Use of non-conventional yeast improves the wine aroma profile of Ribolla Gialla

  • Sofia DashkoEmail author
  • Nerve Zhou
  • Tinkara Tinta
  • Paolo Sivilotti
  • Melita Sternad Lemut
  • Kajetan Trost
  • Amparo Gamero
  • Teun Boekhout
  • Lorena Butinar
  • Urska Vrhovsek
  • Jure Piskur
Fermentation, Cell Culture and Bioengineering


Consumer wine preferences are changing rapidly towards exotic flavours and tastes. In this work, we tested five non-conventional yeast strains for their potential to improve Ribolla Gialla wine quality. These strains were previously selected from numerous yeasts interesting as food production candidates. Sequential fermentation of Ribolla Gialla grape juice with the addition of the Saccharomyces cerevisiae T73 Lalvin industrial strain was performed. Zygosaccharomyces kombuchaensis CBS8849 and Kazachstania gamospora CBS10400 demonstrated positive organoleptic properties and suitable fermentation dynamics, rapid sugar consumption and industrial strain compatibility. At the same time, Torulaspora microellipsoides CBS6641, Dekkera bruxellensis CBS2796 and Dekkera anomala CBS77 were unsuitable for wine production because of poor fermentation dynamics, inefficient sugar consumption and ethanol production levels and major organoleptic defects. Thus, we selected strains of K. gamospora and Z. kombuchaensis that significantly improved the usually plain taste of Ribolla wine by providing additional aromatic complexity in a controlled and reproducible manner.


Non-conventional yeasts Alcoholic fermentation Organoleptic properties Carbon metabolism 



This work was supported by the Slovenian Research Agency (project number J4-4300) and by the EU ITN “Cornucopia” project (FP7, grant agreement GA264717), Creative Core programme (AHA-MOMENT) contract no. 3330-13-500031, co-supported by RS-MIZS and European Regional Development Fund Research. We would like to thank Justin Fay for providing useful advice and corrections during the writing process. We greatly appreciate the access to the yeast strains provided by CBS-KNAW Fungal Biodiversity Centre ( We are especially grateful to the late Jure Piškur, who was our main inspiration and project leader and who is greatly missed.

