Yeast community associated with the solid state fermentation of traditional Chinese Maotai-flavor liquor
Introduction
Maotai-flavor liquor is a symbolic drink in China just as whisky in Scotland, and brandy in France (Fan et al., 2011, Xu and Ji, 2012). It is famous for its soy sauce-like and roasted aroma style. This is due to a unique and complicated spontaneous fermentation process, which includes Daqu (the starter) making, stacking fermentation and liquor fermentation stages (Fig. 1). In the Daqu making stage, the temperature of Daqu increases as the fermentation processes, and the maximum temperature reaches about 60 °C. In stacking fermentation, the mixtures of ground Daqu and steamed sorghum are piled up as a cone on the ground for stacking fermentation, and are terminated when the temperature on the top of the stack reaches about 50 °C. Then the fermented grain (Zaopei) is put into the underground cubic pits, and sealed for liquor fermentation. After 30 days of fermentation, the ethanol content in Zaopei reaches about 4%–6% (w/w). Then Zaopei is distilled and liquor is collected, and the yield of liquor is about 13.4% (v/w) with the average alcohol content of 60% (v/v). It is a statically solid-state fermentation, and qualities of liquors distilled from different layers of the pit are different, which are called top (soy sauce flavor), middle (ethanol-sweet) and bottom (ester fragrant) liquors, separately.
Such a solid state and spontaneous fermentation process accumulates a specific microorganism's community, including fungi, yeasts and bacteria. Previous studies have shown that the stacking and liquor fermentation processes are microbiologically and biochemically complex, but they have not been studied in depth (Wang et al., 2011, Wu et al., 2009, Wu et al., 2012). Fungal species are significant in the stacking fermentation, where they produce the amylolytic enzymes to degrade the starch material in the raw material, sorghum, to fermentable sugars such as glucose and maltose (Wang et al., 2008, Wu et al., 2009). These then become substrates for alcoholic fermentation and flavor production by yeasts and bacteria. Lactic acid bacteria are the main producers of lactic acid and provide substrates for esterification of yeasts (Wang et al., 2011, Wu et al., 2009). Yeasts represent the most important group of microorganisms in the fermentation process, since they contribute significantly to the fermentation rate, product flavor and quality. Therefore, learning yeast community structures and dynamics during the fermentation process would shed light on the yeast function and the fermentative mechanism.
So far, yeast community structures in many fermentation alcoholic beverages have been investigated. For instance, yeast community structure and dynamics in wine fermentation have been clearly described. Generally, Hanseniaspora (Kloeckera), Candida and Metschnikowia are the main species initiating the wine fermentation, but they begin to decline and die off after mid-fermentation, and Saccharomyces cerevisiae becomes predominant and continues the fermentation until its completion (Fleet, 2003). However, for Chinese Maotai-flavor liquor, the long-period (2–7 days of stacking fermentation and 30 days of liquor fermentation) and unique simultaneous saccharification and fermentation with the solid-state fermentation process give birth to a more complex yeast community structure in this liquor, which is also significant to be revealed.
The yeast communities of Chinese Maotai-flavor liquor have been investigated since the 1960's (Xu and Ji, 2012, Wang et al., 2011). Five yeast genera were revealed during the ripening of Daqu and these were Saccharomyces, Hansenula, Candida, Pichia, and Torulaspora (Wang et al., 2008). Using a nested PCR-denaturing gradient gel electrophoresis technique, Liu et al. (2012) reported contributions from Hanseniaspora uvarum and Candida allociferrii during the shaping, ripening and drying of Daqu. Nine yeast species were isolated from the stacking and liquor fermentation stages and identified by 26S rDNA sequencing as S. cerevisiae, Zygosaccharomyces bailii, Pichia membranifaciens, Schizosaccharomyces pombe, Rhodotorula mucilaginosa, Kazachstania exigua, Debaryomyces hansenii, Issatchenkia orientalis (Pichia kudriavzevii) and Galactomyces geotrichum (Wu et al., 2012). However, the yeast community structure and dynamics, as well as the relationship between yeast and fermentation parameters, have not been systematically investigated.
To improve the quality of Maotai-flavor liquor and the efficiency of its production, a more detailed study of the association of yeasts with the production process is required. In this study we reported a detailed investigation of the yeast species associated with the Daqu, stacking and liquor stages of the fermentation process.
Section snippets
Sampling
Sampling was carried out in Guizhou Province, China. Within this factory, two separate liquor making processes were investigated with samples taken for each process from the Daqu, stacking fermentation and liquor fermentation stages. The results were the average of the data sets from the two separate fermentation processes.
Samples of Daqu were collected from Daqu powders before they were used for stacking fermentation. As shown in Fig. 2, samples of Zaopei in stacking fermentation were
Yeast community structure and dynamic analyses in Daqu and stacking fermentation
All the isolated yeast species were divided into 9 types by WLN medium and PCR–RFLP analysis of the 5.8S ITS rDNA region. They were assigned to the species S. cerevisiae, Z. bailii, P. membranifaciens, S. pombe, R. mucilaginosa, K. exigua, D. hansenii, P. kudriavzevii, and G. geotrichum based on the sequencing of D1/D2 domains of the 26S rDNA genes (Wu et al., 2012).
With the unique colony properties (texture, surface, margin, elevation and color) of different yeast species on WLN medium, yeast
Conclusion
It is a unique yeast community structure in the Chinese Maotai-flavor liquor making process. A total of 9 yeast species were discovered in this process. There were only four dominant yeast species in both stacking and liquor fermentation, including S. cerevisiae, Z. bailii, P. membranifaciens and S. pombe. Their dominance, indicating that they might play important roles in liquor fermentation, should be further investigated.
The yeast community in different fermentation stages played different
Acknowledgments
This work was supported by the National High Technology Research and Development Program of China (2012AA021301, 2013AA102108), the National Natural Science Foundation of China (31000806), the Cooperation Project of Jiangsu Province Among Industries, Universities and Institutes (BY2010116), and the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-06).
References (18)
- et al.
Partial 26 s rDNA restriction analysis as a tool to characterise non-Saccharomyces yeasts present during red wine fermentations
International Journal of Food Microbiology
(2005) Yeast interactions and wine flavour
International Journal of Food Microbiology
(2003)- et al.
Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes
FEMS Yeast Research
(2004) - et al.
Microorganisms in Daqu: a starter culture of Chinese Maotai-flavor liquor
World Journal of Microbiology and Biotechnology
(2008) - et al.
Moutai (Maotai): production and sensory properties
- et al.
Identification of yeasts by RFLP analysis of the 5.8 s rRNA gene and the two ribosomal internal transcribed spacers
International Journal of Systematic and Evolutionary Microbiology
(1999) - et al.
Quantification of volatile compounds in Chinese soy sauce aroma type liquor by stir bar sorptive extraction and gas chromatography–mass spectrometry
Journal of the Science of Food and Agriculture
(2011) - et al.
Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8 s rRNA gene and the two internal transcribed spacers
FEMS Yeast Research
(2003) - et al.
Determination of microbial diversity in Daqu, a fermentation starter culture of Maotai liquor, using nested PCR-denaturing gradient gel electrophoresis
World Journal of Microbiology and Biotechnology
(2012)
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