Screening for the production of extracellular hydrolytic enzymes by non-Saccharomyces wine yeasts
van Rensburg Institute for Wine Biotechnology and Department of Viticulture and Oenology, University of Stellenbosch, Private Bag X1, 7602 Matieland (Stellenbosch), South Africa.
Aims: The objective of this study was to investigate what types of enzymes are being produced by non-Saccharomyces yeasts isolated from grapes in South Africa vineyards and clarified grape juice. These enzyme profiles could pave the way for attributing specific effects in wine to some of these enzymes produced by so-called wild yeasts associated with grape must.
Methods and Results: In this study 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases β-glucanases, lichenases, β-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clarified grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except β-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species.
Conclusion: This study clearly revealed the potential of non-Saccharomyces wine yeasts to produce a wide range of useful extracellular enzymes during the initial phase of wine fermentation.
Significance and Impact of the Study: Enzymes produced by indigenous yeasts associated with grapes and juice might be harnessed to catalyse desired biotransformations during wine fermentation.
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The microflora of grapes is highly variable, with a predominance of the low ethanol tolerant strains of Hanseniaspora, Kloeckera and various species of Candida. The more ethanol-tolerant Saccharomyces cerevisiae are present only at low numbers (Peynaud and Domercq 1959; Fleet et al. 1984; Heard and Fleet 1985; Lema et al. 1996). The influence that all of these yeasts will have on the flavour of wine depends on several factors, such as the method of grape harvest, transport, vinification factors such as temperature, extent of juice clarification, use of sulphur dioxide, etc. Yeast cell concentration in freshly prepared must is typically 103–105 cfu ml–1, but after processing may vary from near sterility to > 106 yeast cfu ml–1. This highly variable content of yeast populations may contribute to chemical and flavour changes that accompany fermentation. The early stages of most wine fermentations, whether they develop naturally or after inoculation with S. cerevisiae, are characterized by the significant growth of indigenous species of non-Saccharomyces yeast (Fleet 1992). Prominent among these are species of Kloeckera, Cryptococcus, Hanseniaspora, Candida, Pichia and Hansenula (Fleet et al. 1984). While the controlled growth of these indigenous species may in some circumstances enhance the chemical complexity and sensory quality of wines, there are other circumstances in which their contribution may result in spoilage (Fleet 1992). Several authors have shown that using different starter cultures and indigenous yeast produces wines with significant differences in chemical composition (for a review see Lambrechts and Pretorius 2000). Recently, three studies illustrated the impact that the growth of non-Saccharomyces yeast can have on the sensory character of wines (Egli et al. 1998; Henick-Kling et al. 1998; Soden et al. 2000). Some of these differences might be ascribed to several researchers showing that in contrast to Saccharomyces species, the non-Saccharomyces yeasts produce and secrete several enzymes (esterases, glycosidases, lipases, β-glucosidases, proteases, cellulases, etc.) to the periplasmic space and the medium, where they may interact with grape precursor compounds to produce aroma active compounds, and thus play an important role in varietal aroma (Charoenchai et al. 1997).
Enzymes play a definitive role in the production of wine, which could be seen as the product of enzymatic transformation of the grape juice (for a review see Van Rensburg and Pretorius 2000). The enzyme activities do not only originate from the grape itself, but also from yeast and other microorganisms. The winemaker now reinforces and extends the action of these endogenous enzymes by the use of exogenous, industrial enzyme preparations (Fleet 1993). The production of extracellular hydrolytic enzymes by indigenous yeast could be significant and needs to be understood and managed to the benefit of wine production. Moreover, wine yeast could be potential sources for the commercial production of enzymes to be used in the process of winemaking (Charoenchai et al. 1997). Saccharomyces cerevisiae, the principal wine yeast, is not recognized as a significant producer of extracellular enzymes, although a few strains have recently been reported to degrade polygalacturonate (McKay 1990). There is little information on the production of extracellular enzymes by non-Saccharomyces wine yeast, although some strains of Kloeckera apiculata show extracellular protease activity (Lagace and Bisson 1990; Dizy and Bisson 2000). Various authors have reported glycosidase production by S. cerevisiae and the potential for these enzymes to enhance wine flavour (Delcroix et al. 1994). Glycosidase activity has been reported in strains of Candida, Pichia and Hanseniaspora (Vasserot et al. 1989). A number of sources have been tested for glycosidic activity that may be used under the limiting conditions wine offer. These sources include plants, fungi, bacteria and yeasts. As these glycosidases are to be exploited to release potential aroma, they must satisfy a few prerequisites. These consist of: specificity, pH optimum, and glucose as well as ethanol tolerance.
