The population of wine yeasts during spontaneous must fermentation was characterized by direct 5.8S-ITS rDNA region amplification without previous plate isolation or enrichment. RFLP analysis was applied to each of the amplification products detected, and the corresponding yeast identifications were made. The method provides a fast and direct way of determining yeast population present during wine fermentation.Populacija vinskih kvasaca tijekom spontane fermentacije mošta karakterizirana je izravnom amplifikacijom regije 5,8S-ITS rDNA bez prethodne izolacije na ploči ili povećavanja količine. Primijenjena je RFLP analiza na svakom utvr|enom amplifikacijskom produktu te je provedena odgovarajuća identifikacija kvasca. Postupak omogućava brz i izravan put utvrđivanja populacije kvasca prisutnog u fermentaciji vina

Ana Briones

Juan Carlos Espinosa

Juan Ubeda

Monica Fernandez-Gonzalez

English

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UDC 57.083.18:663.12 scientific noteISSN 1330-9862(FTB-1135)Identification of Wine Yeasts by PCR-RFLPwithout Previous Isolation on PlateJuan Carlos Espinosa, Monica Fernandez-Gonzalez, Juan Ubeda and Ana Briones*Departamento de Química Analítica y Tecnología de Alimentos, Facultad de Ciencias Químicas,Universidad de Castilla-La Mancha, Avda. Camilo José Cela s/n., E-13071 Ciudad Real, SpainReceived: February 5, 2002Accepted: April 10, 2002SummaryThe population of wine yeasts during spontaneous must fermentation was characteri-zed by direct 5.8S-ITS rDNA region amplification without previous plate isolation or en-richment. RFLP analysis was applied to each of the amplification products detected, andthe corresponding yeast identifications were made. The method provides a fast and directway of determining yeast population present during wine fermentation.Key words: wine yeasts, identification, PCR-RFLPIntroductionSubstantial growth of non-Saccharomyces yeaststakes place in the early stages of fermentation and maycontribute to some special characteristics of wine (1,2).The yeasts of the genera Kloeckera, Hanseniaspora, Can-dida, Pichia, and others all grow during the early stagesof fermentation. Afterwards, as ethanol concentrationsrise, Saccharomyces cerevisiae begins to predominate andis responsible for alcoholic fermentation. Identificationof the yeasts involved in fermentation process requiresplate isolation, enrichment and cultivation of differentstrains. Nevertheless, in the last few years new methodshave been developed to characterize wine yeasts di-rectly without the need for enrichment or isolation (3,4).The strains at the species level can be identified us-ing »conventional methods« based on biochemical andphysiological characteristics (5) or molecular techniques(6). PCR-RFLP has been used to characterize wine yeastspresent in the fermentation process after isolation of theyeasts (7).In this study we identified the yeast species presentin a sample collected from an industrial wine fermenta-tion vat using PCR amplification and RFLP analysis ofthe DNA purified directly from the sample, withoutplate isolation.Materials and MethodsSamplingThe sample studied was withdrawn from a singlevat of white must (capacity: 2 105 L) at an early stageof fermentation. Commercial dry yeasts had never beenused before as starters at the wine cellar located in theLa Mancha region in Spain.A volume of 100 mL of must was aseptically takenfrom the fermentation vat and transported to the labora-tory in a sterile vessel with glycerol (15 % v/v). Thesample was frozen and stored at –40 °C until analysis.After thawing and homogenization of the mustsample, a 12 mL aliquot was centrifuged (4 500 rpm,Hettich model 1617 rotor, 4 °C, 10 min), and the sedi-ment was washed carefully three times in sterile waterand then further three times in TE buffer (Tris HCl 10mM, EDTA 1 mM, pH=8) to yield a final volume of 12157J. C. ESPINOSA et al.: Identification of Wine Yeasts by PCR-RFLP, Food Technol. Biotechnol. 