Active Dry Yeast Strains - Applied and Environmental Microbiology

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1992, p. 2948-2953

Vol. 58, No. 9

0099-2240/92/092948-06$02.00/0 Copyright X) 1992, American Society for Microbiology

Molecular Monitoring of Wine Fermentations Conducted by Active Dry Yeast Strains AMPARO QUEROL,1 ELADIO BARRIO,2 TOMAS HUERTA,1 AND DANIEL RAMON3* Departamento de Microbiologia1 and Departamento de Genetica,2 Facultad de Ciencias Biol6gicas, Universidad de Valencia, 46100 Buriasot, and Unidad de Bioingenieria, Instituto de Agroquimica y Tecnologia de Alimentos, Consejo Superior de Investigaciones Cientificas, Jaime Roig 11, 46010 Valencia,3 Spain Received 6 April 1992/Accepted 6 July 1992

A simple and rapid method of yeast strain characterization based on mitochondrial DNA restriction analysis applied to the control of wine fermentations conducted by active dry yeast strains. This molecular approach allows us to understand several important aspects of this process, such as the role of the active dry yeast strain and that of the natural Saccharomyces cerevisiae flora during vinification. In this paper, we demonstrate that the inoculated strain is really responsible for the fermentation but does not suppress significant development of natural strains during the first stages. During this early period, natural strains could have important effects on wine flavor. was

The quality of wines is a direct consequence of the evolution of the microbial flora of the must during fermentation. A number of different strategies have been used for determining the population kinetics during wine fermentation. Nevertheless, these attempts have been restricted owing to the difficulty in distinguishing among the different Saccharomyces cerevisiae strains present in the nonsterile must. Some approaches have used the killer phenotype to examine the population dynamics of either killer-sensitive or killer-resistant strains in both sterile media and musts (7, 13). Similarly, other markers have been used, such as the galactose fermentation phenotype (13). These kinds of strategies are applicable only under sterile laboratory conditions, not in the wineries, where the nonsterile must contains a complex S. cerevisiae flora with killer-sensitive, killer-resistant, galactose-fermenting, or non-galactose-fermenting strains (1, 6, 10, 11). The use of natural or induced mutants resistant to certain drugs (e.g., chloramphenicol or oligomycin) has been exploited by other investigators (17). In this case, the nonsterile must is inoculated with the resistant strain and its growth is examined during the fermentation process. However, all of these methods provide a biased view of the problem, because it is not possible to evaluate the role of the natural S. cerevisiae strains present in the must. During the last few years, we have been studying the wines produced in the Mediterranean region of Alicante (Spain). All fermentations conducted in that region are traditional vinification processes, characterized by an elevated temperature of fermentation and high initial sugar concentrations (11). By the use of foreign wine active dry yeasts it is not possible to obtain a satisfactory development of the fermentation process, probably because of the special fermentation conditions. To overcome this problem, we have isolated and characterized an S. cerevisiae strain (namely, T73) from Alicante musts that, used as an active dry yeast, produces an excellent wine (10). The use of our selected strain in the winemaking process required the development of techniques that could clearly *

differentiate the inoculated yeast strain from the rest of the wild yeast strains present in the must (9). As most of the strains belong to the same species, S. cerevisiae, they could not be identified by classical microbiological methods. Several techniques based on molecular polymorphisms have been used recently for wine yeast strain characterization (for

Hinf

Rsal ;> __">> _m>ii_> _.

m___.^mX~_m

FIG. 1. mtDNA patterns of the most common strains present in the inoculated fermentation at winery A. DNA was extracted and digested with RsaI and Hinfl by the method of Querol et al. (10). Lanes m correspond to a mixture of lambda phage DNA digested with HindIII and with HindIII-EcoRI, used as size markers. Roman numerals correspond to the mtDNA patterns given in Table 1. Pattern I corresponds to that of strain T73, used for inoculation.

Corresponding author. 2948

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MOLECULAR MONITORING OF WINE FERMENTATIONS

TABLE 1. Frequencies of the mtDNA patterns obtained in each sampling of the inoculated carried out at winery A

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(Al) and control (AC) fermentations

Frequency (%) of the mtDNA pattern in the following samples on day:

Patterna

I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX XXI XXII

XXIII XXIV XXV XXVI XXVII XXVIII XXIX XXX

XXXI XXXII XXXIII XXXIV XXXV XXXVI XXXVII XXXVIII XXXIX XL XLI

0

2

6

4

21

All

AC1

A12

AC2

A13

AC3

A14

AC4

(Al5)h

0.00 4.54 2.27 2.27 2.27 6.82 2.27 4.54 4.54 4.54 6.82 4.54

0.00 4.54 6.82 6.82 20.45 11.36 2.27 4.54 4.54 4.54 2.27 4.54 2.27 2.27 2.27 2.27 4.54 4.54 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

