabilization in fruit juice, beer, and wine processing (187).

A myriad of phenolic compounds are found in musts and wine; these compounds range from simple hydroxybenzoic acid and cinnamic acid derivatives to more complex molecules, such as catechins, anthocyanins, flavonols, flavanones, and tannins (178). Such compounds are responsible for the desirable attributes of color, astringency, flavor, and aroma of wine, as well as unwanted attributes, including browning, flavor and aroma alterations, and some forms of haze, which are a consequence of enzymatic and chemical oxidoreduction in white musts and wines. In vinification two main approaches have been taken to combat oxidative decolorization and flavor alteration (madeirization). One of these approaches is inhibition of enzymes in the must by using sulfur dioxide as a reductant and inhibitor; alternatively, the polyphenol substrate content of white wine is reduced by limiting maceration of the must. Where introduction has not been avoided or reduced, polyphenols are removed from the must or wine with fining agents, such as polyvinylpolypyrrolidone, gelatin, casein, and egg albumin, in addition to bentonite (187). More recently, chitosan, a polymeric adjuvant, was used as a fining agent and was found to be comparable to potassium caseinate in terms of wine stabilization (260).

Treatment of the must with enzymes such as laccases, tyrosinases, tannases, and peroxidases has been considered an alternative to treatment with physical-chemical adsorbents (302). Laccases are thought to be the most promising enzymes, because they have broader specificity for phenolic compounds, are more stable at the pH of must and wine, and are less affected by sulfur dioxide (32). With these enzymes, wine stabilization is achieved through prefermentative treatment to bring about oxidation of polyphenol substrates normally involved in the madeirization process. The oxidized products polymerize and precipitate, and they are subsequently removed by conventional clarifiers and filtration. To date, laccases from lignin-degrading fungi, such as Trametes versicolor, Coriolus versicolor, and Agaricus bisporus, and tyrosinases from mushrooms have been used in wine processing with promising results (32). When the enzymes were applied either singularly or in combination and immobilized on supports ranging from metal chelate affinity matrices to molecular sieves, good results were observed in terms of polyphenol removal, retention of enzyme activity, and reuse of the carrier support (see reviews in references 63 and 187).

The occurrence of (poly)phenoloxidases in LAB, particularly those commonly associated with winemaking, is also of interest. The work of Benz and coworkers (17) and Lamia and Moktar (150) suggests that these enzymes are present in fermentative LAB. Specifically, L. lactis is able to reduce humic acids (17), a constituent of soil humus containing substituted phenols and polyphenols (137), which implies that a polyphenoloxidase is involved. Lamia and Moktar (150) demonstrated that L. plantarum grown on diluted olive mill wastewater induced the depolymerization of polyphenols (presumably by the action of tannases) and inhibited the polymerization of simple phenolic compounds which occurs at lower postfermentation pH values.

Studies of wine LAB, particularly O. oeni, have revealed that the growth and rate of MLF of these organisms are influenced by phenolic compounds. Gallic acid and anthocyanins, metabolized by growing cells, have a positive effect on growth and malolactic activity, whereas tannins are inhibitory (288). Other workers have observed that growth of O. oeni is inhibited by phenolic acids, including p-coumaric, caffeic, ferulic, p-hydroxybenzoic, protocatechuic, gallic, vanillic, and syringic acids, while growth of Lactobacillus hilgardii was stimulated (31). These findings are indicative of the evolution of polyphenoloxidases (and possibly tannases) from some of these organisms. If such activities do exist, their contribution to browning or decolorization is yet to be determined, but it might be expected to be most relevant to species that grow in grape juice early in fermentation. This could include LAB which are inoculated early, such as Lactobacillus, or contaminating LAB. Spectrophotometric assays analogous to those described for plant polyphenoloxidases, in which substrates such as catechol, catechin, and p-courmaric acid are used (109), should facilitate extended surveys of wine LAB for such activities.


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LIPASES
 
Wine lipids can be derived from the grape berry (96, 186) or can be released from yeast during autolysis (224). Grape lipids can originate from a number of sources within the berry, including the skin, seeds, and berry pulp. The lipid profiles of each of these sources have been shown to be different, due to variation in both the concentration and the fatty acid composition of neutral lipids, glycolipids, and phospholipids (186). The grape lipid profile also varies with grape maturation (14), climate (136), and variety; red varieties tend to have greater total lipid concentrations than white varieties (96). Yeast autolysis following fermentation releases many different types of lipids, including tri-, di-, and monoacylglycerols and sterols, in amounts and proportions which vary with the yeast strain (224). Such lipids are known to influence not only the sensory profile of sparkling wine but also foam characteristics (224).

The action of lipases on wine lipids could yield a range of volatile compounds, including fatty acids. The low aroma thresholds of fatty acids allows them to contribute to wine aroma, but since their odors are described as vinegar, cheesy, and sweaty, their impact might not be desirable (75). A more positive contribution to the aroma profile of wine can develop when volatile compounds such as esters, ketones, and aldehydes are derived from these fatty acids (36).

The lipolytic activity of wine LAB has not been thoroughly investigated, but preliminary work with nonwine substrates suggests that some LAB may produce lipases. In a study of LAB isolated from wines, Davis et al. (47) observed lipase activity in several strains of L. oenos (O. oeni) and one species of Lactobacillus. By contrast, a more recent study failed to find lipolytic activity in wine isolates comprising 32 Lactobacillus strains, two Leuconostoc strains, and three Lactococcus strains (127). The lipolytic activity of LAB has been more extensively researched in other areas of food production. In dairy foods, lipases can contribute to flavor and processability (279). On the basis of this work, LAB are now generally acknowledged to be weakly lipolytic (93, 142), and their lipases display substrate specificity which is both strain and species dependent (141). By utilizing numerous substrates and various degrees of cell fractionation, several such studies have provided information about activities in genera that are of interest in winemaking, namely, Lactobacillus and Pediococcus (Table 3). A broader review of the lipolytic activity of all dairy LAB was produced by Collins et al. (42). Because lipases are located extracellularly or are associated with the whole cells, LAB (Table 3) have the potential to influence the wine lipid content when they are grown in grape juice or wine. The ability of any of these enzymes to attack membranes of yeast and grape cells and to influence wine aroma remains to be determined.


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  		TABLE 3. Lipase activities of species of LAB that can be found in association with grape juice or winea


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CONCLUSIONS
 
A considerable amount of research has been conducted to determine the enzymatic properties of LAB specifically isolated from wine or, more commonly, the enzymatic properties of analogous species isolated from other foods or beverages. From this work it is clear that these organisms possess an extensive collection of enzymatic activities, many of which have the potential to influence wine composition and therefore the processing, organoleptic properties, and quality of wine. In many cases the precise nature and extent of this influence has yet to be delineated for the LAB that are associated with winemaking and grow under the conditions encountered during grape juice fermentation and wine maturation. Ideally, the potential for these organisms that has been highlighted in this review will stimulate fuller characterization of wine LAB so that at the very least these strains will be able to be applied by the winemaker in a more informed manner. In other cases, these organisms may serve as a source for the preparation of enzyme extracts that are better able to function under the harsh and changing environmental conditions of wine fermentation.


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ACKNOWLEDGMENTS
 
We thank Sakkie Pretorius for his critical reading of the manuscript.

Preparation of this review was undertaken as part of project UA 01/04 supported by Australia's grapegrowers and winemakers through their investment body, the Grape and Wine Research Development Corporation, with matching funds from the Australian Government. Additional support was kindly provided by Lallemand Australia. A.G. is partially supported by a Walter, Carew and Richard Reynella Fellowship.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Agriculture and Wine, The University of Adelaide, PMB1, Glen Osmond, SA 5064, Australia. Phone: 61 8 8303 6651. Fax: 61 8 8303 7415. E-mail: vladimir.jiranek{at}adelaide.edu.au. Back


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