Journal of Applied Bacteriology 1986,60,477-481 2 I93/09/85

Evaluation of selective media for enumeration of yeasts during wine fermentation

G . M . H E A R D & G.H. FLEET School of Food Science and Technology, The University of New South Wales, PO Box I , Kensington, New South Wales, Australia, 2033

Received 23 September 1985 and accepted 19 February 1986

H E A R D , G.M. & FLEET, G . H . 1986. Evaluation of selective media for enumer- ation of yeasts during wine fermentation. Journal of Applied Bacteriology 60, 477- 481.

The growth of individual species of yeasts during wine fermentations was measured by plating wine samples on malt extract, ethanol sulphite and lysine agars. Colonies of Saccharomyces cereuisiae dominated on plates of malt extract agar and sometimes masked the presence of other non-Saccharomyces species. Lysine agar suppressed the growth of S. cereuisiae and enabled the enumeration of non-Saccharomyces species such as Kloeckera apiculata, Candida stellata and Saccharomycodes ludwigii. The growth of non-Saccharomyces yeasts on ethanol sulphite agar was variable.

In traditional wine-making, fermentation develops naturally and is caused by the growth of yeast species that occur as contaminants of the grape and winery equipment. In more modern practices, the fermentation may be induced by inoculation of the grape juice with a starter culture, generally a species of Sac- charomyces. Many qualitative studies have described the succession of yeast species that develops during fermentation and these have been reviewed by Kunkee & Amerine (1970); Kunkee & Goswell (1977); Benda (1981) and Lafon-Lafourcade & Ribereau-Gayon (1984). Alcohol-tolerant species of Saccharomyces, espe- cially Saccharomyces cereuisiae, eventually dominate the fermentation but, during the first 3 to 4 days, there may be significant growth of species of Kloeckera, Hanseniaspora, Candida, Hansenula and Pichia. It is now believed that this initial flora may contribute significant sensory characteristics to the wine and, conse- quently, it has become increasingly important to have better knowledge of the kinetics of growth of individual yeast species during fermentation (Fleet et al. 1984).

The isolation and enumeration of yeasts from fermenting wines are usually done by plating

wine samples on grape juice, malt extract or other nutrient agar. These are non-selective media that allow colonies of all yeast species associated with the fermentation to grow. A major limitation, however, is that these media tend to provide counts of the dominant species only. Thus, during the initial stages of wine fer- mentation, when species of Hanseniaspora and Kloeckera dominate the flora, it is difficult, if not impossible, to find colonies of Sac- charomyces spp. on the plating media even though they may be present in the wine at low 1Cvels. Similarly, these media do not provide reliable data on the counts of Hanseniaspora, Kloeckera or other non-Saccharomyces species in wine samples taken after the first 2 to 3 days of fermentation because such yeasts are present in much lower numbers than the then dominant Saccharomyces species. To overcome this limi- tation, it is necessary to use plating media that will selectively suppress growth of the dominant species, without inhibiting those present in low numbers.

Recently, Kish et al. (1983) described the use of a nutrient plating agar containing ethanol and sodium metabisulphite, which suppress the growth of species of Hanseniaspora and Kloeck-

G. M . Heard and G. H . Fleet era. With this medium, it was possible to follow the growth of Saccharomyces species during the early stages of wine fermentation.

In the brewing industry, lysine agar (Walters & Thiselton 1953; Lin 1975) is used to selec- tively enumerate non-Saccharomyces species of yeasts when they occur in the presence of large numbers of Saccharomyces species. This medium contains lysine as the sole nitrogen supply and exploits the fact that this amino acid cannot be assimilated by Saccharomyces species. Lysine agar would appear to be a most suitable medium for selectively enumerating non- Saccharomyces species of wine yeasts although, as yet, it has not been used for this purpose in the wine industry.

In this paper malt extract agar, ethanol sul- phite agar and lysine agar are compared for the enumeration of yeasts during the fermentation of several wines.

Materials and Methods

W I N E SAMPLES

Wines were produced under commercial condi- tions at wineries in New South Wales. The wines were fermented by inoculation with starter cultures of Saccharornyces cereuisiae but in all cases a portion of the grape juice was left uninoculated and allowed to ferment naturally. Samples of the wines were taken at daily inter- vals for the enumeration of yeasts.

E N U M E R A T I O N , ISOLATION A N D IDENTIFICATION OF YEASTS

Each wine sample was diluted in 0.1% peptone, if necessary, and then 0.1 ml volumes spread inoculated over the surface of plates of malt extract agar (MEA), ethanol sulphite agar (ESA) and lysine agar. All media and ingredients were purchased from Oxoid. Ethanol sulphite agar contained (g or m/l in distilled water): glucose, 20; peptone, 5; yeast extract, 5; agar, 15; ethanol, 120; sodium metabisulphite, 0.15; pH 5.5 and was prepared according to Kish et al. (1983).

After inoculation, plates were incubated at 20"-25"C for 5 d and examined for yeast colo- nies. Individual colony types were counted and representatives of each type were isolated and purified by restreaking on MEA. Isolates were

identified according to the procedures and keys of Kreger van Rij (1984) and Barnett et al. (1983).

Results

Figures la, b and c show the growth of yeast species in a naturally fermenting white, Semillon wine. When the non-selective medium, MEA, was used for yeast enumeration (Fig. la), it was found that Saccharomyces cerevisiae eventually dominated the fermentation but there was sig- nificant growth of Candida krusei and C. coilicu- losa during the first 7 d. After the first 2 d the Candida species were observed only on plates inoculated with lower dilutions of the wine and could be easily overlooked because of crowding by S . cereuisiae. Similar results were obtained when ESA was used for yeast enumeration (Fig. lb) although the counts for C . krusei and C. colliculosa were slightly different from those on MEA. No other species were recovered from the wine using these two media. When the same wine was plated on lysine agar (Fig. lc), the growth of S . cerevisiae and, to some extent, that of C . colliculosa was suppressed. The presence of two other yeasts, Kloeckera apiculata and Sac- charomycodes ludwigii, was revealed. Sac- charomyces cerevisiae appeared only as pinpoint colonies on lysine agar.

