Rev. Fac. Agron. (LUZ). 1998, 15: 249-255

Yeasts isolated from fermenting juice extracted from white-wine grape varieties in Zulia state, Venezuela

Aislamiento de levaduras de jugo fermentado extraído de variedades de uvas de vino blanco en el Estado Zulia, Venezuela

Recibido el 02-10-1997lAceptado el 26-02-1998
1. Laboratorio de Alimentos. Dpto de Química. Facultad Experimental de Ciencias. La Universidad del Zulia. Apartado 526. Maracaibo, ZU 4005. Venezuela. 061-598062. FAX 061-414745.

S. Araujo¹, A. Ferrer¹, B. Sulbarán de Ferrer¹, C. Nava¹, G. Ojeda de Rodríguez¹ y R. A. Nava R

Abstract

Resumen

La producción de vinos en el Estado Zulia, Venezuela, se ha venido incrementando continuamente en los últimos años. Entre las metas a alcanzar está la de producir vinos con características regionales, y una de las alternativas es la de utilizar levaduras endógenas bien adaptadas al medio. Para ello se aislaron 191 levaduras de jugo fermentado extraído de uvas blancas de las variedades French colombard y Ugni blanc, 43 de las cuales fueron identificadas. Se utilizaron técnicas en placa y caldo para el estudio de la morfología, aspecto de colonia, producción de pigmento y formación de pseudomicelio y micelio verdadero. Para la identificación de levaduras se estudió la formación de ascosporas, junto con fermentación de azúcares y asimilación de sustratos carbonados y nitrogenados. Los géneros encontrados en ambas variadades fueron Candida, Pichia, Brettanomyces, Zygosacharomyces y Saccharomyces. Kloeckera y Schizosaccharomyces aparecieron solo en la variedad French colombard. Todas las especies de levaduras encontradas están relacionadas con la manufactura de vino, algunas con propiedades indeseables y otras deseables, entre las cuales las especies de Saccharomyces rouxii y S. cerevisiae lucen las más promisorias.
Palabras claves: mosto de uvas, French colombard, Ugni blanc, Candida, Pichia, Brettanomyces Zygosaccharomyces, Saccharomyces, Kloeckera, Schizosaccharomyces, aislamiento de levaduras, identificación de levaduras.

Introduction

Wine production is becoming an important industry in several South America countries such as Argentina, Chile, Brazil and Peru. Venezuela began growing grapes several years ago. Currently, about 700 ha are grown and more than 3,200 kg grapes/ha are produced per year in Zulia State. About 5,000 bottles of wine/year were produced in the Tropical Viticulture Development Center in a small-scale plant (6). Since then, the wine production in Zulia State has increased to 600,000 bottles/wine per year (7). Endogenous yeasts are required for producing wine in specific environments with regional wine characteristics (14,25). Selection of strains well-adapted to performance under winemaking conditions is of the utmost importance, because winemaking practice varies from one region to another, as does wine pH and temperature (9). The objective of this work is the isolation of endogenous yeasts, adapted to our environment, some of which might be candidates to produce wine with "native" enological characteristics, and that satisfy world and Venezuela tastes. Currently, commercial strains of Saccharomyces cerevisiae are used. This kind of yeast is the most used for wine production in the world (11).

Materials and methods

Sampling. 20 kg of each French colombard and Ugni blanc white-wine grape varieties, grown in Zulia State, were randomly and aseptically taken from 105 to 110 day old grapevines. Grape berries were crushed in a mortar. The resultant must was sieved and 50 mL aliquots were dispersed in 125 mL Erlenmeyer flasks with cotton caps. They were allowed to ferment at room temperature (25-28°C) for up to 21 days.

Yeast isolation and identification. Samples from different fermentation-time flasks were streaked on 0.025% chloramphenicol Malt Agar plates (8). The inverted plates were incubated at 25°C for 48 h. Morphologically-different colonies were picked, purified, transferred to Malt Extract Agar slants and kept at 4°C until used. Growth, colony aspect and pigment production were studied on Malt Extract Agar and cellular morphology, grouping, size and reproduction on Malt Extract Broth (18). The Dalmau's plate technique (18) was employed to study the formation of pseudomycelium and true mycelium. Ascospore formation was studied in Gorodkowa Agar (Difco) at 25°C for 8 weeks (3) and spores were visualized with the Schaffer-Fulton's modification of the Wirtz method (18). Fermentation of sugars (2% glucose, galactose, sucrose, maltose and lactose, and 4% raffinose) was studied in Yeast Extract Medium (Difco) test tubes containing Durham tubes at 25-28°C up to 15 days (18). Carbon and nitrogen assimilation was studied on Agar media (Merck) as auxonograms (3). Substrates used appear in Table 2. Fermentation and assimilation tests were carried out by triplicate and high grade sugars and positive and negative control yeasts were used. The keys reported by Lodder (19), Barnett & Pankhurst (2), Belin (5) and Kreger-Van Rij (18) were used for identification, and the taxonomic designations were updated according to Barnett et al. (3).

Results and discussion

A total of 78 colonies from F. colombard and 113 from U. blanc were isolated, 23 and 20 of which, respectively, were identified.

