title: Development of internal transcribed spacer regions amplification restriction fragment length polymorphism method and its application in monitoring the population of Zygosaccharomyces rouxii M2 in miso fermentation.
abstract: Recently, the use of the dry yeast of Zygosaccharomyces rouxii M2 for miso (soybean paste) fermentation has been established. A molecular monitoring method was developed and validated in this study to analyze the population of Z. rouxii M2 during the fermentation. The method was based on the restriction patterns of internal transcribed spacer (ITS) regions of the rDNA using HaeIII and HhaI. Among the homologous ITS regions of Z. rouxii strains, Z. rouxii M2 produced diagnostic bands by which it can be differentiated from the other strains used. The specific restriction bands were due to the difference in nucleotide sequence of two different copies of ITS of Z. rouxii M2. Both ITS copies showed 94% sequence similarity but a 13-bp nucleotide substitution and a 19-bp deletion were found in the ITS1 region. Phylogenetic trees were constructed based on ITS and 18S rDNA sequences and it was found that the ITS sequences provide better resolution for the classification of Z. rouxii M2. Since Z. rouxii M2 is a promising strain for use in miso fermentation as a dry starter, the method developed is significant in terms of industrial application in monitoring the growth of Z. rouxii M2 in miso fermentation.
authors.affiliation: Laboratory of Microbial Resources and Ecology, Graduate School of Agriculture, Hokkaido University, Kita 9 Jo, Nishi 9 Chome, Kita-ku, Sapporo, Hokkaido 060-8589, Japan.
authors.forename: I Nengah
authors.initials: IN
authors.lastname: Sujaya
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Tamura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Tanaka
authors.forename: Tazusa
authors.initials: T
authors.lastname: Yamaki
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ikeda
authors.forename: Nao
authors.initials: N
authors.lastname: Kikushima
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yata
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yokota
authors.forename: Kozo
authors.initials: K
authors.lastname: Asano
authors.forename: Fusao
authors.initials: F
authors.lastname: Tomita