Zuo et al. Annals of Microbiology (2020) 70:57 Annals of Microbiology https://doi.org/10.1186/s13213-020-01598-1 ORIGINAL ARTICLE Open Access Evaluation of bacterial diversity during fermentation process: a comparison between handmade and machine-made high-temperature Daqu of Maotai-flavor liquor Qiancheng Zuo, Yongguang Huang* and MinGuo Abstract Purpose: High-temperature Daqu is a traditional fermentation starter that is used for Chinese Maotai-flavor Baijiu production. Although the bacteria in high-temperature Daqu are known to be responsible for developing the quality and flavor of Baijiu during the fermentation process, there is little information on the properties of the bacteria during the fermentation of high-temperature Daqu, especially machine-made high-temperature Daqu. This has limited the development of the Maotai-flavor Baijiu industry, particularly with regard to the mechanized production of Maotai-flavor Baijiu. Methods: Illumina MiSeq high-throughput sequencing was applied to study bacterial compositions during the fermentation of handmade and machine-made high temperatures. Results: The results show that bacterial diversity in machine-made Daqu was similar but higher than that in handmade Daqu at the end of fermentation, and there was no significant difference between the methods with regard to the dominant genera and their dynamic changes during fermentation. Rhizobium, Bacillus, Thermoactinomyces, Weissella, Lactobacillus, and Saccharopolyspora were the dominant genera during the fermentation of both Daqus, although the relative abundance of these dominant genera differed between the two methods. Interestingly, the machine-made Daqu contained a higher relative abundance of Bacillus than handmade Daqu at all fermentation times. Bacillus is the most important functional bacteria in the fermentation of Maotai- flavor Baijiu, suggesting that mechanical-molding methods could be applied to industrial Maotai-flavor Daqu production. Conclusion: These results suggest that mechanical-molding methods could be applied to industrial Maotai-flavor Daqu production, which could be helpful for industrial Maotai-flavor Baijiu production and the development of fermentation technology. Keywords: Maotai-flavor Daqu, