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Direct profiling of the yeast dynamics in wine fermentations
FEMS Microbiology Letters 189 (2000) 81^87
www.fems-microbiology.org
Direct pro¢ling of the yeast dynamics in wine fermentations L. Cocolin 1 , L.F. Bisson, D.A. Mills * Department of Viticulture and Enology, University of California, Davis, One Shields Ave., Davis, CA 95616-8749, USA Received 17 February 2000; accepted 30 May 2000
Abstract We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml31 . PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Fermentation ; Denaturing gradient gel electrophoresis ; Microbial ecology ; Ribosomal RNA ; Wine
1. Introduction Wine fermentations are typically carried out by a complex evolution of microorganisms involving both yeasts and bacteria. Numerous studies have examined the succession of yeasts and bacteria that occurs during the fermentation of non-sterile musts [1^7]. In general, yeasts predominate during the alcoholic fermentation, where the low pH and nutritional content of the juice itself selects for yeast growth. A diverse population of yeasts including species of Kloeckera, Metschnikowia, Candida, Hanseniaspora and Saccharomyces are often present in the initial stages of most wine fermentations [8]. The non-Saccharomyces yeasts typically grow for several days before the fermentation is dominated by one or more Saccharomyces cerevisiae strains along with a concurrent increase in ethanol concentration [9]. The predominance of S. cerevisiae in this setting is a likely result of its high ethanol tolerance as compared with other yeasts present in the wine environment [8,10,11]. Non-Saccharomyces yeasts such as Kloeckera sp., Metschnikowia sp., Candida sp. and Hansenula sp.
* Corresponding author. Tel. : +1 (530) 754 7821; Fax: +1 (530) 752 0382; E-mail : [email protected] 1
Present address: Department of Food Science, University of Udine, 33100 Udine, Italy
have been shown to persist in wine fermentations, albeit at a lower level than S. cerevisiae strains [3,12,13]. To date, most studies on the microbial constituents of fermenting wine have employed classical microbiological methods of enrichment and isolation to di¡erentiate the various yeast and bacterial strains [8^10]. Upon isolation, the accumulated strains were characterized taxonomically using traditional microbiological methods [14] or molecular methods [15^19]. The necessity of enrichment and isolation for strain characterization, in addition to being time-consuming, is fraught with potential biases [20]. While this approach has proven useful, it often fails to characterize those microorganisms for which culturing is problematic or impossible [21]. Recent advances in the ¢eld of molecular microbial ecology have brought forth a variety of new tools to directly assess the microbial diversity present in natural habitats [22] without the need for microbial enrichment. One common strategy is to sample the DNA (or RNA) of a mixed microbial community and use it as a template for both the assessment of community structure and to reveal individual constituents [23]. Typically, these strategies examine key molecular loci, such as ribosomal RNAs (rRNAs) from which phylogenetic relationships to known microorganisms can be inferred [22]. This tactic has the advantage of eliminating the necessity for strain isolation, thereby negating the potential biases inherent in microbial enrichment. Indeed, studies which have employed such direct analysis have repeatedly demonstrated a tremendous
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variance between cultivated and naturally-occurring species, thereby dramatically altering perceptions on the true microbial diversity present in various habitats [24]. Recently, denaturing gradient gel electrophoresis (DGGE) has been employed to di¡erentiate rRNA genes directly puri¢ed from complex microbial communities [25]. To date, polymerase chain reaction (PCR)-DGGE has been employed primarily to examine bacterial diversity (or less frequently, fungal diversity) in various natural habitats [26]. In this report we evaluated PCR-DGGE for direct characterization of the yeast population dynamics inherent in laboratory wine fermentations. 