40 (2) 157–160 (2002)* Corresponding author; Fax: ++34 92 62 95 318; E-mail: abriones@qata-cr.uclm.esmL. The washed cells were used for genomic DNA iso-lation as described by Rose et al. (8). Both the sterile wa-ter and the TE buffer were chilled to 4 °C. 1 ng ofgenomic DNA was employed as the optimal amount forthe amplification reactions.PCRThe Internal Transcribed Spacers (ITS1 and ITS4)described by White et al. (9), which amplify the ITS-5.8Sregion on the rDNA region, were used as primers(MGW, Biotech, Germany). The amplification reactionwas performed in a GeneAmp model 2400 PCR System(Perkin-Elmer Corp., Norwalk, Connecticut, USA) underthe following conditions: a 50 L reaction mixture wasmade up of 1.25 U AmpliTaq Gold DNA polymerase(Applied Biosystems Division, Foster City, CA, USA),0.5 M of each of the primers, ITS1 (19-mer 5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (21-mer 5’-TCCTCCGCTTTATTGATATGC-3’), 0.2 mM of each dNTP, 1 AmpliTaq Gold buffer II, 2.5 mM MgCl2 and 1 ng oftemplate DNA. The mixture was put through an initialdenaturing cycle of 10 min at 95 °C, which was followedby 40 cycles comprising 1 min at 95 °C, 2 min at 58 °C,and 3 min at 72 °C, with a final extension step of 10 minat 72 °C (10).The amplification products were separated by elec-trophoresis in a 1.5 % (w/v) agarose gel with 1 TAEusing a 100 base pairs (bp) ladder (Biotools, Madrid,Spain) DNA molecular marker.The different amplification products were purifiedby isolating each band from the agarose gel using theGENECLEAN II KitTM (Bio 101, Carlsbad, CA, USA) ac-cording to the manufacturer’s instructions.Restriction analysisTo identify the yeasts, the amplified products weretreated with two restriction enzymes (HinfI and HaeIII).In some cases the use of DdeI was necessary to discrimi-nate both species. Reaction conditions were created ac-cording to the supplier's (Roche Diagnostics, S.L., Barce-lona, Spain) instructions. The restriction fragments werechecked by electrophoresis in 1.5 % w/v agarose gelplus ethidium bromide (0.5 g/mL).Results and DiscussionDNA from the sample was obtained and amplifiedas indicated in the Materials and Methods section. Sixdifferent amplification products with lengths between400 and 900 bp were discriminated on agarose gel elec-trophoresis (Fig. 1). Each band was extracted from thegel and carefully isolated using GENECLEAN II TM forfurther analysis. The isolation was verified and an en-hanced determination of amplification product size wascarried out (Fig. 2). Afterwards, each of the amplifica-tion products underwent restriction analysis using theenzymes HinfI and HaeIII separately (Fig. 3).The size of the different restriction fragments ob-tained from the six amplification products is shown inTable 1. The identification of the yeasts was achieved bycomparing the patterns obtained in this study withthose described by Esteve-Zarzoso et al. (11). Hansenia-158 J. C. ESPINOSA et al.: Identification of Wine Yeasts by PCR-RFLP, Food Technol. Biotechnol. 40 (2) 157–160 (2002)Fig. 1. Agarose gel with PCR products of an amplification ofthe 5.8S-ITS rDNA obtained without plate isolating. Lanes M:Marker 100 bp (1031, 900, 800, 700, 600, 500, 400, 300, 200, 100, 80).Lane S: Sample of the mustFig. 2. Agarose gel with PCR products of an amplification ofthe 5.8S-ITS rDNA obtained after treatment with GeneCleanII. Lanes M: Marker 100 bp (1031, 900, 800, 700, 600, 500, 400, 300,200, 100, 80), Lanes 1–6: Different amplification products (Lane 1:S.cerevisiae, Lane 2: H. uvarum, Lane 3: K. thermotolerans, Lane 4:Not identified, Lane 5: C. vinaria, Lane 6: C. stellata)spora uvarum was differentiated from H. guilliermondiiwith the use of DdeI. On the other hand one strain couldnot be identified (Lanes 10–12 in Fig. 3).As described in this paper, PCR amplification of5.8S-ITS rDNA carefully obtained from fermenting mustsamples may be a good method for fast identification ofthe yeasts involved