The growth of yeasts during fermentation of a white, Riesling wine that was produced by inoc- ulation with a starter culture of S . cereuisiae is shown in Figs Id, e and f. According to counts obtained on MEA, two yeast species contrib- uted to the fermentation (Fig. Id). These were the dominant yeast, S . cereuisiae, and K . apicul- ata that was not recovered after 7 d fermenta- tion. Kloeckera apiculata was completely inhibited on ESA (Fig. le). Plating of the wine on lysine agar revealed that K. apiculata sur- vived for 11 d fermentation and that another yeast, C. stellata, was also present during the first day of fermentation.

Further evidence of the merits of using lysine agar for enumerating yeasts associated with wine fermentations is presented in Fig. 2. Figures 2a and b show the growth of S . cerevi- siae and K . apiculata during the fermentation of a red Shiraz wine. Although K. apiculata was isolated on MEA along with S. cerevisiae, its longer survival during fermentation was record- ed by the counts on lysine agar. In addition to

Selective media for wine yeasts

P d ' 479

" F ' C 1 -

1 2 3 4 5 6 7 8 910 0 I 2 3 4 5 6 7 8 91011

Fermentation time ( d )

Fig. 1. Growth of yeasts in (a, b and c) a naturally fermenting white, Semillon wine and (d, e and fJ a white, Riesling wine undergoing inoculated fermentation. 0, Candida colliculosa; A, Candida krusei; ., Candidu stel- lata; e, Kloeckeru apiculata; A, Saccharomycodes ludwigii; 0, Saccharomyces cerevisiae. (a and d), Counts on malt extract agar; (b and e), counts on ethanol sulphite agar; (c and f), counts on lysine agar.

S. cerevisiae, K . apiculata and C . stellata con- tributed to the fermentation of a red Malbec wine (Figs 2c and d). Only S. cereuisiae and K . apiculata were found when samples of the wine were plated onto MEA. Kloeckera apiculata was not found on this medium after 4 d fermenta- tion. Candida stellata and K . apiculata were recovered from this wine up until 7 d fermenta- tion when wine samples were examined by plating on lysine agar.

Hansenula anomala, a yeast often associated

with the early stages of wine fermentation, was not isolated from the wines examined in this study and, presumably, did not contribute to the ecology of fermentation in these cases. The ability to recover this species on lysine agar, however, was demonstrated by inoculating and fermenting sterile grape juice with a mixture of S. cerevisiae, C . stellata, H . anomala and K . apiculata. Hansenula anomala was recovered from this culture up until 4 d fermentation, by plating on lysine agar.

480 G . M . Heard and G . H . Fleet

9 P a ' p C )

-

Fermentation time ( d )

Fig. 2. Growth of yeasts in (a and b) a red Shiraz wine undergoing inoculated fermentation and (c and d) a naturally fermenting red Malbec wine. ., Candida stellata; 0, Kloeckera apiculata; 0, Saccharomyces cerevisiae. (a and c), Counts on malt extract agar; (b and d), counts on lysine agar.

Discussion

This study has revealed the limitations of using a single non-selective medium, such as MEA, for the isolation and enumeration of yeast species that grow during natural and inoculated wine fermentations. Although MEA provides reliable information about the growth of Saccharomyces type yeasts during fermentation, it does not give an accurate measurement of the non- Saccharomyces species of yeasts.

Lysine agar proved to be a suitable medium for measuring the growth of non-Saccharomyces yeasts during 'wine fermentation. Its merits were demonstrated most convincingly in those wines where some species, such as K . apiculata, Sac- charomycodes Iudwigii and C. stellata, were not detected by plating on MEA but were detected after plating on lysine agar. The contribution of C. stellata to wine fermentations has been reported recently by Fleet et al. (1984) who also noted that this species grows slowly on MEA, forming very small colonies which are easily overlooked. On lysine agar, C. stellata produces distinct colonies that permit reliable and accu- rate measurement of its behaviour during fer- mentation. Lysine agar also gave a more

accurate indication of the fate of K . apiculata during fermentation. According to earlier studies (Kunkee & Goswell 1977; Benda 1981; Fleet et al. 1984) and our present results using MEA as enumeration medium, this species does not exhibit extended survival and usually dies off during the early stages of fermentation. Our results with lysine agar suggest that this species survives in the wine a little longer than pre- viously thought.

Ethanol sulphite agar was developed by Kish et al. (1983) to suppress the growth of non- Saccharomyces type yeasts and to allow more accurate measurement of Saccharomyces species during the early stages of natural fermentation. In our experience, this medium offered little advantage over MEA for enumeration of S . cerevisiae. The behaviour of ESA in suppressing the growth of non-Saccharomyces yeasts was inconsistent and this probably relates to the technical difficulties of maintaining an accurate concentration of ethanol in the medium.

In conclusion, our study has described the merits of using lysine agar as a selective medium for measuring the growth of non-Saccharomyces species of yeasts during wine fermentation. This medium, used in conjunction with MEA, will

Selective media for wine yeasts 48 1

provide more accurate, quantitative information about the growth of individual yeast species during natural and inoculated wine fermenta- tions, thereby enabling a better understanding of how they might affect wine quality.

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