2. Materials and methods 2.1. Yeast strains and growth conditions The yeast strains used in this study : S. cerevisiae (UCD#522), Metschnikowia pulcherrima (UCD#125), Candida ethanolica (UCD#7) and Kloeckera apiculata (UCD#1000) were provided from the Culture Collection of the Department of Viticulture and Enology, University of California, Davis, CA, USA. The strains were grown in YEPD broth (1% yeast extract, 2% bacteriological peptone, 2% glucose) at 30³C. 2.2. Laboratory fermentations Fermentations were run in 10-l bench-top fermenters (New Brunswick Scienti¢c Co., Edison, NJ, USA) at a temperature of 25³C. Fermentations were carried out in 6 l of Triple M medium [27], or Triple M medium containing half the normal concentration of `yeast nitrogen base w/o amino acids and ammonium sulfate' (YNB ; Difco Laboratories, Sparks, MD, USA). Triple M medium was employed to allow easy modulation of medium nutrients. Previously we observed alterations in yeast population dynamics by changes in the YNB component of Triple M medium (D.A. Mills, unpublished data). Yeast fermentations were run anaerobically. No attempt was made to control or monitor dissolved oxygen. One-ml aliquots from individual overnight cultures of S. cerevisiae, K. apiculata, C. ethanolica and M. pulcherrima, grown in Triple M medium, were mixed and used to inoculate the fermentations resulting in a ¢nal concentration of about 104 ^105 cells ml31 of each strain. During the fermentation, 40-ml samples were collected at least twice a day. Serial dilutions were made in a solution of 8% NaCl, 1% peptone, plated, in duplicate, on WL Nutrient agar (Difco Laboratories) and incubated at room temperature (V22³C) for 4^5 days. The di¡erent yeast strains were enumerated on the basis of the di¡erent color and colony morphology [28]. The fermentations were followed for about 16 days. Glucose, fructose and ethanol were assayed in duplicate using enzymatic analysis kits from Boehringer
Mannheim (Indianapolis, IN, USA) as described by the manufacturer. 2.3. DNA puri¢cation For the isolation of DNA from yeast controls and Triple M medium [27], DNA was puri¢ed using standard procedures [29] with the following modi¢cations. Depending on the broth cell density, 2^20 ml fermentation broth was centrifuged at 16 000Ug for 10 min at 4³C and the cell pellet (approximately 100 Wl volume) was resuspended in 200 Wl of breaking bu¡er (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA pH 8). The cells were homogenized in a bead beater instrument (Fast Prep1, Bio101, USA) with 0.3 g of glass beads (0.5 mm in diameter; BioSpec Products, Bartlesville, OK, USA) in the presence of 200 Wl phenol/chloroform/isoamyl alcohol (50:48:2). Two hundred Wl TE (10 mM Tris, 1 mM EDTA pH 7.6) was added and the bead/cell mixture was centrifuged for 10 min at 16 000Ug at 4³C after which the aqueous phase was collected. The DNA was precipitated with 2.5 volumes of 100% ethanol and centrifuged at 16 000Ug at 4³C for 10 min and the pellet washed with 70% ethanol, dried and resuspended in 50 Wl of sterile distilled water containing 2 IU RNase (Sigma, USA). The sample was then incubated at 37³C for 30 min before storage at 320³C. 2.4. DNA ampli¢cation and primers Approximately 250 nucleotides of the 5P- end region of the 26S rRNA gene was ampli¢ed by PCR using the primer NL1, 5P-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCC ATA TCA ATA AGC GGA GGA AAA G-3P (the GC clamp sequence is underlined) [30] and a reverse primer LS2, 5P-ATT CCC AAA CAA CTC GAC TC-3P (corresponding to nucleotide positions 266 to 285 on the S. cerevisiae 26S RNA gene (GenBank accession number M19229)). PCR was performed in a ¢nal volume of 50 Wl containing 10 mM Tris^HCl, 50 mM KCl, 1.5 mM MgCl2 , 0.2 mM each dATP, dCTP, dGTP and dTTP, 0.2 mM of the primers, 1.25 IU TaqDNA polymerase (Perkin Elmer, Cetus, USA) and 2 Wl of the extracted DNA (approximately 10 ng). The reactions were run for 30 cycles: denaturation was at 95³C for 60 s, annealing at 52³C for 45 s and extension at 72³C for 60 s. An initial 5 min denaturation at 95³C and a ¢nal 7 min extension at 72³C were used. Products were analyzed on 2% agarose gels containing 0.5 Wg ml31 ethidium bromide, visualized under UV light and photographed with the Multimage1 Light Cabinet (Alpha Innotech Corporation, San Leandro, CA, USA). 2.5. DGGE analysis The DCode1 Universal Mutation Detection System
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(Bio-Rad, Hercules, CA, USA) was used for sequence speci¢c separation of PCR products. PCR samples were applied directly onto 8% (w/v) polyacrylamide gels in a running bu¡er containing 40 mM Tris-acetate, 2 mM Na2 EDTAWH2 O, pH 8.5 (TAE) and a denaturing gradient from 30 to 60% of urea and formamide. The electrophoresis was performed at a constant voltage of 120 V for 4 h with a constant temperature of 60³C. After electrophoresis the gels were stained in 1.25UTAE containing SYBR Green (reconstituted according to the manufacturers directions ; Molecular Probes, Eugene, OR, USA) and photographed under UV transillumination. 