Characterization of fungal community and dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine by means of multiple culture-independent methods

Abstract

The objective of this study was to investigate the fungal community and dynamics during the traditional fermentation of Wuyi Hong Qu (black-skin-red-koji) glutinous rice wine using culture-independent approaches based on nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE), 18S ribosomal RNA (rRNA) gene clone libraries and species-specific quantitative PCR (qPCR). Both of nested PCR-DGGE and 18S rRNA gene clone libraries revealed that the predominant microorganisms varied in different brewing phases. The relative proportions of some fungal species (such as Monascus purpureus, Rhizopus oryzae, Pichia guilliermondii and Saccharomycopsis fibuligera) detected at the early brewing stage decreased as the fermentation progressed, while Saccharomyces cerevisiae became the dominant species at the later stage. The present study revealed that PCR-DGGE fingerprint and 18S rRNA gene clone libraries presented the similar results. Nevertheless, the differences of the fungal species detected by the two different molecular biological methods demonstrated that the combined approach of nested PCR-DGGE and 18S rRNA gene clone libraries would give a more comprehensive profile of the fungal dynamics than either alone. Finally, species-specific qPCR was also performed to quantify some dominant fungal species detected by nested PCR-DGGE and 18S rRNA gene clone libraries. Results showed that R. oryzae increased sharply and dominated during the early brewing period, but decreased continuously from day 10 to the end of the traditional brewing. S. fibuligera and P. guilliermondii were the dominant yeast species at early stage, while S. cerevisiae increased sharply during the early phase and became the primary species at the end of the traditional brewing process. This study provides the first detailed evaluation of fungal community and dynamics in Wuyi Hong Qu glutinous rice wine traditional brewing process using multiplex culture-independent methods, which would show great value in scientifically understanding of the brewing mechanism and be useful for the selection of the suitable and beneficial fungal strains to improve the wine quality.