Polar, bipolar and multipolar budding were observed in all but for one strain of Schizosaccharomyces. Among the identified isolates, 40 formed sediment, 10 ring and 9 pellicle. 51.1% of the yeasts formed pseudomycelium, being 39.5 % mycotorula type and the rest (11.6%) mycocandida-type. Pichia stipitis strains formed both types of pseudomycelium and they were found in their asexual state (Candida shehatae). Some yeasts required from 6 to 8 weeks to sporulate. 17 of the yeasts (39.53%) were sporogeneous and belonged to the genera Pichia, Zygosaccharomyces, Saccharomyces and Schizosaccharomyces.

Fermentation tests (table 1) revealed that yeasts that did not ferment glucose, did not ferment any other sugar and they were 23.3% of the total. Most of the yeasts (58.1%) fermented 3 or 4 sugars, being Saccharomyces and Brettanomyces strains among them. However, only the former are considered suitable for wine making. The latter produce undesirable acids (19). Glucose (table 2) was the most fermented sugar (76.7% of the yeasts fermented it). It was followed by galactose (60.5%), maltose (58.1%), sucrose (32.6%) and raffinose (9.3%). No yeast fermented lactose. Although a lactose-fermenting strain usually ferments galactose (18), it did not occur on the contrary.

Table 1. Fermentative ability of the yeast.¹

¹:Numbers below F. colombard and U. blanc are isolation codes of the yeasts (1-23: F. colombard, 1-20: U. blanc). *: Glucose.

Table 3 shows the percentage of yeasts that metabolized each of the carbon and nitrogen substrates. As it was expected, glucose was used by all the strains. Sucrose, galactose, mannitol, maltose and xylose were assimilated by a great number of yeasts while erythritol, dulcitol, rhamnose and inulin, were the least assimilated. Peptone was used by 98% of the yeasts and nitrate by only 2%.

Table 4 shows the identification and the frequency of appearance of the yeasts. Candida, Brettanomyces, Pichia, Zygosaccharomyces and Saccharomyces were isolated from both grape varieties. Kloeckera and Schizosaccharomyces did not appear in the U. blanc variety. Non-identified yeasts account for 6,98%. All the genera found in this work, have been previously reported as related to wine making (18, 19, 24).

Amerine et al. (1) reported that Kloeckera and Saccharomyces genera are the most frequent in grapes all over the world. According to Heard & Fleet (17), S. cerevisiae was the dominant yeast in Australian musts. However, they found a significant growth of K. apiculata (K. lindneri), C. stellata, and C. pulcherrima. The first two species were also found in this work, although Candida sp. predominated. This genus also predominated in Majorca-wine grapes, specifically C. stellata (20). Yeasts belonging to this genus lack good enological properties, since they only grow well in low alcohol wines, are strongly oxidative and most of them do not ferment glucose. Therefore, they would not produce a good wine. On the other hand, Saccharomyces, Debaryomyces, Kluyveromyces, Pichia, Hanseniospora, Saccharomycodes and Torulopsis (recently designated as Candida) have been found in France (16) and Candida, Kloeckera, Pichia, Saccharomyces and Torulospora were the predominant yeasts in fresh grape musts of the Utiel-Requena Region of Spain (22). Several of these genera may be observed in Table 4. A strain of Schizosaccharomyces pombe was also found in this work. This yeast was found in 40% of Sicilia grape musts studied by Florenzano et al. (12), and it is considered a typical component of grape microflora of that region. Candida, Kloeckera, Pichia and Brettanomyces yeasts have been related to wine souring (15,21) so that they should be avoided in the musts. In addition, Fleet (10) reported that Kloeckera and Candida species can grow with Saccharomyces cerevisiae and even exceed its population, mainly at cool fermentation temperatures, changing the organoleptic properties of the wine.

Table 2. Percentage of yeasts that fermented the sugars.

Table 3. Percentage of yeasts that assimilated the substrates.¹

1: Peptone and nitrate were used for nitrogen assimilation; the others for carbon assimilation.

Table 4. Yeasts identification.¹

1: Numbers are isolation codes. *: Found in its asexual state as C. shehatae (Barnett et al. 1990).

The genera isolated in grapevines grown in Zulia State (western Venezuela) reported in this work, have also been found in other regions of the country (8,13,14). Garassini (13) also reported that the best wines were produced with Saccharomyces sp. According to Peynaud (23), they produce higher ethanol concentrations, lower quantities of volatile acids and better flavor and aroma. In addition, most of commercial wine strains belong to this genus (4). Therefore, S. rouxii and S. cerevisiae strains isolated in this study (strains N^o 18, 19, 20 from F. colombard and 5 from U. blanc) will be tested for wine production.

Conclusions

Yeasts found in this work are typical of grapes and fermenting musts. Most of them showed strong fermentation and assimilation ability. Glucose, followed by galactose, maltose and sucrose, were the most fermented sugars. Glucose was assimilated by all yeasts. Among the seven genera isolated, five were found in both grape varieties, although there were differences in species. Many yeasts present in the must have been related to wine souring. However, Saccharomyces rouxii and S. cerevisiae species look very promising and will be tested in wine making.

Literature cited

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