3. Results 3.1. Di¡erentiation of wine yeast genera by PCR-DGGE Numerous studies have demonstrated the use of rRNA sequence analysis for di¡erentiating microbial species [31^ 33]. Recently a variety of yeast species were di¡erentiated by analysis of partial sequences of the large subunit rDNA [34]. These studies examined the 5P end of the large subunit rDNA encompassing the D1 and D2 expansion domains [35]. To amplify a portion of this region for the di¡erentiation of wine yeasts by DGGE, we used the previously designed NL-1 forward primer [30] and a new LS2 reverse primer (see Section 2). These primers amplify a product of approximately 250 bp covering most of the D1 expansion loop [36]. As indicated in Fig. 1, NL-1/ LS2 ampli¢cation products from a variety of wine-associated yeast genera can be easily di¡erentiated by DGGE however, they do not di¡erentiate di¡erent species of the same genera. 3.2. PCR-DGGE pro¢le of model fermentations To evaluate PCR-DGGE for pro¢ling the microbial successions inherent in wine fermentations, we examined mixed culture fermentations of model wine medium inoculated with four yeasts: S. cerevisiae, M. pulcherrima, C. ethanolica and K. apiculata. This mixture of microorganisms was chosen for several reasons. First, each of these genera is routinely isolated from the early stages of most wine fermentations [8,10]. Second, these four species are easily di¡erentiated on WL plating medium thereby facilitating plate counting of each strain from a mixed culture [28]. Third, NL-1/LS2 ampli¢cation products for all four species are readily separated by DGGE (Fig. 1). The initial fermentation employed standard Triple M medium (fermentation A). Plating results (Fig. 2A) indicated that populations of M. pulcherrima, C. ethanolica and K. apiculata increased dramatically within the ¢rst 50 h (106 to 107 cfu ml31 ) while the growth of S. cerevisiae lagged slightly behind reaching similar levels within 75 h. Populations of M. pulcherrima and K. apiculata began to
Fig. 1. DGGE pro¢les of ampli¢ed 26S rRNA D1 regions obtained from di¡erent wine-related yeast species. Lane 1, Brettanomyces spp. (UCD#734) ; lane 2, Torulaspora delbrueckii (ATCC #36030); lane 3, Hansenula saturnis (UCD#20); lane 4, Pichia anomala (UCD#646); lane 5, Candida vini (UCD#36) ; lane 6, Candida ethanolica (UCD#7); lane 7, Metschnikowia pulcherrima (UCD#125); lane 8, Kloeckera apiculata (UCD#1000) ; lane 9, Saccharomyces cerevisiae (UCD#522) ; lane 10, Saccharomyces cerevisiae (UCD#713); lane 11, Saccharomyces uvarum (UCD#169) ; lane 12, Zygosaccharomyces bailii (UCD#795) ; lane 13, Saccharomyces bayanus (UCD#89); lane 14, Saccharomyces bisporus (UCD#134). The bands common to all isolates (labeled ssDNA) are single stranded DNA artifacts that were not in£uenced di¡erentially by the gradient [46].
decline after approximately 100^130 h coinciding with the accumulation of ethanol at 15^22 g l31 . Neither M. pulcherrima nor K. apiculata could be observed on plating medium after 240 h. C. ethanolica was shown to reach the highest concentration (V107 cfu ml31 ) and persisted throughout the fermentation. The population of C. ethanolica only began to decrease with a coincident increase of S. cerevisiae near the end of the fermentation. Interestingly, the available glucose was not completely consumed within 400 h in this fermentation. As shown in Fig. 2B, the PCR-DGGE pro¢le of fermentation A accurately monitored the presence or absence of speci¢c strains throughout the fermentation. All four strains could be detected during the early stages of the fermentation. Moreover, as populations of M. pulcherrima and K. apiculata began to fall below 103 cfu ml31 , at approximately 240 h into the fermentation, the corresponding DGGE band disappeared (Fig. 2B). In general the DGGE bands of M. pulcherrima and K. apiculata persisted for several hours beyond our ability to detect viable cells of either strain on plating media. This could be due to the cells entering a viable non-culturable (VNC) state or could possibly re£ect the presence of M. pulcherrima and K. apiculata DNA in the fermentation as a result of cell lysis. Previous work in our laboratory indicated that elimina-
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Fig. 2. (A) Microbial growth pro¢les, glucose, fructose and ethanol contents of fermentation A (complete Triple M medium). Abbreviations : S.c., S. cerevisiae ; C.e., C. ethanolica ; K.a., K. apiculata; M.p., M. pulcherrima. (B) DGGE pro¢les of fermentation A. Lane labels indicate the time of fermentation sampling (h). Abbreviations : K.a., K. apiculata; S.c., S. cerevisiae ; M.p., M. pulcherrima; C.e., C. ethanolica ; ssDNA, single-stranded DNA. Band identity was determined on the basis of co-migration with NL1-LS2 ampli¢cation products from individual yeast controls (removed from ¢gure).
