The Collection of Saccharomyces Strains Isolated from the Different Soil-Climatic Zones of Eastern Georgia

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The Collection of Saccharomyces Strains Isolated from the Different Soil-Climatic Zones of Eastern Georgia

L. Amiranashvili(1), N. Gagelidze(2), Kh. Varsimashvili(3), L. Tinikashvili(4), E. Kirtadze(5)

(1) S. Durmishidze Institute of Biochemistry and Biotechnology, 10th km, D. Agmashenebeli alley, Tbilisi, Georgia, [email protected]





ABSTRACT The well known regions of viticulture in Georgia, Kakheti and Qartli are characterized by temperate, corresponding to dry continental climate. The multiplicity of soil-climatic zones, rich in fruits and grapevine varieties determines the diversity of fermentative yeasts with different indices of alcoholic fermentation and pesticide resistance. Over 150 yeast strains of the genus Saccharomyces were isolated from Kakheti and Qartli regions at the stages of blossoming, early ripeness, and complete ripeness.

In 50 strains, the intensity of alcoholic fermentation exceeded 12%. The yeast screening has shown that the doses of three different widespread pesticides introduced into the vineyard have various impacts on the yeast growth. The highest toxicity is characteristic for Sakozeb M-45. Only 5% of tested yeasts grew intensively on Sakozeb M-45, 13% – on Cihom Blue and 40% – on Neoram. Some strains resistant to all three pesticides were also revealed. RESÜMEE Georgiens beste Traubenregionen von Kachetien und Kartli zeichnen sich durch das gemäßigte, trockene und kontinentale Klima aus. Die Vielfalt von Boden-Klima-Zonen einerseits sowie Obst- und Weintraubensorten andererseits ist gute voraussetzung für großes Spektrum von Gärungshefen mit unterschiedlichen Gärungsparametern und Stabilität gegen die Pestiziden. Von uns wurde in Kachetien und Kartli in der Blute-, Halbreife- und Vollreifephasen der Weintrauben mehr als 150 verschiedene Stammen von Saccharomyces-Familien isoliert. Davon übertraf in 50 Stammen die Intensität von alkoholischer Gärung die 12%-Marke. Die Untersuchungen haben gezeigt, dass die Wirkung von den in Weinberg einzusetzenden Pestiziden auf das Wachstum von Hefen ist unterschiedlich. Mit höchster Toxizität lässt sich Sakozeb M-45 charakterisieren. Intensives Wachstum wurde nur in 5%

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von untersuchten Hefen auf Sakozeb M-45 festgestellt, auf Cihom-blue 13% und auf Neoram 40%.

INTRODUCTION Viticulture and winemaking are among the chief and traditional fields of Georgian economics descending from the ancient ages (Javakhishvili, 1986; Ketskhoveli et al., 1960). The well-known regions of viticulture in Georgia Kakheti and Qartli are characterized by temperate corresponding to continental dry climate. Cinnamonic forest, raw humus calcareous, alluvial calcareous, alluvial forest calcareous, chestnut, and black soils are found in above mentioned viticulture regions. The multiplicity of soil-climatic zones and grapevine varieties determine the diversity of fermentative yeasts, which are characterized by specific physiological properties and high metabolic potential (Kirtadze, Kurdadze, 1992). Southern Caucasus is considered as homeland of yeasts (Jackson, 2000).

Unique endemic varieties of grapevine together with national technologies of winemaking perform Georgia a unique wine producing country. Besides, while producing of ecologically clean wine, vine diseases revealing at different stages of vegetation should be taken into consideration. During the entire period of vegetation, vine culture is treated several times by different pesticides. Part of the pesticides penetrates into grape juice, and as a result of fermentation appears in wine material, essentially influencing its quality. Thus, nowadays production of ecologically clean wine remains one of the most important problems of modern wine industry (Cabras et al., 1999; 2001).

In natural populations of yeasts indigenous strains adapted to pesticides and heavy metals are revealed (Kadagishvili et al., 2006; Machavariani et al., 2006). Presumably, exactly such strains are characterized by oxidative degradation of pesticides. Alcoholic fermentation, conducted by applying of their metabolic potential is a way to obtain ecologically clean products.

The goal of the present work is isolation of yeast strains belonging to the genus Saccharomyces at various stages of vine vegetation from the regions of Eastern Georgia (Kakheti, Qartli) and selection of yeasts capable of high alcoholic fermentation and pesticide detoxification potential among them.

MATERIALS AND METHODS The samples were taken from phylosphere and rizosphere of different cultivars (cv) of Vitis vinifera: cv Rkatsiteli, cv Manavis mtsvane, cv Aladasturi, cv Saperavi, cv Goruli mtsvane, cv Gorula.

The sampling was conducted at the stages of blossoming, early ripeness and complete ripeness and from the sediments of wine produced in peasant farms.

