Yeast diversity of traditional alcohol fermentation starters for Hong Qu glutinous rice wine brewing, revealed by culture-dependent and culture-independent methods

Abstract

Hong Qu glutinous rice wine, one of the most popular traditional rice wines in China, is brewed from glutinous rice with the addition of two traditional fermentation starters – Hong Qu (hóng qū) and Yao Qu (yào qū). The aim of this study was to investigate the yeast flora present in these fermentation starters using a combination of culture-dependent and -independent molecular biological methods.Molecular identification of a total of 500 yeast isolate from 10 kinds of representative wine fermentation starters (5 Hong Qu and 5 Yao Qu) was achieved by a combination of polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis of the internal transcribed spacer region ITS1-5.8S-ITS2 and sequencing of the D1/D2 domain of the 26S rRNA gene. The PCR–RFLP method generated 13 different ITS/RFLP profiles. Sequencing identification allowed identifying 13 different ITS/RFLP profiles into 12 different species of yeast belong to eight different genera (Pichia, Saccharomyces, Candida, Cryptococcus, Rhodotorula, Sporobolomyces, Rhodosporidium and Saccharomycopsis). Species diversity was thus considerable: Saccharomycopsis fibuligera was the most commonly isolated specie, followed by Saccharomyces cerevisiae. On the other hand, the yeast diversity associated with wine starters for Hong Qu glutinous rice wine was also investigated through culture-independent method using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) patterns and sequencing of the DNA bands. The results revealed by culture-independent method were almost the same as that of culture-dependent methodology. However, several yeast species can not be detected by traditional microbiological procedures but can be detected using PCR–DGGE analysis. Similarly, some species could only be detected by culture-dependent method. For example, Sporobolomyces nylandii was detected existing in wine starter samples (Q7 and Q10) through culture-dependent method, but not detected in wine starters for Hong Qu glutinous rice wine through fungal PCR–DGGE assay. Therefore, it is recommended that polyphonic approaches based on culture-dependent and culture independent methods seem the best strategy to get a more comprehensive picture of the microbial diversity in traditional wine starters. These results enrich our knowledge of rice wine-related yeasts, and can be used to promote the development of the traditional brewing industry.