Due to the importance of yeast biodiversity to the wine industry, a comprehensive long-term research programme has been launched by several researchers from the Wine and Fermentation Technology Division at the ARC Infruitec-Nietvoorbij Research Institute and the Institute for Wine Biotechnology at the University of Stellenbosch (for a review see Pretorius et al. 1999). As part of this programme, the natural distribution of non-Saccharomyces strains in the vineyards of the Western Cape in South Africa was determined. To further characterize the isolated yeast strains, the aims of this paper were to determine the ability of these yeasts to produce extracellular pectinases, proteases, β-glucanases, β-glucosidases, cellulases, hemicellulases, cellobiases as well as enzymes involved in the degradation of starch. The ability of the indigenous wine yeast to produce H2S was also investigated.
MATERIALS AND METHODS
The 245 wine yeast strains used in this study (Table 1) were obtained from the Wine and Fermentation Technology Division, ARC Infruitech-Nietvoorbij. The yeast strains are indigenous strains isolated from four wine production regions of the Western Cape, South Africa, i.e. Constantia (isolates C), Robertson (isolates M), Slanghoek (isolates O) and Stellenbosch (isolates R). The yeast strains are further subdivided into those isolated from vineyards (group 1) and from clarified grape juice prior to fermentation (group 2).
Yeast isolates used in this study
Media and screening procedures
All yeast isolates were grown on YPD plates (containing 1% yeast extract, 2% peptone, 2% glucose and 2% agar) and then replica plated to the mediums described below to test for extracellular enzyme activity.
Yeasts were screened for polygalacturonase production by the method described by McKay (1988) with some modifications. Yeasts were replica plated onto polygalacturonate agar medium containing 1·25% polygalacturonic acid (Sigma), 0·68% potassium phosphate (pH 3·5), 0·67% yeast nitrogen base (YNB, Difco), 1% glucose and 2% agar. The plates were incubated for 5 d at 30°C. The colonies were rinsed off the plates with distilled water before staining the plates with 0·1% Ruthenium Red. Colonies showing a purple halo were identified as positive.
Extracellular protease production was determined by replica plating yeast colonies onto YPD plates containing 2% casein (BDH laboratories). The plates were incubated for 5 d at 30°C. A clear zone around the colony identified protease activity.
Production of β-glucanase activity was determined by replica plating the yeast onto YPD plates containing 0·2% barley β-glucan (Sigma) and YPD plates containing 0·2% lichenan (Sigma). The plates were incubated for 5 d at 30°C. Colonies were rinsed off the plates with distilled water before staining the plates with 0·03% Congo Red (Teather and Wood 1982). A clear zone around the colony identified glucanase activity.
β-Glucosidase activity was determined by replica plating the yeast onto selective medium (SC) containing 0·67% yeast nitrogen base (YNB, Difco), 0·5% arbutin (ICN) and 2% agar. The pH of the media was adjusted to 5 before autoclaving. Two millilitres of a filter-sterilized 1% ammonium ferric citrate solution was added to 100 ml media before pouring the plates. The plates were incubated for 5 d at 30°C. Colonies showing activity were identified by the discoloration of the media to a brown colour.
Cellulase production was determined by replica plating the yeast onto YPD plates containing 0·4% carboxymethylcellulose (CMC, Sigma) and YPGE plates (containing 1% yeast extract, 2% peptone, 3% glycerol and 2% ethanol) containing 0·4% CMC. The plates were incubated for 5 d at 30°C. The colonies were rinsed off the plates with distilled water before staining the plates with 0·03% Congo Red, followed by destaining with 1 M NaCl (Teather and Wood 1982).
Yeasts were screened for hemicellulase activity by replica plating onto SC plates containing 0·2% Remazol Brilliant Blue Xylan (RBB-Xylan, Sigma) and YPGE plates containing 0·2% RBB-xylan. The plates were incubated for 5 d at 30°C. Colonies showing activity were identified by a clear zone around the colony. Production of cellobiase activity was assessed on SC plates containing 2% cellobiose as the only carbon source. Growth on the plates indicated cellobiase activity.
