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J. Gen. App!. Microbiol., 40, 135-142 (1994)
EFFECT OF TEMPERATURE ON ETHANOLPRODUCTIVITY AND RESISTANCE OFCRYOPHILIC WINE YEASTSt
MUNEKAZU KISHIMOTO,* AKINARI OSHIDA,' TAKASHI SHINOHARA,'EIICHI SOMA, AND SHOJI GOTO'
Technology Department, Sapporo Wines Ltd., Shibuya-ku, Tokyo 150, Japan'The Institute of Enology and Viticulture, Yamanashi University, Kofu 400, Japan(Received September 28, 1993; Accepted March 9, 1994)
Effects of incubation temperature and ethanol concentration on cellviability of two strains of cryophilic wine yeasts YM-84 and YM-126 werecompared with those of mesophilic wine yeasts W3 and OC-2. Thecryophilic wine yeasts showed certain cell viability in the presence of 10%ethanol at 8°C, the same as the mesophilic wine yeasts. At 25°C,however, viable cell numbers of these cryophilic wine yeasts were de-creased, and this tendency became more marked with the presence ofethanol. Comparison of the composition of fatty acid in cells, YM-84 andYM-126 showed higher proportions of myristic acid to fatty acid, inaddition to higher ratios of C16 fatty acids to C,8 fatty acids. Thesephenomena suggest that these strains have different adaptation mecha-nisms against temperatures. Five strains of cryophilic wine yeasts selectedin our culture collection showed the same fermentation velocity at the lowtemperature of 7°C as did YM-84 and YM-126, and at 28°C their ethanolyields were reduced. From these results, it can be concluded that goodfermentability at low temperatures accompanied by production of lowamounts of ethanol at intermediate temperatures is one of the fermenta-tion characteristics observed among cryophilic wine yeasts.
Generally, the fermentation process in white wine making is controlled at lowtemperatures to obtain high quality products. Therefore, a yeast strain thatpossesses good fermentability at low temperatures is desired. So far, Hara et al. (2)and Kusewicz (7) reported some improvement regarding cryophilic wine yeasts.However, little has been reported about biochemical and fermentation characteris-
This paper was presented at the Annual Meeting of the Japan Society for Bioscience, Biotech-nology, and Agrochemistry in Sendai on April 2, 1993.Present address and address reprint requests to: Dr. Munekazu Kishimoto, Katsunuma Win-ery, Sapporo Wines Ltd., Watazuka, Katsunuma, Higashi-yamanashi-gun, Yamanashi 409-13, Japan.
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136KISHIMOTO et al.VOL. 40
tics of cryophilic wine yeasts. The study of these characteristics is important toestablish a firm definition of cryophilic wine yeasts, and to select and improve usefulcryophilic wine yeasts more efficiently.We (6) previously reported that in comparison with the mesophilic wine yeastsW3 and OC-2, two strains of cryophilic wine yeasts, YM-84 and YM-126, showedgood fermentability at low temperatures (7 and 13°C); however, at intermediatetemperatures (22-30°C), their fermentation ceased prematurely and their ethanolyields were reduced, and this tendency became more marked as temperature wasincreased.In this study, we examined effects of incubation temperature and ethanol uponcell viability of cryophilic wine yeasts with the assumption that the fermentationcharacteristic which produce low amounts of ethanol at intermediate temperatureswould be due to a decrease in cell viability. We also estimated fermentation velocityand ethanol productivity of the strains selected as cryophilic wine yeasts.
MATERIALS AND METHODS
Strains. Two strains of cryophilic wine yeasts, Saccharomyces uvarum YM-84(RIFY 1114) and YM-126 (RIFY 1218), which were selected for the previousreport (6), were tested. Two strains of mesophilic wine yeasts, S. cerevisiae W3(RIFY 1001) and OC-2 (IAM 4274), which are generally used in wine making inJapan, were used as reference strains. Five strains of cryophilic wine yeasts (Table2) selected from the culture collection at Yamanashi University (RIFY) by theprevious method (6) were submitted to the growth and fermentation tests.Fermentation tests. Fermentation tests were performed in 200 ml of PYG-F(polypeptone 0.75%, yeast extract 0.45%, glucose 18%, pH 4.5) medium accord-ing to the previous report (6). For fermentation tests under the presence ofethanol, PYG-F liquid medium containing 10% ethanol and 8% glucose were used.Fermentation temperatures were controlled at 8 or 25°C. Fermentation velocitieswere expressed by the reduction of weight due to C02 evolution. Ethanol con-centrations were measured by gas chromatography at the end of fermentation.Ethanol resistance tests. Ethanol resistance tests were carried out by themethod of Gotoh et al. (1). After two days of cultivation in YPD (glucose 2%,polypeptone 2%, yeast extract 1%) liquid medium at 25°C, the cells were washedwith sterilized water. They were then resuspended in an acetate buffer (pH 4.2)containing the indicated concentrations (0, 5, 10 and 15%) of ethanol and 1% ofglucose, and incubated at 8 or 25°C. Viable cell numbers were counted on YPDplate time-dependently.Preparation of total cellular lipids and analysis of fatty acid composition. Totallipids of the yeasts were obtained from fresh cells grown on PYG-F liquid mediumaccording to Naganuma et al. (8) using a Tower-shaped electric mixer (TaiyoMix-Tower Model A-14). A chloroform : methanol mixture (2: 1 v/v) was used asan extraction solvent. To determine the fatty acid composition, fatty acid methyl
 
1994 Characteristicsf CryophilicWineYeasts 137 esters were prepared by the HCl-methanol. Gas chromatography was carried outaccording to Kaneko et al. (4). Fatty acid composition was expressed as apercentage of the peak area.RESULTS ND DISCUSSIONEffects of ethanol on fermentationThe results of fermentation are shown in Fig. 1. Among all four strains, totalcell numbers were increased to approximately 2 X 10' cells/ml after 20 days offermentation at 8°C. Six to 7 g of C02 evolution was observed from 200 ml ofmedium at the final stage of fermentation with little variation of activity. Ethanol
Fig. 1. Time courses of fermentation of cryophilic and mesophilic wine yeasts.Fermentations were carried out on PYG-F liquid medium containing 10% ethanoland 8% glucose at 8°C (A) and 25°C (B). Cryophilic wine yeast, YM-84 (D) andYM-126 (`~ ). Mesophilic wine yeast, W3 (0) and OC-2 (0).
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