tion of the YNB component (Difco Laboratories) of Triple M medium resulted in a complete lack of growth of yeast strains (data not shown). This reagent contains a variety of vitamins, trace elements and salts that aid in the propagation of yeast [37]. In an e¡ort to pro¢le an altered microbial growth pattern, an additional fermentation was carried out using modi¢ed Triple M medium in which the content of the YNB component was included at one half the normal concentration (fermentation B). As indicated in Fig. 3A, the overall pattern of microbial growth in fermentation B is roughly similar to that in fermentation A. M. pulcherrima, C. ethanolica and K. apiculata grow rapidly to high cell densities (V107 cfu ml31 ) within 50 h and both M. pulcherrima and K. apiculata could not be detected on plating media as the ethanol concentration in the fermentation reached 20^30 g l31 . The pattern of microbial growth is di¡erent from fermentation A, however, in that the populations of M. pulcherrima, K. apiculata and C. ethanolica were extinguished more rapidly. M. pulcherrima and K. apiculata became undetectable approximately 50^100 h earlier (respectively) as compared to fermentation A. Moreover, C. ethanolica did not persist throughout 400 h of fermentation, becoming undetectable after V340 h. The likely cause for the
early exit of M. pulcherrima, C. ethanolica and K. apiculata is the more vigorous ethanol production (most likely produced from S. cerevisiae) in fermentation B. Surprisingly, glucose was completely consumed within 400 h in fermentation B as compared to fermentation A. One possible rationale for this enhanced conversion by S. cerevisiae is the more rapid disappearance of M. pulcherrima, C. ethanolica and K. apiculata thereby reducing competition for limiting nutrients and/or reducing the production of inhibitory compounds by these yeasts. As seen in Fig. 3B, the DGGE pro¢le clearly monitored the altered microbial population changes that took place in fermentation B. DGGE bands corresponding to K. apiculata and M. pulcherrima disappeared after 138 and 174 h (respectively), coinciding with the inability to observe these strains on plating media (Fig. 3A). The disappearance of C. ethanolica is also clearly represented by the lack of a cognate band after 333 h of fermentation. 4. Discussion Wine fermentations represent an amalgam of microbiological activities derived from both starter and non-starter
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Fig. 3. (A) Microbial growth pro¢les, glucose, fructose and ethanol contents of fermentation B (Triple M medium with half strength YNB). Abbreviations: S.c., S. cerevisiae ; C.e., C. ethanolica; K.a., K. apiculata; M.p., M. pulcherrima. (B) DGGE pro¢les of fermentation B. Lane labels indicate the time of fermentation sampling (h). Abbreviations: see legend to Fig. 2B. Band identity was determined on the basis of co-migration with NL1-LS2 ampli¢cation products from individual yeast controls (removed from ¢gure).
microorganisms [9,10]. The growth of non-starter (or indigenous) microorganisms can signi¢cantly impact the ef¢cacy of starter cultures and even inhibit normal starter function [38]. All previous attempts to characterize the microbial diversity in wine fermentations have employed standard methods of enrichment and isolation to cultivate various microbial constituents before taxonomic identi¢cation [8^10,12]. This estimate of microbial diversity is often problematic, however, since many microorganisms may not grow on standard laboratory media. Moreover, microorganisms may enter a VNC state that may be overlooked in standard plating schemes [22,39]. In this work we demonstrate that PCR-DGGE is a viable alternative to standard plating methods for a qualitative assessment of the microbial constituents in model wine fermentations. PCR-DGGE was shown to be quite sensitive, detecting subtle di¡erences in yeast £ora development and persistence in two fermentations done under slightly di¡erent conditions. In each case there was excellent agreement between the plating data and the presence or absence of a cognate DGGE band. Given the complex nature of multitemplate PCR, the quantitation of DGGE pro¢les is problematic [40^43]. Other groups have attempted to quantitate cell populations by comparing DGGE band intensity to internal stan-
dards [44,45]. While no attempt was made to quantitate individual yeast populations in this work, DGGE bands were observed for populations as low as 103 cells ml31 . Therefore, the presence of a DGGE band represents a yeast population above a minimum threshold value of 103 cells ml31 and thus identi¢es only the predominant yeast populations in these fermentations. Since PCR-DGGE can be performed in a reasonably rapid fashion (1 day) and with minimal sample volume, this method may be useful for characterizing wine microbial diversity in situations where plating may be impractical, such as in studies involving a large number of fermentation replicates. Indeed, a modi¢ed version of the method presented here has been used to obtain discriminatory pro¢les of yeast populations in several commercial white wine fermentations (D.A. Mills, unpublished results). The use of PCR-DGGE should enable enologists to gain a better view of the microbial diversity present in a range of fermentations without the necessity for problematic plating analysis. Acknowledgements This work was supported by grants from the American
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Vineyard Foundation (L.F.B., D.A.M.), the California Competitive Grants Program for Research in Viticulture and Enology (L.F.B., D.A.M.) and the UC-Davis New Faculty Research Program (D.A.M.).
