versión On-line ISSN 1851-7617
Rev. argent. microbiol. vol.42 no.3 Ciudad Autónoma de Buenos Aires jul./set. 2010
MICROBIOLOGÍA INDUSTRIAL Y AMBIENTAL
Effects of using mixed wine yeast cultures in the production of Chardonnay wines
1Departamento en Ciencia y Tecnología de los Alimentos;
2Centro de Estudios en Ciencia y Tecnología de los Alimentos, Universidad de Santiago de Chile. Alameda 3363, Estación Central, Santiago, Chile;
3Departamento de Biotecnología de Alimentos, Instituto de Agroquímica y Tecnología de los Alimentos, Consejo Superior de Investigaciones Científicas. P.O. Box 73, 46100 Burjassot, Valencia, Spain.
*Correspondence. Email: firstname.lastname@example.org
The effect of using mixed cultures of non-Saccharomyces and Saccharomyces cerevisiae yeasts in the physicochemical and sensory qualities of the wines were analyzed in this study. Based on growth curves, sugar consumption and glycerol production in synthetic must, Candida membranifaciens L1805 was selected from a group of four Candidas spp. isolates from Chile and Argentina. This yeast was subsequently used in combination with S. cerevisiae in Chardonnay must. A monoculture of S. cerevisiae was used as control. The wines fermented with mixed cultures had lower volatile acidity and ethanol concentration than the control. Furthermore, the chromatographic analysis showed that the wines from mixed cultures presented differences in the concentration of esters and propanol. These characteristics positively influenced the sensory qualities of the wines produced with mixed cultures, which was reflected in the preference for these wines by a panel of enologists. This study shows that the use of non-Saccharomyces yeasts could be a strategy to obtain distinctive wines using the native microorganisms from each winemaking area.
Key words: Non-Saccharomyces; Wine; Aroma; Flavor; Co-fermentation
Efecto del uso de cultivos mixtos de levaduras en la producción de vinos Chardonnay. En este estudio se analizó el efecto del uso de cultivos mixtos de levaduras no-Saccharomyces y Saccharomyces cerevisiae en las cualidades fisicoquímicas y sensoriales de los vinos. Candida membranifaciens L1805 fue elegida de un grupo de cuatro Candida spp. aisladas de Chile y Argentina, sobre la base de las curvas de crecimiento, el consumo de azúcar y la producción de glicerol en mosto sintético. Posteriormente, esta levadura fue usada en cultivo mixto con S. cerevisiae en mosto Chardonnay. Como control se utilizó un monocultivo de S. cerevisiae. Los vinos producidos por cultivos mixtos tuvieron menor acidez volátil y producción de etanol que los correspondientes al control. Los análisis cromatográficos mostraron que estos vinos presentaron diferencias en la concentración de ésteres y de propanol. Estas características afectaron positivamente las cualidades sensoriales de los vinos, lo cual se reflejó en la preferencia del panel de enólogos. El estudio muestra que el uso de levaduras no-Saccharomyces puede ser una estrategia para obtener vinos diferentes usando microorganismos nativos de cada área vitivinícola.
Palabras clave: No-Saccharomyces; Vino; Aroma; Sabor; Cultivos mixtos
In wine production, yeasts are responsible for transforming the sugar present in the grape must into ethanol, carbon dioxide and hundreds of secondary products that collectively contribute to the qualities of the product (7). Hence, these microorganisms may have a positive or negative influence in the sensory traits of the product. Although non-Saccharomyces yeasts were long considered harmful, evidence in recent years has shown that their use may give complex organoleptic characteristics to the wine, thus increasing its quality (3, 6, 8, 9), because they produce compounds such as glycerol, isoamilic alcohol, succinic acid, acetic acid and propanol that affect the sensory characteristics of the product (4, 8). Experiments carried out by Ciani and Picciotti (5) with white must, using 6 non-Saccharomyces yeasts independently, showed that fermentation with Candida stellata DBVPG4124 produced higher glycerol concentration compared to the other microorganisms used, including Saccharomyces cerevisiae. Moreover, there was also a variation in the concentration of isobutanol and isoamilic alcohol. On the other hand, Toro and Vazquez (14) using Candida cantarelli in mixed cultures with S. cerevisiae obtained wines with 40% more glycerol than those fermented only with S. cerevisiae. In addition, the use of mixed cultures of C. stellata and S. cerevisiae or Debaryomyces vanriji and S. cerevisiae have produced wines with a fruty aroma (3, 6, 8, 9, 12). Based on these promising results, there are already dry yeast mixtures of S. cerevisiae / Kluyveromyces thermotolerans / Torulaspora delbrueckii (Vinifloras® Harmony.nsac, CHR Hansen) and T. delbruekki / S. cerevisiae (Level 2TD, Lallemand Inc.) in the market. With the aim of determining the potential of non-Saccharomyces yeasts from South America, in particular those of the genus Candida, we studied the effect of using mixed cultures of native isolates of Candida spp. and S. cerevisiae in the physicochemical and sensory traits of the wines. C. stellata L1560 and Candida membranifaciens L1805 isolated from Cauquenes (Chile) and C. stellata L2120 isolated from Mendoza (Argentina) were used. Commercial yeasts of S. cerevisiae XL were also used, obtained from DSM Food Specialties. Fermentations were carried out in triplicate in 300 ml of synthetic must (5 g/l tartaric acid, 5 g/l malic acid; 0.3 g/l CaCl2; 1.3 g/l MgSO4 x 7 H2O, 1.2 g/l NH4PO4; 2.5 g/l KOH; 100 g/l fructose; 5 g/l sucrose; 100 g/l glucose; 2 mg/l thyamin; 9 μg/l biotin; 4.6 mg/l nicotinic acid; 400 μg/l pyridoxine hydrochloride; 2 mg/l calcium pantoneate) and inoculated with 1x106 cells/ml, which were incubated for 14 days at 25 °C without shaking. A culture of S. cerevisiae was used as control. The fermentation was followed by count in a Neubauer chamber and quantification of sugar reduction (13). The concentration of glycerol was quantified at the end of the process using the enzymatic kit from Roche (Germany).