Starch degrading activity.
Amylolytic activity was determined on SC plates containing 2% starch (Saarchem). After the plates were incubated for 6 d at 30°C they were placed at 4°C for precipitation. A clear zone around the colony identified yeast containing amylolytic activity. Production of α-amylase was assessed by plating the yeast onto SC containing 40 phadebas (Pharmacia and Upjohn) pills per litre. The plates were incubated for 6 d at 30°C and yeast showing activity were identified by a clear zone around the colony.
The H2S-production potential of the yeasts were determined by plating the yeasts onto a solid grape juice indicator agar (GJIA) (Jiranek et al. 1995) with some modifications. The media contained per litre: 250 ml grape juice, 15 ml succinate (pH 5·1), 735 ml distilled water, 11 g bismuth citrate (Sigma) and 3% agar. The plates were incubated for 6 d at 30°C. A low H2S-producing colony was identified by its white colour whereas a high H2S-producing colony had a black colour.
Yeasts were grown to an O.D. of 1 in YPD broth and the dry biomass determined.
The amylolytic activity was determined by growing the yeast overnight in 5 ml YPD broth at 30°C. The cultures were centrifuged (6 K for 2 min) and the supernatant used for the assay. Samples of culture supernatants (600 μl) were mixed with 200 μl of 0·5 mol l–1 sodium-acetate buffer (pH 3·2) and 200 μl of the substrate (0·5% starch solution; Sigma) and incubated for 60 min at 30°C. The reaction was stopped by boiling the mixture for 2 min. After cooling on ice, the liberated glucose was determined with a Trinder assay kit as recommended by the supplier (Sigma). The units were defined as mmol l–1 g–1 dry cells.
Xylanase activity was determined by growing the yeast overnight in 5 ml YPD broth at 30°C. Samples of culture supernatant (100 μl) were mixed with 900 μl of the substrate [1% xylan (Sigma) dissolved in 0·5 mol l–1 sodium-acetate buffer (pH 3·2)] and incubated for 60 min at 30°C. The liberation of reducing groups was determined by adding dinitrosalicylic acid (DNS) to the mixture (Miller et al. 1960) and boiled for 15 min. The sample was cooled and the absorption measured at 540 nm. The units were defined as nkat units g–1 dry cells.
Glucanase activity was tested in 0·1% barley β-glucan (Sigma) and in 0·2% laminarin (Sigma) solutions. Both solutions were made with 0·5 mol l–1 sodium-acetate buffer pH 3·2. The assays were performed in two parts. One part was used to determine the liberation of glucose with the Trinder kit and the other part was used to determine the liberation of reducing groups by the DNS method.
The isolates investigated were mostly strains of K. apiculata. The second largest group, Candida, contained the most species, 11 compared to the two of Kloeckera. From the 245 isolates, 121 were isolated from the vineyard and 120 from the clarified grape juice. The 245 isolates represented 21 different species of yeast. From the 21 species, 9 were isolated from the vineyard and 13 from the clarified grape juice. These isolates were obtained as representatives of non-Saccharomyces strains found in a single vintage (1997). However, it should be noted that depending on climate and viticultural practices, the population dynamics differ from year to year, thereby influencing the non-Saccharomyces population composition.
Only nine isolates represented by Candida stellata, Candida oleophila, Candida pulcherrima, Candida valida and K. apiculata showed pectolytic activity on plates in our study (Table 2). McKay (1990) reported that the secretion of polygalacturonases by some strains of S. cerevisiae was constitutive and the medium must be supplemented with 1% glucose for enzyme production. For this reason, glucose was included in the composition of the polygalacturonate agar used in the present study. However, it is possible that the presence of glucose in the medium could inhibit the production of these enzymes in some of the isolates.
Production of extracellular enzymes and H2
S by species of non-Saccharomyces
The results in Table 2 indicated that 10 isolates of C. stellata, C. pulcherrima, K. apiculata and one isolate of Debaryomyces hansenii have showed proteolytic activity in our study. Charoenchai et al. (1997) demonstrated that the presence of a readily utilisable nitrogen source repressed extracellular proteases, but in stark contrast these isolates showed activity on YPD-based plates that contained a readily utilisable nitrogen source. However, it is possible that if our medium had lacked amino acids and ammonium sulphate, our results could have been different.
