Identification and characterization of yeasts in brem, a traditional Balinese rice wine I.N. Sujaya 1 , N.S. Antara 2 , T. Sone 3 , Y. Tamura 4 , W.R. Aryanta 2 , A. Yokota 1 , K. Asano 3, * and F. Tomita 3,  1 Laboratory of Microbial Resources and Ecology, Department of Molecular Bioscience, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, North 9, West 9, Sapporo, Hokkaido 060 8589, Japan 2 Laboratory of Microbiology, Study Program for Agricultural Technology, Udayana University, Bali, Indonesia 3 Laboratory of Applied Microbiology, Department of Molecular Bioscience, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, North 9, West 9, Sapporo, Hokkaido 060 8589, Japan 4 Hokkaido Food Processing Research Center, Ebetsu shi, Bunkyodai Midori Machi 589-4, Hokkaido, Japan *Author for correspondence: Tel.: þ81-11-706-4185, Fax: þ81-11-706-4961, E-mail: asanok@chem.agr.hokudai.ac.jp   Present address: The University of the Air, Hokkaido Study Center, North 17, West 8, Sapporo, Hokkaido 060 0817, Japan Received 23 May 2003; accepted 4 September 2003 Keywords: Brem, Candida, Issatchenkia, ITS, PFGE, Pichia, 18S rDNA, Saccharomyces Summary Fifty-one yeast strains isolated from fermented mash of Balinese rice wine, brem, fermented using five different types of starters, ragi tape, were identified on the basis of their internal transcribed spacer (ITS) regions and their 18S rDNA sequences. The results revealed that Saccharomyces cerevisiae (35 strains), Candida glabrata (six strains), Pichia anomala (three strains) and Issatchenkia orientalis (seven strains) were the main yeasts in the fermentation of the rice wine. These yeasts undergo succession during the fermentation in which S. cerevisiae was mostly found as the principal yeast at the end of fermentation. Phylogenetic analysis based on the 18S rDNA sequences of selected strains placed the isolated S. cerevisiae strains in the Saccharomyces sensu stricto group. Karyotype analysis of the S. cerevisiae strains resolved using pulsed field gel electrophoresis (PFGE) showed that the strains are typically associated with different types of starters. Introduction Brem is an alcoholic beverage brewed traditionally in Bali Island, Indonesia, from a mixture of black and white glutinous rice using a dry-starter, which is locally called ragi tape. The steamed rice is liquefied by sprinkling powdered ragi tape on it and incubated for 5 days at room temperature (about 27 °C). Since this process produces a must of fermented rice, called tape in the local language, therefore it is often referred as tape fermentation (Djien 1972; Cronk et al. 1977, 1979; Ardana & Fleet 1989; Steinkraus 1996). The tape-mash then is pressed to obtain the tape-juice and fermented in closed-containers (or tanks) for about 6 months to produce a clear-brownish liquid, brem. This rice wine has a sweet, sour and alcoholic taste. Brem is a produced utilizing the traditional techniques (Steinkraus 1996), resulting in a product with inconsis- tent quality. Though this rice wine has been produced for centuries through successive generations, still not much attention has been given to improving its produc- tion method. Ragi tape is used as inoculum in a production method where liquefaction and alcoholic fermentation occur simultaneously without any effort to control the fermentation. Consequently, the quality of brem varies considerably depending on the microflora of ragi tape. In addition, the taste of brem is too sweet, containing 5–10% (w/v) of residual glucose and with 11–15% (w/v) ethanol (unpublished observation). Numerous researchers have reported the yeasts present in ragi tape and its principal product, tape (Hesseltine 1965, 1983; Dwijosepoetro & Wolf 1970; Ardana & Fleet 1989). Recently, Kuriyama et al. (1997) reported that Saccharomycopsis fibuligera and Pichia anomala were the principal amylolytic and ethanol- producing yeasts, respectively, found in Indonesian cassava-tape and ragi tape. Nevertheless, the identity of the yeasts that grow after the tape fermentation or during the liquid fermentation of brem has never been published. Therefore, this study was aimed at identifying and verifying the diversity of yeasts involved in the alcoholic fermentation of brem. The results should provide the basic information for improving the fer- mentation for a better quality of brem. Journal : WIBI MS Code : 4025 PIPS No. : 5151537 h TYPESET h DISK h LE h CP Dispatch : 29-9-2003 Pages : 8 4 4 World Journal of Microbiology & Biotechnology 20: 000–000, 2003. 1 Ó 2003 Kluwer Academic Publishers. Printed in the Netherlands. UNCORRECTED PROOF! PDF-OUTPUT