Figure 1A shows the growth curves of each isolate. It indicates that the C. stellata L1560 isolate had the greatest growth of the non-Saccharomyces isolates, reaching an average population of 1.8 x 107 cells/ml at the end of the fermentative process, close to the average yeast population of the S. cerevisiae isolate (2 x 107 cells/ml). The C. stellata L2120 isolate grew slowly in the first days of the process, reaching its maximum average population of 4.8 x 106 cells/ml on the eighth day. The C. membranifaciens L1805 isolate had an average population of 1.2 x 107 cells/ml on the eighth day of fermentation, with an average of 8.3 x 106 cells/ml by the end of the process. The sugar consumption in the cultures (Figure 1B) showed that the C. stellata L1560 isolate had a similar behavior to S. cerevisiae XL, obtaining a final concentration of residual sugar of 10.9 g/l on day 14. The use of this non-Saccharomyces isolate is questionable given that in a mixed culture of C. stellata L1560 and S. cerevisiae it may influence the growth of S. cerevisiae. The C. stellata L2120 isolate had a final glycerol concentration of 6.6 ± 0.1 g/l, while the C. stellata L1560 culture obtained a concentration of 8.8 ± 0.16 g/l. The C. membranifaciens L1805 culture had a final concentration of 5.3 ± 0.1 g/l. All the non-Saccharomyces isolates showed a higher glycerol concentration in the media than that produced by S. cerevisiae (3.6 ± 0.1 g/l). Based on the glycerol production, the growth kinetics and the fact that C. membranifaciens L1805 does not predominate with S. cerevisiae, this isolate was used in subsequent studies of natural must fermentations. This fermentation was carried out in triplicate in Chardonnay must maintained at -20 °C for 6 months. The microbiological analysis of the must prior to its use showed a yeast concentration below 10 cfu/ml. For natural must fermentation, C. membranifaciens L1805 was grown in 500 ml of sterilized natural must (heated at 60 °C for 10 min) and incubated at 28 °C for 24 h. The final yeast population was determined by count in a Neubauer chamber. Based on previous works on mixed fermentations (14), the initial cultures were added at a final concentration of 1.5 x 106 cells/ml at 3.5 l of must. Additionally, 2.5 x 104 cells/ml of S. cerevisiae were added, resulting in a 1:60 ratio of S. cerevisiae: non-Saccharomyces. The fermentation control containing only S. cerevisiae was used. Fermentation was carried out at 16 °C for 25 days, with daily homogenization and monitoring by variations in density. When the fermentations were completed, the wines were stored at 4 °C. The wines were analyzed for sugar reduction (13), pH (2), alcohol concentration (2), volatile acidity concentration (2), density (2), sensorial analysis (1) and quantification of aromatic compounds (15). All the experiments were carried out in triplicate and the data were subjected to analysis of variance (ANOVA) with a p ≤ 0.05 (LSD Test). Table 1 shows the physicochemical and chromatography analysis of the wines obtained. The volatile acidity and alcohol concentration of the wine with the mixed culture had lower values in both parameters, which is an interesting result considering that volatile acidity is an indicator of microbiological contamination. In relation to the reduction of alcohol concentration in the mixed culture, there could be competition among the inoculated microorganisms which made the production of this metabolite less efficient. The monoculture of S. cerevisiae alone had a higher concentration of glycerol, which was contrary to what was expected. The levels of sugar consumption in the co-fermentation of C. membranifaciens L1085 / S. cerevisiae and the control assays were similar. The similarity in sugar consumption and the lower levels of glycerol concentration obtained in mixed culture may have occurred because the non- Saccharomyces yeast did not grow and the S. cerevisiae yeast predominated at all times. To verify this observation, 10% of yeast colonies obtained at 2, 10 and 26 days of fermentation were analyzed by RFLP analysis of the ITS region (11). This analysis confirmed that two days after the process began there was only 20% of the C. membranifaciens population, which could be insufficient for glycerol production. It is also possible that there are microcomponents in the natural must, not considered in a synthetic must, that could affect microorganism growth and metabolite production. Consequently, it would be interesting in future studies to analyze the relative doses of S. cerevisiae and non-Saccharomyces to apply. With the aim of determining if this small population of C. membranifaciens influences the sensorial characteristics of the wines, a preference test was carried out (1), where enologists preferred the wines obtained from mixed cultures. Parallelly, the aromatic compounds of the wines were analyzed using the technique described by Viana et al. (15). The concentrations of isoamilic alcohol, 2-phenylethanol, isobutanol, benzyl acetate, ethyl acetate, isobutyl acetate, isoamyl acetate, diethyl succinate, 2-phenylethanol acetate, 2-phenylethanol, isobutane and ethyl lactate did not show differences in the two types of wine obtained. However, significant differences were observed in the concentrations of ethyl caproate, ethyl caprylate and hexyl acetate, all being higher in the control wine (S. cerevisiae). The mixture of ethyl caproate, ethyl caprylate and hexyl acetate has been described as conferring fruity aromas, but an increase of these can also have a negative effect (10). This could be the reason why the enologists preferred wines fermented with mixed cultures.
