Isolation and Phylogenetic Analysis of Two Thermotolerant, Fermentative Yeast Strains from Liquid Tapé Ketan (Indonesian Rice Wine)

Abstract

A total of 34 yeast strains were isolated from rice wines pressed from tapé ketan, Indonesian traditional fermented-glutinous rice. Among them, two strains that grew and fermented above 42°C were selected for further analysis. Fermentation was carried out in medium containing 15% (w/v) glucose at 40°C, and ethanol productivity was measured after 48 h of incubation. The two strains, TB2-1 and TW1-3, were found to have upper temperature limit of 46 and 47°C, and higher ethanol productivity of 5.9% and 6.4% (w/v), respectively. These two yeast strains were identified by traditional and molecular techniques. In the molecular technique, 18S rDNA and internal transcribed spacer 1 (ITS1) rDNA genes were amplified by polymerase chain reaction (PCR) using specific primers. The sequences of these two regions for the two strains were phylogenetically analyzed, and the yeast strains TB2-1 and TW1-3 were identified as Issatchenkia orientalis and Saccharomyces cerevisiae, respectively.Keywords: rice wine, thermotolerant and fermentative yeasts, 18S rDNA, ITS1 rDNA *To whom correspondence should be addressed. E-mail: watanabe@chem.agri.kagoshima-u.ac.jp IntroductionThe production of ethanol by yeast fermentation in tropical countries, especially during the summer season, is not economically feasible because of the high energy input required to cool the fermenters. Thermotolerant yeasts offer potential advantages in the alcohol industry by reducing cooling costs and by having faster fermentation rates, thereby making the process more economical (Kiransree et al., 2000;Pasha et al., 2007). There have been a limited number of attempts to obtain yeasts from non-food sources that are capable of growth and fermentation at or above 40℃, with some researchers genetically modifying fermentative yeasts to be thermotolerant (Hong et al., 2007;Kawamura, 1999). Apart from these approaches, screening of yeasts from various environments, including soil in hot regions (Kiran Sree et al., 2000), appears to be the major way of identifying suitable strains for commercial production and reducing the cost of the process. To the best of our knowledge, there has been no report on the characteristics of thermotolerant, fermentative yeasts from rice wines. In this study, we used Indonesian rice wines pressed from tapé ketan to identify thermotolerant, fermentative yeasts.Tapé ketan is a traditional Indonesian fermented food prepared from glutinous rice, which has been steamed, inoculated, and allowed to ferment for 24 to 48 h or longer at ambient temperatures (25 to 30℃) (Cronk et al., 1979). The alcohol content of tapé ketan inoculated with different starter cultures ranges from 2.6 to 3.7% (v/v) after fermentation for 48 h and 4.2 to 7.4% (v/v) after 96 h (Law et al., 2011). In tapé ketan fermentation, reducing sugars increase from less than 1% to approximately 5% in 24 h and reach a maximum concentration, 16 to 17%, between 36 and 48 h (Cronk et al., 1977).Rice wines like the liquid portions of tapé ketan are popular alcoho130lic beverage in Asian ...

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