in wine fermentation, yielding infor-mation without need of cultivation or time-consumingplate isolation methods.ConclusionThe method proposed is effective in characterizingthe yeast population and detecting potentially undesir-able yeasts, such as spoilage yeasts present in earlystages of wine fermentation, and can be completed injust a few hours, PCR amplification being the mosttime-consuming step. Nevertheless further studies arenecessary to evaluate the reproducibility of the method.AcknowledegmentsThis research was supported by the Ministerio deCiencia y Tecnología (INIA project RM01-003)References1. M. Ciani, G. Picciotti, Biotechnol. Lett. 17 (1995) 1247– 1250.2. P. Romano, G. Suzzi, P. Domizio, F. Fatichenti, Antonie vanLeeuwenhoeck, 71 (1997) 239–242.3. L. Cocolin, L. F. Bisson, D. A. Mills, FEMS Microbiol. Lett.189 (2000) 81–87.4. M. Fernández-González. J. C. Espinosa, J. F. Úbeda, A. I.Briones, Syst. Appl. Microbiol. 24 (2001) 634–638.159J. C. ESPINOSA et al.: Identification of Wine Yeasts by PCR-RFLP, Food Technol. Biotechnol. 40 (2) 157–160 (2002)M M1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Fig. 3. Agarose gel with PCR products from amplification of the 5.8S-ITS region of rDNA from six different species (Lanes1,4,7,10,13,16). Digestion products of the amplificates obtained using the restriction enzymes HinfI (Lanes 2,5,8,11,14,17) and HaeIII (La-nes 3,6,9,12,15,18). Lanes M: Marker 100 bp (1031, 900, 800, 700, 600, 500, 400, 300, 200, 100, 80) (Lanes 1–3: Saccharomyces cerevisiae; Lanes4–6: H. uvarum, Lanes 7–9: K. thermotolerans, Lanes 10–12: Not identified, Lanes 13–15: C. vinaria, Lanes 16–18: C. stellata)Table 1. Summary of the molecular profiles and their possible identification. Size (bp) of the amplifica-tion products (AP) and of the corresponding restriction fragmentsLane APRestriction fragmentsYeast identificationHaeIII HinfI1 880 320+230+180+150 365+365+155 Saccharomyces cerevisiae2 750 750 350+200+180 *Hanseniaspora uvarum3 700 310+210+95+95 350+345 Kluyveromyces thermotolerans4 650 330+140+85 360+340 Unidentified5 560 460+100 250+200+100 Candida vinaria6 475 475 240+235 Candida stelliata*Hanseniaspora uvarum was differentiated from H. guilliermondii with the use of DdeI(300, 180, 95, 90, 85 bp)5. J. A. Barnett, R. W. Payne, D. Yarrow: Yeast: Characteristicsand Identification, 3rd ed., Cambridge University Press,Cambridge (2000).6. A. Querol, E. Barrio, T. Huerta, D. Ramón, Appl. Environ.Microbiol. 58 (1992) 2948–2953.7. D. Granchi, M. Bosco, A. Messini, M. Vincenzini, J. Appl.Microbiol. 87 (1999) 949–956.8. M. D. Rose, F. Winston, P. Hieter: Methods in Yeast Gene-tics: A Laboratory Course Manual, Cold Spring Harbor Labo-ratory Press, New York (1990) p. 198.9. T. J. White, T. Bruns, S. Lee, J. Taylor: Amplification anddirect sequencing of fungal ribosomal RNA genes forphylogenetics. In: PCR Protocols: A Guide to Methods andApplications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J.White (Eds.), Academic Press, London (1990) pp. 315–322.10. M. Fernández, J. Úbeda, A. Briones, Int. J. Food Microbiol.59 (2000) 29–36.11. B. Esteve-Zarzoso, C. Belloch, F. Uruburu, A. Querol, Int.J. Syst. Bacteriol. 49 (1999) 329–337.Identifikacija vinskih kvasaca postupkom PCR-RFLPbez prethodne izolacije na plo~iSa`etakPopulacija vinskih kvasaca tijekom spontane fermentacije mo{ta karakterizirana je iz-ravnom amplifikacijom regije 5,8S-ITS rDNA bez prethodne izolacije na plo~i ili pove}ava-nja koli~ine. Primijenjena je RFLP analiza na svakom utvr|enom amplifikacijskom produk-tu te je provedena odgovaraju}a identifikacija kvasca. Postupak omogu}ava brz i izravanput utvr|ivanja populacije kvasca prisutnog u fermentaciji vina.160 J. C. ESPINOSA et al.: Identification of Wine Yeasts by PCR-RFLP, Food Technol. Biotechnol. 40 (2) 157–160 (2002)

Identifikacija vinskih kvasaca postupkom PCR-RFLP bez prethodne izolacije na ploči

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