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Direct pro¢ling of the yeast dynamics in wine fermentations L. Cocolin 1 , L.F. Bisson, D.A. Mills * Department of Viticulture and Enology, University of California, Davis, One Shields Ave., Davis, CA 95616-8749, USA Received 17 February 2000; accepted 30 May 2000
Abstract We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml31 . PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Fermentation ; Denaturing gradient gel electrophoresis ; Microbial ecology ; Ribosomal RNA ; Wine
1. Introduction Wine fermentations are typically carried out by a complex evolution of microorganisms involving both yeasts and bacteria. Numerous studies have examined the succession of yeasts and bacteria that occurs during the fermentation of non-sterile musts [1^7]. In general, yeasts predominate during the alcoholic fermentation, where the low pH and nutritional content of the juice itself selects for yeast growth. A diverse population of yeasts including species of Kloeckera, Metschnikowia, Candida, Hanseniaspora and Saccharomyces are often present in the initial stages of most wine fermentations [8]. The non-Saccharomyces yeasts typically grow for several days before the fermentation is dominated by one or more Saccharomyces cerevisiae strains along with a concurrent increase in ethanol concentration [9]. The predominance of S. cerevisiae in this setting is a likely result of its high ethanol tolerance as compared with other yeasts present in the wine environment [8,10,11]. Non-Saccharomyces yeasts such as Kloeckera sp., Metschnikowia sp., Candida sp. and Hansenula sp.
* Corresponding author. Tel. : +1 (530) 754 7821; Fax: +1 (530) 752 0382; E-mail : [email protected] 1
Present address: Department of Food Science, University of Udine, 33100 Udine, Italy
have been shown to persist in wine fermentations, albeit at a lower level than S. cerevisiae strains [3,12,13]. To date, most studies on the microbial constituents of fermenting wine have employed classical microbiological methods of enrichment and isolation to di¡erentiate the various yeast and bacterial strains [8^10]. Upon isolation, the accumulated strains were characterized taxonomically using traditional microbiological methods [14] or molecular methods [15^19]. The necessity of enrichment and isolation for strain characterization, in addition to being time-consuming, is fraught with potential biases [20]. While this approach has proven useful, it often fails to characterize those microorganisms for which culturing is problematic or impossible [21]. Recent advances in the ¢eld of molecular microbial ecology have brought forth a variety of new tools to directly assess the microbial diversity present in natural habitats [22] without the need for microbial enrichment. One common strategy is to sample the DNA (or RNA) of a mixed microbial community and use it as a template for both the assessment of community structure and to reveal individual constituents [23]. Typically, these strategies examine key molecular loci, such as ribosomal RNAs (rRNAs) from which phylogenetic relationships to known microorganisms can be inferred [22]. This tactic has the advantage of eliminating the necessity for strain isolation, thereby negating the potential biases inherent in microbial enrichment. Indeed, studies which have employed such direct analysis have repeatedly demonstrated a tremendous
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variance between cultivated and naturally-occurring species, thereby dramatically altering perceptions on the true microbial diversity present in various habitats [24]. Recently, denaturing gradient gel electrophoresis (DGGE) has been employed to di¡erentiate rRNA genes directly puri¢ed from complex microbial communities [25]. To date, polymerase chain reaction (PCR)-DGGE has been employed primarily to examine bacterial diversity (or less frequently, fungal diversity) in various natural habitats [26]. In this report we evaluated PCR-DGGE for direct characterization of the yeast population dynamics inherent in laboratory wine fermentations. 