Lichenan is a linear, 1,3–1,4-β-D-glucan with a structure similar to that of barley and oat β-D-glucan. Lichenan has a much higher proportion of 1,3- to 1,4-β-D-linkages than the other two glucanes. The ratio of 1,4- to 1,3-β-D-linkages is approximately 2 : 1. Only C. stellata, C. pulcherrima and K. apiculata isolates showed lichenase activity (Table 2).
β-Glucosidase activity can be measured by using β-glucoside analogues, 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG) or ρ-nitrophenyl-β-D-glucopyranoside (p-NPG). However, these substrates are unreliable because 1,3-glucanase activity (which most yeasts contain) can also hydrolize these substrates. In this case it is more reliable to use a substrate like arbutin. Colonies showing activity can be identified by the discoloration of the media to a brown colour. In a preliminary study when p-NPG was used, it appeared that most of the strains contained glucosidase activity (data not shown), but when the assay was repeated with arbutin none of the strains under our test conditions showed glucosidase activity. It was speculated that the presence of glucose could have a considerable effect on the production of β-glucosidases. However, even when omitting glucose, only some weak β-glucosidase activity was detected.
Only 11 isolates of C. stellata, C. pulcherrima and K. apiculata showed some cellulase activity, but what is interesting is that C. pulcherrima showed only activity on YPD-based plates that contained glucose (Table 2). On the YPGE-based plates no activity was observed for C. pulcherrima.
Most isolates of C. stellata, C. hellenica and K. apiculata showed activity on plates, but only a few isolates of C. sorbosa, C. lambica, C. pulcherrima showed activity on plate assays (Table 2). In contrast, more Candida species, Pichia farinosa and P. kluyveri were able to degrade glucan in liquid assays (Table 3). Candida stellata (18028 units) and K. apiculata (23153 units) were the two highest glucose producers. When we tested for the production of reducing groups C. stellata produced 190 nkat units and K. apiculata 306 nkat units.
Amylase, glucanase, lichinase and xylanase activity of supernatant of non-Saccharomyces
wine yeasts grown in liquid cultures
One isolate of C. stellata showed strong activity on only YPGE-based plates, while one isolate of C. pulcherrima showed only little activity on YPGE-based plates. Two isolates of C. oleophila showed little activity on YPGE, while one of these isolates showed also activity on YPD plates. One isolate of K. apiculata showed little activity on only YPD-based plates. In liquid assays K. apiculata (1550 units) and C. stellata (510 units) produced the highest xylanase activities.
Starch hydrolytic enzymes
Although the degradation of starch is not important from an oenological perspective, most of the isolates were able to degrade starch. It must be noted that the amylolytic activity observed was not α-amylase activity as no activity was observed on phadebas plates which is specific for α-amylase activity (data not shown). This ability may be more important for whisky fermentations. In contrast, Charoenchai et al. (1997) did not find any amylolytic wine yeast in their study. It is tempting to speculate that this can be due to the low percentage of substrate (0·1 g l–1) that they used in their plates.
Although most strains produce high amounts of H2S, there were a few that did not produce H2S (Table 2). Since the production of H2S by wine yeasts leads to off odours in the wine, the H2S-producing potential of the non-Saccharomyces yeasts was also tested under oenological conditions. Media containing bismuth citrate has proven to be effective for the selective cultivation and differentiation of Candida spp. from pathological samples (Nickerson 1953). Growth on indicator media provides a visual measure of genetically determined maximal activity of sulfite reductase of any given strain and consequently, its potential to produce H2S (Jiranek et al. 1995). Colonies which produce hydrogen sulphide become discoloured in a concentration-dependent manner, ranging from white, through brown, to black. The simplicity of hydrogen sulphide detection inherent with this media has resulted in the adoption of this technique by scientists for the rapid presumptive identification of low or non-H2S-producing wine yeast (Rupela and Tauro 1984).