In order to isolate yeasts from samples, the methods of limited dilution and enrichment culture were used. In addition, samples of phylosphere were directly placed on Petri dishes on the surface of solid nutrient medium. The perfect medium for yeasts, g/l – KH2PO4 – 2; MgSO4× 7H2O – 1; (NH4)2SO4 – 1; glucose – 20; yeast extract – 2, (agar – 20), pH 6.5 (Zakharov et al.,, 1984) was used both in solid and liquid forms to isolate yeasts. The incubation of yeasts on solid medium was performed at 25°C in thermostat for 2-3 days. The incubation of yeasts in liquid medium was conducted in 750 ml flasks at rotary shaker (180 revolutions per minute) at 25°C for 3 days. After multiple purification and plating, pure

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cultures of yeasts were obtained. The growth intensity of yeast strains in liquid medium was measured by spectrophotometer at wavelength of 660 nm.

In order to determine the intensity of alcoholic fermentation, the yeast biomass was placed into the medium of the same composition under anaerobic conditions; the amount of glucose was equal to 23%. The fermentation was conducted at 25% in thermostat for two weeks. To estimate the concentration of alcohol accumulated during fermentation, the amount of residual glucose was determined (Dahlgvist, 1961). The concentration of ethyl alcohol was established in terms of the amount of glucose applied by yeasts (К=0.58). Ethyl alcohol was determined at HPLC by distillation method– MA-E-AS312-01-TALVOL.

The pesticides: Sakozeb M-45R (reactant – Mankoceb 800 g/kg), Neoram (reactant – copper chloroxide 377 g/kg), Cihom Blue (reactant – Cineb 340 g/kg and copper oxicloride – 170 g/kg) sanctioned by the Ministry of Agriculture of Goergia were used for research. The amount of pesticieds introduced into the media was determined by the standards of their charges. Particularly, Sakozeb M-45 – 3,5 g/l, Neoram – 4 g/l, Cihom Blue – 5 g/l. The cultivation of yeasts was conducted on solid nutrient media containing one of the above mentioned pestisides at 25°C in thermostat for a week. The yeast growth intensity was estimated visually by 4-point system: +++ – intensive growth; ++ – limited growth; + – supressed growth; - – growth inhibition. RESULTS AND DISCISSION From phylosphere and rizosphere of different cultivars (cv) of Vitis vinifera (cv Rkatsiteli, cv Manavis mtsvane, cv Aladasturi, cv Saperavi, cv Goruli mtsvane, cv Gorula) spread in the regions of Eastern Georgia (Qartli and Kakheti) have been isolated the following pure yeast cultures:

• 9 pure yeast cultures at blooming stage • 70 pure yeast cultures at the stage of early ripeness • 67 pure yeast cultures at the stage of complete ripeness • 37 pure cultures from the sediments of wine produced in peasant farms (Fig. 1).

Fig. 1. Pure yeast cultures isolated from wine sediments and different stages of vine vegetation: a – blooming, b –early ripeness, c – complete ripeness

At the stages of early ripeness and complete ripeness, the yeasts were mainly isolated from

phylosphere; the amount of isolated yeasts is in correlation with concentration of sugar in grapes.

The screening to determine pesticide (Sakozeb M-45, Neoram, Cihom Blue) detoxification potential of yeasts freshly isolated at the different stages of vine vegetation has shown that the influence of required treating dosage on yeast growth and development is different. The

a b c

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highest toxicity is characteristic to Sakozeb M-45. Only 45 strains of tested yeasts were grown in Sakozeb M-45-containing medium. Among them 7 strains exposed intensive growth. In case of Neoram, 114 strains grew; among them 58 strains exposed intensive growth. 80 strains grew at Cihom Blue-containing medium; 19 strains revealed intensive growth, i.d. only 5% of 146 strains isolated at different stages of vine vegetation are characterized by intensive growth on Sakozeb M-45, 13% – on Cihom Blue and 40% – on Neoram (Fig. 2).

16 strains out of 37 ones isolated from wine sediment grew at Sakozeb M-45-containing medium. Among them 2 strains displayed intensive growth. In case of Neoram, 26 strains grew; 6 strains revealed intensive growth. 24 strains grew on Cihom Blue-containing medium. Among them 3 strains displayed intensive growth.

Fig. 2. Different growth intensity of yeast strains on the media containing various pesticides: a – Sakozeb M-45, b–Neoram, c– Cihom Blue

The yeast cultures while growing on Neoram-containing medium decolorize the nutrient

medium. In some cases, blue color of the nutrient medium caused by the pesticide proceeds to yeast biomass. Presumably, the ions stipulating color of the nutrient medium penetrate to biomass and color it (Fig.3).

Fig. 3. Color change of Neoram-containing medium caused by yeast strains

Sakozeb M-45 and Cihom Blue influence on form and color of yeast colony. In some cases, the smooth surface of yeast colonies transform into rugose one (Fig. 4: a, b); sometimes so-called secondary growth takes place (Fig. 4: c).

a b c

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Fig.4. The impact of Sakozeb M-45 (a) and Cihom Blue (b, c) on the form of yeast colonies

The screening of freshly isolated strains at different stages of vine vegetation resistant to the

pesticides was conducted according to high fermenting potential. The fermentation results for strains isolated at blooming stage show that none of them

displayed high intensity of fermentation and accumulated amount of alcohol was equal to 2-4%.