Figure 1. Growth of yeasts (A) and sugar concentration (B) during fermentation of Candida spp. isolates in synthetic must. S. cerevisiae XL was used as control.
Table 1. Chemical analysis and volatile compounds of the wines obtained with mixed cultures and yeast monocultures(1)
(1)Means of the same row with the same superscript letters are not significantly different (p > 0.05).
(2)Expressed as acetic acid.
While the chemical parameters did not show any differences between the control wine and those obtained from mixed cultures, variations were observed in the composition of the aromatic compounds, which could be correlated to the preferences of the enologists. The search for new non-Saccharomyces isolates allows to provide wines with distinctive qualities, and our continent has great potential owing to the wide diversity of yet unstudied yeasts that can serve as differentiators of our musts.
1. Anzaldúa-Morales A. In: Editorial Acribia. La evaluación sensorial de los alimentos en la teoría y la práctica. Zaragoza, España, 1994, p. 68-9, 131. [ Links ]
2. Bordeu E, Scarpa J. In: Ediciones Universidad Católica de Chile, Textos Universitarios Facultad de Agronomía e Ingeniería Forestal. Análisis químico del vino. Santiago, Chile, 1998, p. 58, 92-5, 125-6, 159-60. [ Links ]
3. Ciani M, Comitini F, Mannazzu I, Domizio P. Controlled mixed culture fermentation: a new perspective on the use of non-Saccharomyces yeasts in winemaking. FEMS Yeast Res 2010; 10: 123-33. [ Links ]
4. Ciani M, Ferrano L. Combined use of immobilized Candida stellata cells and Saccharomyces cerevisaie to improve the quality of wines. J Appl Microbiol 1998; 85: 247-54. [ Links ]
5. Ciani M, Picciotti G. The growth kinetics and fermentation behaviour of some non-Saccharomcyes yeast associated with wine making. Biotechnol Lett 1995; 17: 1247-50. [ Links ]
6. Ferraro L, Fatichenti F, Ciani M. Pilot scale vinification process by immobilised Candida stellata and Saccharomyces cerevisiae. Process Biochem 2000; 35: 1125-9. [ Links ]
7. Fleet G. Yeast interaction and wine flavour. Int J Food Microbiol 2003; 86: 11-22. [ Links ]
8. Garcia A, Carcel C, Dulau L, Samson A, Aguera E, Agosin E, et al. Influence of a mixture culture with Debaryomyces vanriji and Saccharomcyes cerevisiae on the volatiles of a muscat wine. J Food Sc 2002; 67: 1138-43. [ Links ]
9. Jolly N, Augustyn O, Pretorius I. The role and use of non- Saccharomyces yeasts in wine production. S Afr J Enol Vitic 2006; 27: 15-39. [ Links ]
10. Lilly M, Bauer F, Lambrechts M, Swiegers J, Cozzolino D, Pretorius I. The effect of increased yeast alcohol acetyl-transferase and esterase activity on the flavour profiles of wine and distillates. Yeast 2006; 23: 641-59. [ Links ]
11. Llano F, Fernández-Espinar M, Querol A. Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and two ribosomal internal transcribed spacers. Antonie van Leeuwenhoek 2004; 85: 175-85. [ Links ]
12. Soden A, Francis I, Oakey H, Henschke P. Effects of cofermentation with Candida stellata and Saccharomyces cerevisiae on the aroma and composition of Chardonnay wine. Aust J Grape Wine Res 2000; 6: 21-30. [ Links ]
13. Somogyi M. Notes on sugar determination. J Biol Chem 1952; 195: 19-23. [ Links ]
14. Toro M, Vázquez F. Fermentation behaviour of controlled mixed and sequential culture of Candida cantarelli and Saccharomyces cerevisiae wine yeast. World J Microbiol Biotechnol 2002; 18: 347-54. [ Links ]
15. Viana F, Gil J, Genove S, Valle S, Manzanares P. Rational selection of non-Saccharomyces wine yeasts for mixed starters based on ester formation and enological traits. Food Microbiol 2008; 25: 778-85. [ Links ]