Supplementary material

10295_2015_1620_MOESM1_ESM.pdf (124 kb)
Supplementary material 1 (PDF 124 kb). Weight loss during the fermentation of laboratory-scale fermentations performed with D. anomala (CBS77), D. bruxellensis (CBS2796), K. gamospora (CBS10400), T. microellipsoides (CBS6641), Z. kombuchaensis (CBS8849). The steeper is the curve the higher is fermentation dynamic of the sample
10295_2015_1620_MOESM2_ESM.pdf (349 kb)
Supplementary material 2 (PDF 349 kb). PCA plot with most influential loadings of six yeast species based on the major volatiles formed during the fermentation process. Fermentation samples produced with DA - D. anomala (CBS77), DB - D. bruxellensis (CBS2796), KG - K. gamospora (CBS10400), TM - T. microellipsoides (CBS6641), ZK - Z. kombuchaensis (CBS8849)
10295_2015_1620_MOESM3_ESM.pdf (279 kb)
Supplementary material 3 (PDF 279 kb). Results of sensory evaluation of young wines using hedonic scale method. 1 – least preferred sample, 10 – most preferred one. (a) evaluation of “smell” attribute in six samples of the young wines. (b) evaluation of “color” attribute in six samples of the young wines. Fermentation samples produced with DA - D. anomala (CBS77), DB - D. bruxellensis (CBS2796), KG - K. gamospora (CBS10400), TM - T. microellipsoides (CBS6641), ZK - Z. kombuchaensis (CBS8849)
10295_2015_1620_MOESM4_ESM.pdf (349 kb)
Supplementary material 4 (PDF 348 kb). PCA plot demonstrating patterns of variations of sensory attributes. (a) Z. kombuchaensis (CBS8849) and K. gamospora(CBS10400) are mostly impacting aroma attribute (in red), in less extent attributes of color (in blue) and taste (in green). (b) wines produced with K. gamospora are more distinguishable from S. cerevisiae Lalvin T73 produced wines in comparison to Z. kombuchaensis samples
10295_2015_1620_MOESM5_ESM.pdf (104 kb)
Supplementary material 5 (PDF 104 kb). Levels of sugars and ethanol in the pilot scale fermentation samples fermented with K. gamospora (CBS10400), Z. kombuchaensis (CBS8849) and S. cerevisiae Lalvin T73 after 350 h of fermentation. Statistical groups determined using LSD test
10295_2015_1620_MOESM6_ESM.pdf (376 kb)
Supplementary material 6 (PDF 376 kb). Comparative physiology of the control strain, S. cerevisiae Lalvin T73 andK. gamospora (CBS10400), Z. kombuchaensis (CBS8849).Yields (ethanol, acetate, biomass, glycerol and pyruvate) were calculated during the exponential phase as a function of glucose consumed. Corresponding consumption and production rates were calculated during the same time intervals on minimal media supplied with 2% glucose in batch fermentations. a Maximum specific growth rate. b Yield coefficients per gram of glucose consumed (g−1); Yse, yield of ethanol; Ysx, yield of biomass; Ysp, yield of pyruvate; Ysac, yield of acetate; Ysg, yield of glycerol. c Specific consumption rate per gram of biomass per hour (mmol g−1 h−1); qGlucose, glucose consumption rate; qO2, oxygen consumption rate. d Specific production rates per hour per gram of biomass (mmol g−1 h−1);qEthanol, ethanol production rate; qCO2, carbon dioxide consumption rate. e Respiratory Quotient ; ratio of carbon dioxide production rate as per oxygen consumption rate
10295_2015_1620_MOESM7_ESM.pdf (59 kb)
Supplementary material 7 (PDF 58 kb). List of strains used in the study
10295_2015_1620_MOESM8_ESM.pdf (68 kb)
Supplementary material 8 (PDF 67 kb). Ethanol, glucose, and fructose measurements of the samples taken after 60 h, 150 h, and 300 h. Data was processed using one-way ANOVA (n.s.: not significant; *: P<0.05 (*); **: P<0.01; ***: P<0.001)
10295_2015_1620_MOESM9_ESM.pdf (169 kb)
Supplementary material 9 (PDF 169 kb). (a) Concentrations of volatiles expressed as 2-octanol using chromatographic peak area ratios at the end of laboratory scale fermentations. (b) Concentrations of volatiles expressed as 2-octanol using chromatographic peak area ratios at the end of pilot scale fermentations
10295_2015_1620_MOESM10_ESM.pdf (88 kb)
Supplementary material 10 (PDF 87 kb). Sensory descriptors of individual compounds
10295_2015_1620_MOESM11_ESM.pdf (383 kb)
Supplementary material 11 (PDF 382 kb). Raw data of sensory attributes with statistical data attached. Data was processed using one-way ANOVA (n.s.: not significant; *: P<0.05 (*); **: P<0.01; ***: P<0.001). Groups determined using LSD test
10295_2015_1620_MOESM12_ESM.pdf (63 kb)
Supplementary material 12 (PDF 63 kb). Raw data from HPLC analysis of basic metabolites from pilot scale fermentation samples fermented with K. gamospora (CBS10400), Z. kombuchaensis (CBS8849) and S. cerevisiae Lalvin T73. Statistical analysis attached, groups determined using LSD test