6.52 6.52 0.00 0.00 0.00 2.17 0.00 0.00 0.00 0.00 19.56 0.00 0.00 0.00 10.87 15.22 0.00 0.00 0.00 2.17 0.00 4.35 10.87 2.17 2.17 4.35 2.17 2.17 4.35 2.17 0.00 0.00 0.00 2.17 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 16.28 0.00 37.21 2.32 13.85 0.00 0.00 0.00 2.32 0.00 0.00 18.60 2.32 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.65 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.32

21.28 8.51 0.00 0.00 4.26 4.26 0.00 4.26 0.00 0.00 14.89 0.00 0.00 0.00 0.00 21.28 0.00 0.00 0.00 0.00 0.00 0.00 2.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.13 4.26 2.13 2.13 2.13 4.26 2.13 0.00 0.00 0.00 0.00

0.00 0.00 0.00 20.51 5.13 43.59 0.00 0.00 20.51 0.00 0.00 0.00 10.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

38.30 2.13 2.13 0.00 0.00 2.13 0.00 0.00 0.00 0.00 25.53 0.00 0.00 0.00 0.00 10.64 0.00 0.00 2.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 6.38 0.00 0.00 0.00 0.00 0.00 2.13 2.13 0.00 0.00

0.00 0.00 2.13 24.19 4.88 41.46 2.44 0.00 12.19 0.00 0.00 0.00 7.32 4.88 0.00 0.00 2.44 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

68.09 4.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.26 0.00 0.00 0.00 2.13 14.89 2.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

2.27 2.27 20.45 13.64 4.54 4.54 2.27 2.27 2.27 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.13 0.00 0.00 2.13 0.00

a Roman numerals correspond to the same patterns in both fermentations. b AC5 was not investigated.

a comparative study, see the works of Degre et al. [3] and Querol et al. [9]). These consist of analysis of protein patterns (16), DNA sequence polymorphisms (3), electrophoretic karyotyping (18), and mitochondrial DNA (mtDNA) restriction analysis (4, 8, 18). Nevertheless, the complexity of these techniques renders their industrial application difficult. Recently, we have developed a fast, reliable, and economic method to detect yeast mtDNA restriction patterns (9) which does not require sophisticated equipment and personal skill and allows differentiation among yeast strains isolated from the same must. In the present work, we describe the use of this last technique at the industrial level in order to demonstrate the gradual dominance of the T73 strain in inoculated fermentations, as well as to study the evolution of the whole S. cerevisiae flora present in the fermentation process. These results allowed us to know the answers to such classical

questions in the wine industry as: (i) is only the inoculated strain responsible of the fermentation process? (ii) how many strains appear in an inoculated fermentation? and (iii) which strains play an important role in the vinification process? MATERIALS AND METHODS Wine fermentations. The wines were produced from grapes harvested during the 1991 vintage in two different wineries located in Valencia (Spain). In each case, the must was supplemented with sulfur dioxide (30 mg/liter) and was separated in two different tanks of 15,000 liters. One tank was inoculated (20 g of lyophilized yeast per 100 liters of must) with the previously selected T73 S. cerevisiae strain (Lallemand Inc., Montreal, Quebec, Canada), and the other one was not inoculated, as a control. Inocula were rehy-

APPL. ENvIRON. MICROBIOL.

QUEROL ET AL.

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TABLE 2. Frequencies of the mtDNA patterns obtained in each sampling of the inoculated (BI) and control (BC) fermentations carried out at winery B Frequency (%) of the mtDNA pattern in the following samples on day: Patterna

I II III IV V VI

VII VIII IX X XI

XII XIII XIV XV

XVI XVII XVIII XIX XX

XXI XXII XXIII XXIV XXV XXVI XXVII XXVIII XXIX XXX XXXI XXXII XXXIII XXXIV

8

5

3

0

18

BIl

BC1

BI2

BC2

BI3

BC3

B14

BC4

B15

BC5

0.00 29.38 2.26 2.26 2.26 18.08 2.26 9.09 4.54 4.54 2.26 6.82 9.09 2.26 2.26 2.26 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 32.50 12.50 0.00 10.00 0.00 0.00 0.00 0.00 0.00 0.00 17.50 2.50 0.00 0.00 0.00 2.50 7.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.50 2.50 2.50 2.50 2.50 0.00 0.00