2. Materials and methods 2.1. Yeast strains and growth conditions The yeast strains used in this study : S. cerevisiae (UCD#522), Metschnikowia pulcherrima (UCD#125), Candida ethanolica (UCD#7) and Kloeckera apiculata (UCD#1000) were provided from the Culture Collection of the Department of Viticulture and Enology, University of California, Davis, CA, USA. The strains were grown in YEPD broth (1% yeast extract, 2% bacteriological peptone, 2% glucose) at 30³C. 2.2. Laboratory fermentations Fermentations were run in 10-l bench-top fermenters (New Brunswick Scienti¢c Co., Edison, NJ, USA) at a temperature of 25³C. Fermentations were carried out in 6 l of Triple M medium [27], or Triple M medium containing half the normal concentration of `yeast nitrogen base w/o amino acids and ammonium sulfate' (YNB ; Difco Laboratories, Sparks, MD, USA). Triple M medium was employed to allow easy modulation of medium nutrients. Previously we observed alterations in yeast population dynamics by changes in the YNB component of Triple M medium (D.A. Mills, unpublished data). Yeast fermentations were run anaerobically. No attempt was made to control or monitor dissolved oxygen. One-ml aliquots from individual overnight cultures of S. cerevisiae, K. apiculata, C. ethanolica and M. pulcherrima, grown in Triple M medium, were mixed and used to inoculate the fermentations resulting in a ¢nal concentration of about 104 ^105 cells ml31 of each strain. During the fermentation, 40-ml samples were collected at least twice a day. Serial dilutions were made in a solution of 8% NaCl, 1% peptone, plated, in duplicate, on WL Nutrient agar (Difco Laboratories) and incubated at room temperature (V22³C) for 4^5 days. The di¡erent yeast strains were enumerated on the basis of the di¡erent color and colony morphology [28]. The fermentations were followed for about 16 days. Glucose, fructose and ethanol were assayed in duplicate using enzymatic analysis kits from Boehringer
Mannheim (Indianapolis, IN, USA) as described by the manufacturer. 2.3. DNA puri¢cation For the isolation of DNA from yeast controls and Triple M medium [27], DNA was puri¢ed using standard procedures [29] with the following modi¢cations. Depending on the broth cell density, 2^20 ml fermentation broth was centrifuged at 16 000Ug for 10 min at 4³C and the cell pellet (approximately 100 Wl volume) was resuspended in 200 Wl of breaking bu¡er (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA pH 8). The cells were homogenized in a bead beater instrument (Fast Prep1, Bio101, USA) with 0.3 g of glass beads (0.5 mm in diameter; BioSpec Products, Bartlesville, OK, USA) in the presence of 200 Wl phenol/chloroform/isoamyl alcohol (50:48:2). Two hundred Wl TE (10 mM Tris, 1 mM EDTA pH 7.6) was added and the bead/cell mixture was centrifuged for 10 min at 16 000Ug at 4³C after which the aqueous phase was collected. The DNA was precipitated with 2.5 volumes of 100% ethanol and centrifuged at 16 000Ug at 4³C for 10 min and the pellet washed with 70% ethanol, dried and resuspended in 50 Wl of sterile distilled water containing 2 IU RNase (Sigma, USA). The sample was then incubated at 37³C for 30 min before storage at 320³C. 2.4. DNA ampli¢cation and primers Approximately 250 nucleotides of the 5P- end region of the 26S rRNA gene was ampli¢ed by PCR using the primer NL1, 5P-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCC ATA TCA ATA AGC GGA GGA AAA G-3P (the GC clamp sequence is underlined) [30] and a reverse primer LS2, 5P-ATT CCC AAA CAA CTC GAC TC-3P (corresponding to nucleotide positions 266 to 285 on the S. cerevisiae 26S RNA gene (GenBank accession number M19229)). PCR was performed in a ¢nal volume of 50 Wl containing 10 mM Tris^HCl, 50 mM KCl, 1.5 mM MgCl2 , 0.2 mM each dATP, dCTP, dGTP and dTTP, 0.2 mM of the primers, 1.25 IU TaqDNA polymerase (Perkin Elmer, Cetus, USA) and 2 Wl of the extracted DNA (approximately 10 ng). The reactions were run for 30 cycles: denaturation was at 95³C for 60 s, annealing at 52³C for 45 s and extension at 72³C for 60 s. An initial 5 min denaturation at 95³C and a ¢nal 7 min extension at 72³C were used. Products were analyzed on 2% agarose gels containing 0.5 Wg ml31 ethidium bromide, visualized under UV light and photographed with the Multimage1 Light Cabinet (Alpha Innotech Corporation, San Leandro, CA, USA). 2.5. DGGE analysis The DCode1 Universal Mutation Detection System
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(Bio-Rad, Hercules, CA, USA) was used for sequence speci¢c separation of PCR products. PCR samples were applied directly onto 8% (w/v) polyacrylamide gels in a running bu¡er containing 40 mM Tris-acetate, 2 mM Na2 EDTAWH2 O, pH 8.5 (TAE) and a denaturing gradient from 30 to 60% of urea and formamide. The electrophoresis was performed at a constant voltage of 120 V for 4 h with a constant temperature of 60³C. After electrophoresis the gels were stained in 1.25UTAE containing SYBR Green (reconstituted according to the manufacturers directions ; Molecular Probes, Eugene, OR, USA) and photographed under UV transillumination. 