Although our study clearly indicated that non-Saccharomyces strains are capable of producing pectolytic activity, in the study of Charoenchai et al. (1997) none of the 23 strains tested showed any pectolytic activity. Pectic enzymes are mainly produced by moulds and bacteria, but are also produced by some yeasts. Significant pectolytic activity was found in Saccharomyces fragilis (Kluyveromyces fragilis) and Candida tropicalis, whereas Saccharomyces thermantitonum, Torulopsis kefyr and Torulopsis lactosa have weaker activity (Luh and Phaff 1951). Pectinesterases were detected in Debaryomyces membranaefaciens var. hollandius, Endomycopsis olmeri var. minor, Candida krusei, Hansenula, Rhodotorula and Zygopichia (Bell and Etchells 1956). Polygalacturonase activity was found in Candida silvae, Candida norvegensis, Geotrichum candidum, Pichia guilliermondii, Pichia membranaefaciens, Torulopsis candida and Trichosporum cutaneum (Call and Emeis 1978; Sanchez et al. 1984; Ravelomanana et al. 1986). Furthermore, several Saccharomyces species were also reported to have polygalacturonase activity, including S. carlsbergensis, S. chevalieri, S. cerevisiae, S. oviformis, S. uvarum and S. vini (Kotomina and Pisarnitskii 1974; Sanchez et al. 1984). Bell and Etchells (1956) reported weak pectolytic activity for S. cerevisiae, whereas Luh and Phaff (1951) reported that S. cerevisiae cultures tested had no noticeable effect on pectin.
It was later claimed that certain strains of S. cerevisiae have the ability to degrade polygalacturonic acid in the presence of glucose (McKay 1990). Recently, a single culture of S. cerevisiae was isolated that supposedly produces pectinesterase, polygalacturonase and pectin lyase (Gainvors et al. 1994). None of these enzymes have been purified nor their genes cloned. Blanco et al. (1994) reported that at least 75% of oenological strains tested showed limited pectolytic activity. Endopectate degrading enzymes occurred primarily in the growth medium, as is the case for most other yeast species. Synthesis of pectic enzymes was reported to be constitutive, providing the glucose concentration in the medium did not exceed 2%. A higher concentration of glucose led to the total inhibition of these pectolytic activities. Interestingly enough, the pectolytic activity was found to be significantly lower with growth on glucose as carbon source than with galactose. Subsequently, Blanco et al. (1998) speculated that all S. cerevisiae strains contain a promoter-less polygalacturonase gene or else a nonfunctional one. This structural polygalacturonase-encoding gene (PGU1) from S. cerevisiae IM1-8b was eventually cloned and sequenced. The predicted protein comprises 361 amino acids, with a signal peptide between residues 1 and 18 and two potential glycosylation points in residues 318 and 330. The putative active site is a conserved histidine in position 222. This S. cerevisiae polygalacturonase shows 54% homology with the fungal polygalacturonases and only 24% homology with its plant and bacterial counterparts. PGU1 is present in a single gene copy per haploid genome and it is detected in all strains, regardless of their phenotype. The expression of PGU1 gene in several strains of S. cerevisiae revealed that the polygalacturonase activity depend on the plasmid used and also on the genetic background of each strain but in all cases the enzymatic activity increased.
Proteolytic activity of yeasts has been studied in relation to protein haze reduction in beer and wine. Our study has confirmed that several species of indigenous wine yeasts can produce proteolytic activity. The vacuolar protease A plays an important role during the autolysis process, which occurs in wines kept on yeast lees during ageing. However, because of the particular conditions found in wine, only a few proteases are active (Lurton 1987). Eight yeast strains were identified which consistently exhibited proteolytic activity in model wine solutions. These included Candida olea, C. flavus, K. apiculata, Metschnikowia pulcherrima, Pichia pinus, Torulopsis magnolia, Torulopsis monosa and Yarrowia lipolytica. Protease activity was maintained in actual wine solutions, but at somewhat reduced levels. Incubation of a protease concentrate from K. apiculata with Chenin blanc and Chardonnay wines showed some degradation of wine proteins. Lagace and Bisson (1990) demonstrated that extracellular proteolytic activities produced by C. olea, C. lipolytica, Cryptococcus flavus, K. apiculata and C. pulcherrima could be correlated with their ability to reduce wine haze. In a recent study Dizy and Bisson (2000) demonstrated that strains of Kloeckera and Hanseniaspora produced the most proteolytic activity in grape juice, and affected the protein profile of the finished wines. Protease activity produced did not significantly reduce the heat/chill haze forming potential of the wines, however. In some cases, the haze level formed was greater in the fermentations with high proteolytic activity.