35.52 33.30

0.00 36.36 9.09 0.00 9.09 0.00

37.50 17.50 0.00 0.00 0.00 10.00 0.00 2.50

0.00 38.09 9.52 0.00 21.43 0.00 0.00 0.00 0.00 2.38 0.00 2.38

41.60 16.64 0.00 0.00 0.00 14.56 0.00 12.48 0.00 2.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.16 0.00 0.00 4.16 2.08 2.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 47.62 11.9 0.00 14.28 0.00

89.46 4.26 0.00 0.00 0.00 2.13 0.00 2.13 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 64.28 30.95 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 4.44 6.66 0.00 8.88 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.44 4.44 2.22 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 4.54 2.72 9.09 0.00 0.00 0.00 4.54 6.82 11.36 2.72 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.72 2.72

XXXV a Roman numerals correspond to the same patterns in both fermentations.

drated prior to inoculation according to the instructions of the supplier. The fermentations were conducted under the same conditions (20 to 25°C) for various times (18 to 21 days). The organoleptic characteristics of the four wines were tested by trained experts. Microbiological analysis. Samples were taken periodically during fermentation for the isolation and enumeration of yeasts. Aliquots (0.1 ml each) of several dilutions were spread onto plates of malt extract agar (Oxoid). Plates were incubated at 28°C for 5 days. Yeast colonies were isolated and characterized by the method of Barnett et al. (2), and only those corresponding to species of the S. cerevisiae group that proved to be responsible for the fermentation process (1) were analyzed. mtDNA isolation and restriction analysis. At each time point when a sample was taken, 50 independent isolates obtained as colonies were analyzed for mtDNA. This number is statistically significant in accordance with Snedecor and Cochran (15). A total of 950 colonies were analyzed, and their mtDNA patterns were determined by the method of Querol et al. (9), as follows. Yeast cells were grown in an overnight culture of 5 ml of YEPD (1% yeast extract, 2%

0.00 0.00 2.50 0.00 5.00 0.00 0.00 0.00 2.50 0.00 2.50 5.00 2.50 2.50 5.00 2.50 2.50 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 2.38 9.52 11.91 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00

2.38 1.14 0.00 0.00 0.00 7.14 9.52 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 4.76 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

peptone, 2% glucose). Cells were spun down in a microcentrifuge and suspended in 0.5 ml of 1 M sorbitol-0.1 M EDTA, pH 7.5. Then they were transferred to a 1.5-ml microcentrifuge tube, to which 0.02 ml of a solution of Zymolyase 60 (2.5 mg/ml) was added. Tubes were incubated at 37°C for 30 to 60 min in order to obtain spheroplasts, and their release was optionally monitored. Spheroplasts were pelleted for 1 min in a microcentrifuge and suspended in 0.5 ml of 50 mM Tris-HCl-20 mM EDTA, pH 7.4. After suspension, 0.05 ml of 10% sodium dodecyl sulfate was added and the mixture was incubated at 65°C for 30 min. Immediately thereafter, 0.2 ml of 5 M potassium acetate was added and the tubes were placed on ice for 30 min. Then they were centrifuged at maximum speed in a microcentrifuge for 5 min. Supernatant was transferred to a fresh microcentrifuge tube, and the DNA was precipitated by adding 1 volume of isopropanol. After incubation at room temperature for 5 min, the tubes were centrifuged for 10 min. The DNA was washed with 70% ethanol, vacuum dried, and dissolved in 50 ,ul of TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Two microliters of DNA was digested with two restriction endonucleases that recognize 4 bp (RsaI) and 5 bp (Hinfl) according to the instruc-

VOL. 58, 1992


FREQUENCY (%)

(o/l)

REDUCIG SU

du/ml

2951

1 .OOOE+08

A10000000

1000000

0

5

10

15

20

25

0

5

10

TIME (days) 1-I4+11 lU

9

IN

=~~- ~100000 20 25

15

TIME (days)

Iv

v

-V

-REDUCING SUGARS

-4

T73

250

1 ,OOOE+09

200

1,000E+08 1 50

100

10000000 50

0

5

10

15

20

TIME (days)

1000000

C

0

5

10

15

20

TIME (days) -

I

|

II111 I

o

IV

FIG. 2. Growth of the most frequently detected yeast strains present in the inoculated fermentations carried out at wineries A (A) and B (B). Pattern I corresponds to that of strain T73, used for inoculation.