3. Results 3.1. Di¡erentiation of wine yeast genera by PCR-DGGE Numerous studies have demonstrated the use of rRNA sequence analysis for di¡erentiating microbial species [31^ 33]. Recently a variety of yeast species were di¡erentiated by analysis of partial sequences of the large subunit rDNA [34]. These studies examined the 5P end of the large subunit rDNA encompassing the D1 and D2 expansion domains [35]. To amplify a portion of this region for the di¡erentiation of wine yeasts by DGGE, we used the previously designed NL-1 forward primer [30] and a new LS2 reverse primer (see Section 2). These primers amplify a product of approximately 250 bp covering most of the D1 expansion loop [36]. As indicated in Fig. 1, NL-1/ LS2 ampli¢cation products from a variety of wine-associated yeast genera can be easily di¡erentiated by DGGE however, they do not di¡erentiate di¡erent species of the same genera. 3.2. PCR-DGGE pro¢le of model fermentations To evaluate PCR-DGGE for pro¢ling the microbial successions inherent in wine fermentations, we examined mixed culture fermentations of model wine medium inoculated with four yeasts: S. cerevisiae, M. pulcherrima, C. ethanolica and K. apiculata. This mixture of microorganisms was chosen for several reasons. First, each of these genera is routinely isolated from the early stages of most wine fermentations [8,10]. Second, these four species are easily di¡erentiated on WL plating medium thereby facilitating plate counting of each strain from a mixed culture [28]. Third, NL-1/LS2 ampli¢cation products for all four species are readily separated by DGGE (Fig. 1). The initial fermentation employed standard Triple M medium (fermentation A). Plating results (Fig. 2A) indicated that populations of M. pulcherrima, C. ethanolica and K. apiculata increased dramatically within the ¢rst 50 h (106 to 107 cfu ml31 ) while the growth of S. cerevisiae lagged slightly behind reaching similar levels within 75 h. Populations of M. pulcherrima and K. apiculata began to
Fig. 1. DGGE pro¢les of ampli¢ed 26S rRNA D1 regions obtained from di¡erent wine-related yeast species. Lane 1, Brettanomyces spp. (UCD#734) ; lane 2, Torulaspora delbrueckii (ATCC #36030); lane 3, Hansenula saturnis (UCD#20); lane 4, Pichia anomala (UCD#646); lane 5, Candida vini (UCD#36) ; lane 6, Candida ethanolica (UCD#7); lane 7, Metschnikowia pulcherrima (UCD#125); lane 8, Kloeckera apiculata (UCD#1000) ; lane 9, Saccharomyces cerevisiae (UCD#522) ; lane 10, Saccharomyces cerevisiae (UCD#713); lane 11, Saccharomyces uvarum (UCD#169) ; lane 12, Zygosaccharomyces bailii (UCD#795) ; lane 13, Saccharomyces bayanus (UCD#89); lane 14, Saccharomyces bisporus (UCD#134). The bands common to all isolates (labeled ssDNA) are single stranded DNA artifacts that were not in£uenced di¡erentially by the gradient [46].
decline after approximately 100^130 h coinciding with the accumulation of ethanol at 15^22 g l31 . Neither M. pulcherrima nor K. apiculata could be observed on plating medium after 240 h. C. ethanolica was shown to reach the highest concentration (V107 cfu ml31 ) and persisted throughout the fermentation. The population of C. ethanolica only began to decrease with a coincident increase of S. cerevisiae near the end of the fermentation. Interestingly, the available glucose was not completely consumed within 400 h in this fermentation. As shown in Fig. 2B, the PCR-DGGE pro¢le of fermentation A accurately monitored the presence or absence of speci¢c strains throughout the fermentation. All four strains could be detected during the early stages of the fermentation. Moreover, as populations of M. pulcherrima and K. apiculata began to fall below 103 cfu ml31 , at approximately 240 h into the fermentation, the corresponding DGGE band disappeared (Fig. 2B). In general the DGGE bands of M. pulcherrima and K. apiculata persisted for several hours beyond our ability to detect viable cells of either strain on plating media. This could be due to the cells entering a viable non-culturable (VNC) state or could possibly re£ect the presence of M. pulcherrima and K. apiculata DNA in the fermentation as a result of cell lysis. Previous work in our laboratory indicated that elimina-
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Fig. 2. (A) Microbial growth pro¢les, glucose, fructose and ethanol contents of fermentation A (complete Triple M medium). Abbreviations : S.c., S. cerevisiae ; C.e., C. ethanolica ; K.a., K. apiculata; M.p., M. pulcherrima. (B) DGGE pro¢les of fermentation A. Lane labels indicate the time of fermentation sampling (h). Abbreviations : K.a., K. apiculata; S.c., S. cerevisiae ; M.p., M. pulcherrima; C.e., C. ethanolica ; ssDNA, single-stranded DNA. Band identity was determined on the basis of co-migration with NL1-LS2 ampli¢cation products from individual yeast controls (removed from ¢gure).