  1. 1.
    Bavčar D, Baša Česnik H, Čuš F, Košmerl T (2011) The influence of skin contact during alcoholic fermentation on the aroma composition of Ribolla Gialla and Malvasia Istriana Vitis vinifera (L.) grape wines. Int J Food Sci Technol 46:1801–1808. doi: 10.1111/j.1365-2621.2011.02679.x CrossRefGoogle Scholar
  2. 2.
    Beckner M, Ivey ML, Phister TG (2011) Microbial contamination of fuel ethanol fermentations. Lett Appl Microbiol 53:387–394. doi: 10.1111/j.1472-765X.2011.03124.x PubMedCrossRefGoogle Scholar
  3. 3.
    Bely M, Stoeckle P, Masneuf-Pomarède I, Dubourdieu D (2008) Impact of mixed Torulaspora delbrueckii-Saccharomyces cerevisiae culture on high-sugar fermentation. Int J Food Microbiol 122:312–320. doi: 10.1016/j.ijfoodmicro.2007.12.023 PubMedCrossRefGoogle Scholar
  4. 4.
    Benda I (1982) Wine and brandy. In: Prescott SC, Dunn CG, Reed G (eds) Prescott & Dunn’s industrial microbiology, 4th edn. AVI Publishing Co., Westport, CNGoogle Scholar
  5. 5.
    Blomqvist J, Eberhard T, Schnürer J, Passoth V (2010) Fermentation characteristics of Dekkera bruxellensis strains. Appl Microbiol Biotechnol 87:1487–1497. doi: 10.1007/s00253-010-2619-y PubMedCrossRefGoogle Scholar
  6. 6.
    Boekhout T, Kurtzman CP, O’Donnell K, Smith MT (1994) Phylogeny of the yeast genera Hanseniaspora (anamorph Kloeckera), Dekkera (anamorph Brettanomyces), and Eeniella as inferred from partial 26S ribosomal DNA nucleotide sequences. Int J Syst Bacteriol 44:781–786PubMedCrossRefGoogle Scholar
  7. 7.
    Comitini F, Gobbi M, Domizio P, Romani C, Lencioni L, Mannazzu I, Ciani M (2011) Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae. Food Microbiol 28:873–882. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  8. 8.
    Conant GC, Wolfe KH (2007) Increased glycolytic flux as an outcome of whole-genome duplication in yeast. Mol Syst Biol. doi: 10.1038/msb4100170 PubMedCentralPubMedGoogle Scholar
  9. 9.
    Coutinho R, Branco P, Monteiro M, Malfeito-Ferreira M, Albergaria H (2013) Saccharomyces cerevisiae and Dekkera bruxellensis interactions in alcoholic fermentations: growth and 4-ethylphenol production. MicroBiotec’13: Portuguese Congress of Microbiology and Biotechnology, Aveiro, Portugal, p 94Google Scholar
  10. 10.
    Dairou V, Sieffermann J-M (2002) A comparison of 14 jams characterized by conventional profile and a quick original method, the flash profile. J Food Sci 67:826–834. doi: 10.1111/j.1365-2621.2002.tb10685.x CrossRefGoogle Scholar
  11. 11.
    Dashko S, Zhou N, Compagno C, Piškur J (2014) Why, when, and how did yeast evolve alcoholic fermentation? FEMS Yeast Res 14:826–832. doi: 10.1111/1567-1364.12161 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    De Deken RH (1966) The Crabtree effect: a regulatory system in yeast. J Gen Microbiol 44:149–156PubMedCrossRefGoogle Scholar
  13. 13.
    Van Dijken JP, Scheffers WA (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol Lett 32:199–224. doi: 10.1111/j.1574-6968.1986.tb01194.x CrossRefGoogle Scholar
  14. 14.
    Domizio P, Romani C, Lencioni L, Comitini F, Gobbi M, Mannazzu I, Ciani M (2011) Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation. Int J Food Microbiol 147:170–180. doi: 10.1016/j.ijfoodmicro.2011.03.020 PubMedCrossRefGoogle Scholar
  15. 15.
    Fleet GH (2003) Yeast interactions and wine flavour. Int J Food Microbiol 86:11–22PubMedCrossRefGoogle Scholar
  16. 16.
    Galafassi S, Merico A, Pizza F, Hellborg L, Molinari F, Piškur J, Compagno C (2011) Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions. J Ind Microbiol Biotechnol 38:1079–1088. doi: 10.1007/s10295-010-0885-4 PubMedCrossRefGoogle Scholar
  17. 17.
    Gobbi M, Comitini F, Domizio P, Romani C, Lencioni L, Mannazzu I, Ciani M (2013) Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine. Food Microbiol 33:271–281. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  18. 18.
    Gobbi M, De Vero L, Solieri L, Comitini F, Oro L, Giudici P, Ciani M (2014) Fermentative aptitude of non-Saccharomycescerevisiae wine yeasts for reduction in ethanol content in wine. Eur Food Res, TechnolGoogle Scholar
  19. 19.