REDUCING SUGARS

I

T73

FIG. 3. Growth of yeast strain T73 and reducing sugar disappearance during the inoculated fermentations carried out at wineries A (A) and B (B).

tions of the supplier (Boehringer Mannheim). These enzymes recognize a large number of sites in the yeast nuclear DNA but few sites in the mtDNA. Restriction fragments were separated in 0.8% agarose gel electrophoresis and visualized in a UV transilluminator after ethidium bromide staining (14). RESULTS

During the 1991 vintages, we conducted industrial vinification processes in two different wineries (designated A and

B) in the Valencia region of Spain. In each winery, we used two fermentors of 15,000 liters. In one of them, the selected T73 S. cerevisiae strain (10) was inoculated, and in the other a natural fermentation was conducted as a control. A sensory test of the wines was done with trained judges, who determined that the organoleptic characteristics of the wines produced by inoculated fermentation were better than those of the control wines (80 and 75% of the tests in wineries A and B, respectively). Wine samples were periodically collected during fermen-

2952

QUEROL ET AL.

tation, and the enumeration and identification of the yeast isolates were done by classical microbiological methods. From each sample 50 S. cerevisiae colonies were selected, and their mtDNA restriction patterns were visualized by our method. As an example, Fig. 1 shows the most common mtDNA patterns present in an inoculated fermentation. Tables 1 and 2 represent the frequencies of all of the mtDNA patterns obtained in each sampling of the inoculated and control fermentations. Only pattern I, corresponding to the T73 strain, is found in common at these two wineries. Our method allowed us to detect a large number of mtDNA patterns (41 and 35 patterns in wineries A and B, respectively), that is to say, S. cerevisiae strains. A great diversity of wild strains was detected in the fermentations. Most of these wild strains appeared in both inoculated and noninoculated fermentations (43.9% in winery A and 34.3% in winery B), but only some of them were present in all of the samples at significant frequencies (>10%). Figure 2 shows the growth of the most frequent strains throughout the inoculated fermentation in wineries A and B. As can be seen in the first stages of the processes, only five and three patterns were significantly present at high levels in wineries A and B, respectively. Only after 3 and 4 days of fermentation does the inoculated strain appear more frequently than the wild strains present in the must. As was expected, in the control fermentors, in which the T73 strain was not inoculated, the most frequent wild strains (patterns IV and VI in winery A and patterns II and III in winery B) were present until the end of the process, and hence they could be the agents responsible for those natural fermentations. The predominance of the inoculated strain was evident in both wineries. At the end of the fermentation, the T73 strain (pattern I) represented 68.09 and 89.46% of the total S. cerevisiae isolates from wineries A and B, respectively. Finally, the evolution of the sugar degradation during the inoculated fermentations shows a clear correspondence to the growth of the T73 strain (Fig. 3). This fact clearly indicates that the T73 strain was responsible for the fermentation. DISCUSSION The main problem in using active dry yeast strains in winemaking is our ignorance about the population dynamics of the inoculated S. cerevisiae strain during fermentation, as well as about the role of the wild strains in the must in this process. Even though several different approaches have been made, none could resolve these questions. The application of a new technique based on mtDNA restriction analysis has allowed us to extend our knowledge about the evolution of the inoculated and wild strains during wine fermentation, and hence their respective roles in this process. It is evident from our results that the population dynamics in a wine fermentation is a complex phenomenon. In the first stages of the fermentation, a great diversity of S. cerevisiae strains was observed, but only a few of them were present throughout the whole process. Curiously, the strain diversity was greater in the inoculated fermentation than in the natural one for both wineries. This fact could be explained by assuming that the inoculated strain disturbs the equilibrium of the ecosystem. In fact, some of the most frequent strains in the noninoculated fermentors were also the most common wild strains present in the first stages of the inoculated fermentations. The growth of the inoculated strain could

APPL. ENVIRON. MICROBIOL.