The screening of yeasts isolated at the stages of early ripeness and complete ripeness to reveal high capability of alcohol fermentation showed that the intensity of alcohol fermentation ranged in 7-13%. The fermentation process continued for 16 days. During the first four days, the process was conducted at 30Cº that was unfavorable for fermentation process. As the temperature decreased to 25Cº, the fermentation process was intensified and the amount of accumulated alcohol for 12 days exceeded 9% in strains under the following conditional numbers: 91, 99, and 112. In all tested strains the fermentation process actually completed and alcohol concentration insignificantly changed further (Fig. 5). Some strains under conditional numbers - 91, 112, and 143 are shown in Fig.5.

Fig. 5. The intensity of alcohol fermentation of yeasts isolated at the stages of early ripeness (a) and complete ripeness (b): a - - - 91; - - 95; - - 99; - - 103; -

- 112; b - - - 143; - - 171; - - 188; - - 141; - - 237 (The conditional numbers belong to the yeast strains isolated from the different cultivars of Eastern Georgia: 91 – cv Saperavi, village Anaga, Sighnaghi district, 95 – cv Rkatsiteli, village Vaqiri, Sighnaghi district, 99 – cv Rkatsiteli, village Kardenakhi, Gurjaani district, 103 – village Manavi, cv Rkatsiteli, Sagarejo district, 112 – village Manavi, cv Saperavi, Sagarejo district, 141 – cv Rkatsiteli, village Tibaani, Sighnaghi district; 171 – cv Rkatsiteli, village Chandari, Gurjaani district; 143 – cv Aladasturi, village Tibaani, Sighnaghi district; 188 – cv Saperavi, village Nikozi, Gori district; 237 – cv Saperavi, village Qvemo Khodasheni, Telavi district).

b ca

a b

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It was found that 50 strains among 183 revealed intensive alcoholic fermentation: the concentration of formed ethanol exceeded 12%.

The intensity of alcohol fermentation was especially high in the yeasts isolated from the sediments of wine produced in peasant farms. In most strains, the concentration of alcohol formed by yeasts exceeded 12%; it could be explained by screening of strains tolerant to high alcohol concentrations that naturally takes place in wine.

On the base of the experiments, alcohol fermentation was more intensive in the yeasts isolated from Kakheti region in comparison with that of isolated from Qartli region; it must be the result of natural selection process. Grapes of vine cultivars from Qartli region are characterized by less sugary compared with those of Kakheti region that is clearly reflected on the yield of ethyl alcohol.

CONCLUSIONS As a result of conducted works it might be concluded:

• The yeast collection including 146 strains isolated from Kakheti and Qartli regions at the stages of blooming, early ripeness and complete ripeness and 37 strains isolated from wine sediments has been created.

• Only 5% of strains out of 146 isolated at different stages of vine vegetation are characterized by intensive growth on Sakozeb M-45, 13% – on Cihom Blue and 40% – on Noeram. 6% of strains out of 37 isolated from wine sediments are characterized by intensive growth on Sakozeb M-45, 7% – on Cihom Blue and 16% – on Neoram.

• In the yeasts isolated from Kakheti region, alcoholic fermentation is more intensive in comparison with that of isolated from Qartli region; it must be the result of natural selection process.

• Alcohol fermentation is more intensive in the yeasts isolated from wine sediments, where take place the naturally screening of strains tolerant to high alcohol concentrations.

ACKNOWLIDGMENTS The designated project has been fulfilled by financial support of the Georgian National Science Foundation (Grant #GNSF/ST08/8-497). Any idea in this publication is possessed by the authors and may not represent the opinion of the Georgian National Science Foundation itself.

BIBLIOGRAPHY Cabras P., Angioni A., Garau V., Pirisi F., Farris G., Madau G., Emonti G. 1999. Pesticides in

fermentative processes of wine. J. Agric Food Chem. 47, 9:3854-386. Cabras P., Angioni A., Garau V., Pirisi F., Cabitza F., Pala M., Farris G. 2001. Fenhexamid

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reagent. Biochem. J., 1961, 80, p. 547-551. Jackson. R. 2000. Wine Science: Principles, Practice, Perception. Academic Press. Elsevier. Javakhishvili I. 1986. The History of Georgian Economics. v. II, In: Works in 12 volumes.

Metsniereba, Tbilisi, Ed. D. Chkhikvishvili. Ketskhoveli N. Ramishvili M., Tabidze D. 1960. Georgian Ampelography. Georgian

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Academy of Sciences, Tbilisi. Kirtadze E., Kurdovanidze T. 1992. Biochemical Features of the Secondary Alcohol

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The Collection of Saccharomyces Strains Isolated from the Different Soil-Climatic Zones of Eastern Georgia