    Godoy L, Martínez C, Carrasco N, Ganga MA (2008) Purification and characterization of a p-coumarate decarboxylase and a vinylphenol reductase from Brettanomyces bruxellensis. Int J Food Microbiol 127:6–11. doi: 10.1016/j.ijfoodmicro.2008.05.011 PubMedCrossRefGoogle Scholar
  20. 20.
    Guillaume C, Delobel P, Sablayrolles J-M, Blondin B (2007) Molecular basis of fructose utilization by the wine yeast Saccharomyces cerevisiae: a mutated HXT3allele enhances fructose fermentation. Appl Environ Microbiol 73:2432–2439. doi: 10.1128/AEM.02269-06 PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Hagman A, Säll T, Compagno C, Piskur J (2013) Yeast “make-accumulate-consume” life strategy evolved as a multi-step process that predates the whole genome duplication. PLoS One 8:e68734. doi: 10.1371/journal.pone.0068734 PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Van Hoek P, Van Dijken JP, Pronk JT (1998) Effect of specific growth rate on fermentative capacity of baker’s yeast. Appl Environ Microbiol 64:4226–4233PubMedCentralPubMedGoogle Scholar
  23. 23.
    Jolly NP, Varela C, Pretorius IS (2014) Not your ordinary yeast: non-Saccharomyces yeasts in wine production uncovered. FEMS Yeast Res 14:215–237. doi: 10.1111/1567-1364.12111 PubMedCrossRefGoogle Scholar
  24. 24.
    Jussier D, Dubé Morneau A, Mira de Orduña R (2006) Effect of simultaneous inoculation with yeast and bacteria on fermentation kinetics and key wine parameters of cool-climate Chardonnay. Appl Environ Microbiol 72:221–227. doi: 10.1128/AEM.72.1.221-227.2006 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Kim D-H, Hong Y-A, Park H-D (2008) Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine. Biotechnol Lett 30:1633–1638. doi: 10.1007/s10529-008-9726-1 PubMedCrossRefGoogle Scholar
  26. 26.
    Kurtzman CP, Robnett CJ (1997) Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5′ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 35:1216–1223PubMedCentralPubMedGoogle Scholar
  27. 27.
    Kurtzman CP, Robnett CJ (2003) Phylogenetic relationships among yeasts of the “Saccharomyces complex” determined from multigene sequence analyses. FEMS Yeast Res 3:417–432PubMedCrossRefGoogle Scholar
  28. 28.
    Lambrechts MG, Pretorius IS (2000) Yeast and its importance to wine aroma—a review. South Afr J Enol Vitic, South AfrGoogle Scholar
  29. 29.
    Lawless HTHH (2010) Sensory evaluation of food—principles and practices. Springer Science and Business Media, New YorkCrossRefGoogle Scholar
  30. 30.
    Luca Riccardo Formenti AN (2014) Challenges in industrial fermentation technology research. Biotechnol J. doi: 10.1002/biot.201300236 PubMedGoogle Scholar
  31. 31.
    Øyvind Langsrud TN (1998) A unified framework for significance testing in fractional factorials. Comput Stat Amp Data Anal. doi: 10.1016/S0167-9473(98)90151-7 Google Scholar
  32. 32.
    Pao SS, Paulsen IT, Saier MH (1998) Major Facilitator Superfamily. Microbiol Mol Biol Rev 62:1–34PubMedCentralPubMedGoogle Scholar
  33. 33.
    Piskur J, Langkjaer RB (2004) Yeast genome sequencing: the power of comparative genomics. Mol Microbiol 53:381–389. doi: 10.1111/j.1365-2958.2004.04182.x PubMedCrossRefGoogle Scholar
  34. 34.
    Piskur J, Rozpedowska E, Polakova S, Merico A, Compagno C (2006) How did Saccharomyces evolve to become a good brewer? Trends Genet TIG 22:183–186. doi: 10.1016/j.tig.2006.02.002 CrossRefGoogle Scholar
  35. 35.
    Rainieri S, Pretorius IS (2000) Selection and improvement of wine yeasts. Ann Microbiol 50:15–31Google Scholar
  36. 36.
    Rantsiou K, Dolci P, Giacosa S, Torchio F, Tofalo R, Torriani S, Suzzi G, Rolle L, Cocolin L (2012) Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations. Appl Environ Microbiol 78:1987–1994. doi: 10.1128/AEM.06768-11 PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Ravasio D, Walther A, Trost K, Vrhovsek U, Wendland J (2014) An indirect assay for volatile compound production in yeast strains. Sci Rep 4:3707. doi: 10.1038/srep03707 PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Romano A, Perello MC, Lonvaud-Funel A, Sicard G, de Revel G (2009) Sensory and analytical re-evaluation of “Brett character”. Food Chem 114:15–19. doi: 10.1016/j.foodchem.2008.09.006 CrossRefGoogle Scholar
  39. 39.