The significance of β-glucosidases for the wine industry lies in their potential for releasing flavour compounds from glycosidically bound nonvolatile precursors in wine. A few papers have reported that non-Saccharomyces wine yeasts do contain glucosidase activity but these results are questionable, because of the possibility that the yeast tested may have had no glucosidase activity but rather that 1,3-glucanases were able to hydrolyse the β-glucoside analogues used in most studies. In contrast to the grape glycosidase, yeast glucosidases are not inhibited by glucose. It was reported that although the β-glucosidase from Hansenula sp., isolated from fermenting must, was capable of liberating aroma substances in wine, it was less effective in must (Grossmann et al. 1987). An intracellular β-glucosidase from Debaryomyces hansenii Y-44 was purified and used in the fermentation of Muscat juices (Yanai and Sato 1999). A wine with a considerable increase in the concentration of monoterpenols was produced. The linalool and nerol contents increased especially, by 90% and 116%, respectively. According to some reports, certain strains of S. cerevisiae also possess a β-glucosidase that is located in the periplasmic space of the yeast cell (Darriet et al. 1988; Dubourdieu et al. 1988). This activity appears to be very limited and therefore recent studies have rather focused on non-Saccharomyces yeasts such as Hanseniaspora vinea (Vasserot et al. 1989) and Candida species (Günata et al. 1990). Results obtained so far from studies on yeast glycosidases indeed suggest that specific yeast strains can affect the varietal aroma of wines (Laffort et al. 1989). In a recent study of McMahon et al. (1999) they determined the β-glucosidase production in 32 strains of the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces, Torulaspora and Brettanomyces. Only one Saccharomyces strain exhibited activity, but several of the non-Saccharomyces yeast species showed activity, especially Brettanomyces (eight of the 10 strains tested). However, the majority of the β-glucosidase activity was located in the whole cell fraction, with smaller amounts found in permeabilized cells and released into the growth medium. Only Aureobasidium pullulans hydrolysed glycosides found in grapes.
Hemicellulose constitutes a group of polysaccharides which is associated with cellulose in plant cell walls. These complex carbohydrate polymers contain xylan as their main component and β-1,4-xylans are mainly found in the secondary cell walls of plants, where it acts as one of the major components of woody tissue (Thomson 1993). The degradation of hemicellulose is performed by a complex set of enzymes such as xylanases and could contribute to wine aroma by increasing the amount of monoterpenyldiglycoside precursors in the must. These compounds can be released from the walls of grape cells by the action of these enzymes. Since cellulose and hemicellulose represent the primary structural polysaccharides of the plant cell wall, a portion of them might be released into the wine after acid hydrolysis. This could lead to filtration and clarification problems (Zoecklein et al. 1995). The presence of cellulase and hemicellulase in the wine can solve this problem. Lichenase activity can also play an important role in filterability of a wine, since a major portion of the grape and Botrytis glucans contain 1,3–1,4 linkages. Cellulase activity can play a role in extracting more colour and flavour out of the skins of the grapes. It is also possible that this activity can change the tannin composition of the wine or even shorten the time for fermentation on the skins. Enzyme preparations containing cellulases and hemicellulases, in addition to pectinase activities, are known as macerating enzymes. These preparations are used to improve juice yields by degrading structural polysaccharides that interfere with juice extraction, clarification, and filtration.
When comparing the isolates from the different areas, the C. stellata and K. apiculata strains, isolated from the Constantia area, contain a much more complex enzyme profile. It is well established that wine fermentations, whether they develop naturally or after inoculation with S. cerevisiae, are characterized by the significant growth of indigenous species of non-Saccharomyces yeast. It is also believed and proven that these yeasts must effect the chemical composition and final sensory quality of the wine. Our study has revealed the potential of indigenous wine yeasts to produce a wide range of extracellular enzymes. The findings indicate the need to determine what impact such activities may have on the sensory properties of wine. In ongoing research we are using some of these strains in cocultures with Saccharomyces strains to see what effect these strains have on wine aroma. In preliminary results it seems that the strains with the most enzymatic activities are the ones that have the biggest effect on wine aroma. Therefore, future research should focus on the activity of these enzymes in wine fermentations and a better knowledge of the physiological and metabolical features of non-Saccharomyces yeast is required.
The authors thank the South African wine industry (Winetech), the National Research Foundation (NRF) and THRIP for funding.