tion of the YNB component (Difco Laboratories) of Triple M medium resulted in a complete lack of growth of yeast strains (data not shown). This reagent contains a variety of vitamins, trace elements and salts that aid in the propagation of yeast [37]. In an e¡ort to pro¢le an altered microbial growth pattern, an additional fermentation was carried out using modi¢ed Triple M medium in which the content of the YNB component was included at one half the normal concentration (fermentation B). As indicated in Fig. 3A, the overall pattern of microbial growth in fermentation B is roughly similar to that in fermentation A. M. pulcherrima, C. ethanolica and K. apiculata grow rapidly to high cell densities (V107 cfu ml31 ) within 50 h and both M. pulcherrima and K. apiculata could not be detected on plating media as the ethanol concentration in the fermentation reached 20^30 g l31 . The pattern of microbial growth is di¡erent from fermentation A, however, in that the populations of M. pulcherrima, K. apiculata and C. ethanolica were extinguished more rapidly. M. pulcherrima and K. apiculata became undetectable approximately 50^100 h earlier (respectively) as compared to fermentation A. Moreover, C. ethanolica did not persist throughout 400 h of fermentation, becoming undetectable after V340 h. The likely cause for the
early exit of M. pulcherrima, C. ethanolica and K. apiculata is the more vigorous ethanol production (most likely produced from S. cerevisiae) in fermentation B. Surprisingly, glucose was completely consumed within 400 h in fermentation B as compared to fermentation A. One possible rationale for this enhanced conversion by S. cerevisiae is the more rapid disappearance of M. pulcherrima, C. ethanolica and K. apiculata thereby reducing competition for limiting nutrients and/or reducing the production of inhibitory compounds by these yeasts. As seen in Fig. 3B, the DGGE pro¢le clearly monitored the altered microbial population changes that took place in fermentation B. DGGE bands corresponding to K. apiculata and M. pulcherrima disappeared after 138 and 174 h (respectively), coinciding with the inability to observe these strains on plating media (Fig. 3A). The disappearance of C. ethanolica is also clearly represented by the lack of a cognate band after 333 h of fermentation. 4. Discussion Wine fermentations represent an amalgam of microbiological activities derived from both starter and non-starter
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Fig. 3. (A) Microbial growth pro¢les, glucose, fructose and ethanol contents of fermentation B (Triple M medium with half strength YNB). Abbreviations: S.c., S. cerevisiae ; C.e., C. ethanolica; K.a., K. apiculata; M.p., M. pulcherrima. (B) DGGE pro¢les of fermentation B. Lane labels indicate the time of fermentation sampling (h). Abbreviations: see legend to Fig. 2B. Band identity was determined on the basis of co-migration with NL1-LS2 ampli¢cation products from individual yeast controls (removed from ¢gure).
microorganisms [9,10]. The growth of non-starter (or indigenous) microorganisms can signi¢cantly impact the ef¢cacy of starter cultures and even inhibit normal starter function [38]. All previous attempts to characterize the microbial diversity in wine fermentations have employed standard methods of enrichment and isolation to cultivate various microbial constituents before taxonomic identi¢cation [8^10,12]. This estimate of microbial diversity is often problematic, however, since many microorganisms may not grow on standard laboratory media. Moreover, microorganisms may enter a VNC state that may be overlooked in standard plating schemes [22,39]. In this work we demonstrate that PCR-DGGE is a viable alternative to standard plating methods for a qualitative assessment of the microbial constituents in model wine fermentations. PCR-DGGE was shown to be quite sensitive, detecting subtle di¡erences in yeast £ora development and persistence in two fermentations done under slightly di¡erent conditions. In each case there was excellent agreement between the plating data and the presence or absence of a cognate DGGE band. Given the complex nature of multitemplate PCR, the quantitation of DGGE pro¢les is problematic [40^43]. Other groups have attempted to quantitate cell populations by comparing DGGE band intensity to internal stan-
dards [44,45]. While no attempt was made to quantitate individual yeast populations in this work, DGGE bands were observed for populations as low as 103 cells ml31 . Therefore, the presence of a DGGE band represents a yeast population above a minimum threshold value of 103 cells ml31 and thus identi¢es only the predominant yeast populations in these fermentations. Since PCR-DGGE can be performed in a reasonably rapid fashion (1 day) and with minimal sample volume, this method may be useful for characterizing wine microbial diversity in situations where plating may be impractical, such as in studies involving a large number of fermentation replicates. Indeed, a modi¢ed version of the method presented here has been used to obtain discriminatory pro¢les of yeast populations in several commercial white wine fermentations (D.A. Mills, unpublished results). The use of PCR-DGGE should enable enologists to gain a better view of the microbial diversity present in a range of fermentations without the necessity for problematic plating analysis. Acknowledgements This work was supported by grants from the American
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Vineyard Foundation (L.F.B., D.A.M.), the California Competitive Grants Program for Research in Viticulture and Enology (L.F.B., D.A.M.) and the UC-Davis New Faculty Research Program (D.A.M.).
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