    Rozpędowska E, Hellborg L, Ishchuk OP, Orhan F, Galafassi S, Merico A, Woolfit M, Compagno C, Piskur J (2011) Parallel evolution of the make-accumulate-consume strategy in Saccharomyces and Dekkera yeasts. Nat Commun 2:302. doi: 10.1038/ncomms1305 PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Schifferdecker AJ, Dashko S, Ishchuk OP, Piškur J (2014) The wine and beer yeast Dekkera bruxellensis: the wine and beer yeast Dekkera bruxellensis. Yeast 31:323–332. doi: 10.1002/yea.3023 PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Semchyshyn HM, Abrat OB, Miedzobrodzki J, Inoue Y, Lushchak VI (2011) Acetate but not propionate induces oxidative stress in bakers’ yeast Saccharomyces cerevisiae. Redox Rep Commun Free Radic Res 16:15–23. doi: 10.1179/174329211X12968219310954 CrossRefGoogle Scholar
  42. 42.
    Sousa-Dias SGT (1996) Kinetics and regulation of fructose and glucose transport systems are responsible for fructophily in Zygosaccharomyces bailii. Microbiol-Sgm 142:1733–1738. doi: 10.1099/13500872-142-7-1733 CrossRefGoogle Scholar
  43. 43.
    Stone H, Sidel JL (1998) Quantitative descriptive analysis: developments, applications and the future. Food Technol, USAGoogle Scholar
  44. 44.
    Suárez R, Suárez-Lepe JA, Morata A, Calderón F (2007) The production of ethylphenols in wine by yeasts of the genera Brettanomyces and Dekkera: a review. Food Chem 102:10–21. doi: 10.1016/j.foodchem.2006.03.030 CrossRefGoogle Scholar
  45. 45.
    Tiukova IA, Petterson ME, Tellgren-Roth C, Bunikis I, Eberhard T, Pettersson OV, Passoth V (2013) Transcriptome of the alternative ethanol production strain Dekkera bruxellensis CBS 11270 in sugar limited, low oxygen cultivation. PLoS One 8:e58455. doi: 10.1371/journal.pone.0058455 PubMedCentralPubMedCrossRefGoogle Scholar
  46. 46.
    Toro ME, Vazquez F (2002) Fermentation behaviour of controlled mixed and sequential cultures of Candida cantarellii and Saccharomyces cerevisiae wine yeasts. World J Microbiol Biotechnol 18:351–358. doi: 10.1023/A:1015242818473 CrossRefGoogle Scholar
  47. 47.
    Verduyn C, Postma E, Scheffers WA, Van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast Chichester Engl 8:501–517. doi: 10.1002/yea.320080703 CrossRefGoogle Scholar
  48. 48.
    Wedzicha BL (1984) Chemistry of sulphur dioxide in foods. Elsevier Applied Science, London, New YorkGoogle Scholar
  49. 49.
    Wehrens R, Weingart G, Mattivi F (2014) metaMS: an open-source pipeline for GC–MS-based untargeted metabolomics. J Chromatogr B 966:109–116. doi: 10.1016/j.jchromb.2014.02.051 CrossRefGoogle Scholar
  50. 50.
    White T, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Shinsky J, White T (eds) PCR Protoc. Academic Press, Guide Methods Appl, pp 315–322Google Scholar
  51. 51.
    Wolfe K (2004) Evolutionary genomics: yeasts accelerate beyond BLAST. Curr Biol CB 14:R392–R394. doi: 10.1016/j.cub.2004.05.015 CrossRefGoogle Scholar
  52. 52.
    Wolfe KH, Shields DC (1997) Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387:708–713. doi: 10.1038/42711 PubMedCrossRefGoogle Scholar
  53. 53.
    Yamaoka C, Kurita O, Kubo T (2014) Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation. Microbiol Res 169:907–914. doi: 10.1016/j.micres.2014.04.007 PubMedCrossRefGoogle Scholar
  54. 54.
    Green SR, Gray PP (1950) A differential procedure applicable to bacteriological investigation in brewing. Wallerstein Lab. Commun 13:357Google Scholar
  55. 55.
    Gamero Lluna A, de Jong C (2013) Novel yeasts, novel flavours. New Food Mag. 16(3)26–28Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2015

Authors and Affiliations

  • Sofia Dashko
    • 1
    • 2
    Email author
  • Nerve Zhou
    • 2
  • Tinkara Tinta
    • 3
  • Paolo Sivilotti
    • 1
  • Melita Sternad Lemut
    • 1
  • Kajetan Trost
    • 1
    • 4
  • Amparo Gamero
    • 5
  • Teun Boekhout
    • 6
  • Lorena Butinar
    • 1
  • Urska Vrhovsek
    • 4
  • Jure Piskur
    • 1
    • 2
  1. 1.University of Nova GoricaVipavaSlovenia
  2. 2.Lund UniversityLundSweden
  3. 3.National Institute of Biology, Marine Biology StationPiranSlovenia
  4. 4.Fondazione Edmund MachSan Michele All’adigeItaly
  5. 5.NIZO Food Research BVEdeThe Netherlands
  6. 6.CBS-KNAW Fungal Biodiversity CentreUtrechtThe Netherlands

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