favor the development of other minority wild strains by inhibiting the growth of the wild strains responsible for the natural fermentation. There has been considerable controversy over the use of selected pure strains in wine fermentations (12). In natural fermentation, a succession of yeast genera has been observed during the first stages of the process, followed by certain Saccharomyces species, which then dominate in the most active stages of the fermentation and towards the end (1, 6, 11). It has been suggested that this succession of species leads to a more complex aroma of the wine (12). However, some investigators claimed that there are actually disadvantages in using selected yeasts because the inoculated strain could suppress significant development of natural yeast during wine fermentation (12). In contrast, Heard and Fleet (5) postulated that inoculated S. cerevisiae strains could beneficially influence the development of wild Saccharomyces strains by inhibiting the growth of non-Saccharomyces yeasts. The most striking result of the present study is the fact that the inoculated strain competes with natural strains but does not completely suppress their growth until several days after inoculation. In the first stages of the inoculated fermentation, the wild strains were present at a significant level, and their frequencies decreased after 3 to 6 days. During this time, the wild strains may have important influences on wine flavor. On the basis of organoleptic testing of wine, some investigators have claimed advantages for either natural or inoculated fermentations. Now, we can conclude that the products of inoculated fermentations not only have a higher quality as a result of controlled processes (75 and 80% of the trained experts preferred the inoculated wine in wineries B and A, respectively) but also maintain the specific characteristics produced by the natural flora. This work summarizes the application of a new technique of yeast strain characterization based on mtDNA restriction analysis to the control of wine fermentations conducted by active dry yeast strains. We consider it to be an exciting time for enology because it could be invigorated by the application of molecular techniques. In this way, our method could be a new way of asking questions about microbial persistence and interactions in the biotechnological use of yeast strains (wine fermentation, beer brewing, bakery products, active dry yeast production, etc.). ACKNOWLEDGMENTS This work was supported by grant ALI90-0949 from Comisi6n Interministerial de Ciencia y Tecnologia, Spain. A.Q. and E. B. were recipients of fellowships from Conselleria de Cultura, Educacion y Ciencia de la Generalidad Valenciana. We warmly thank A. Latorre and J. P. Beltran for critical reading of the manuscript. REFERENCES 1. Amerine, M. A., H. W. Berg, R. E. Kunkee, C. S. Ough, V. L. U. L. Singleton, and A. D. Webb. 1982. The technology of wine making, 4th ed. AVI Publishers, Westport, Conn. 2. Barnett, J. A., R. W. Payne, and D. Yarrow. 1983. Yeasts: characteristics and identification. Cambridge University Press, New York. 3. Degre, R., D. Y. Thomas, J. Ash, K. Mailhiot, A. Morin, and C. Dubord. 1989. Wine yeast strain identification. Am. J. Enol. Vitic. 40:309-315. 4. Dubordieu, D., A. Sokol, J. Zucca, P. Thalouarn, A. Datte, and M. Aigle. 1984. Identification des souches de levures isolees de vins par l'analyse de leur ADN mitochondrial. Connais Vigne Vin. 21:267-278.

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5. Heard, G. M., and G. H. Fleet. 1985. Growth of natural yeast flora during the fermentation of inoculated wines. Appl. Environ. Microbiol. 50:727-728. 6. Lafon-Lafourcade, S. 1983. Wine and brandy, p. 81-161. In G. Reed (ed.), Biotechnology, vol. 5. Verlag-Chemie, Heidelberg. 7. Longo, E., J. B. Velazquez, J. Cansado, P. Calo, and T. Villa. 1990. Role of killer effect in fermentation conducted by mixed cultures of Saccharomyces cerevisiae. FEMS Microbiol. Lett. 71:331-336. 8. Querol, A., and E. Barrio. 1990. A rapid and simple method for the preparation of yeast mitochondrial DNA. Nucleic Acids Res. 18:1657. 9. Querol, A., E. Barrio, and D. Ram6n. A comparative study of different methods of yeast strain characterization. Syst. Appl. Microbiol., in press. 10. Querol, A., T. Huerta, E. Barrio, and D. Ram6n. 1992. Strain for use as dry yeast in fermentation of Alicante wine: selection and DNA patterns. J. Food Sci. 57:183-185. 11. Querol, A., M. Jimenez, and T. Huerta. 1990. A study on microbiological and enological parameters during fermentation of must from poor and normal grapes harvested in the region of

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Alicante (Spain). J. Food Sci. 55:1603-1606. 12. Reed, G., and T. W. Nagodawithana. 1988. Technology of yeast usage in winemaking. Am. J. Enol. Vitic. 39:83-90. 13. Roziere, C., F. Raginel, C. Sanchez, and P. Strehaiano. 1989. Implantation de levures selectionnees. Etude en site industriel de vinification. Rev. Fr. Oenologie 119:37-41. 14. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 15. Snedecor, G., and W. Cochran. 1956. Statistical methods, 5th ed. Iowa State University Press, Ames. 16. Van Vuuren, H. J. J., and L. Van der Meer. 1987. Fingerprinting of yeast by protein electrophoresis. Am. J. Enol. Vitic. 38:4953. 17. Vezinhet, F. 1985. Le marquage genetique de souches de levures oenologiques. Rev. Fr. Oenologie 97:47-51. 18. Vezinhet, F., B. Blondin, and J. N. Hallet. 1990. Chromosomal DNA patterns and mitochondrial DNA polymorphism as tools for identification of enological strains of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 32:568-571.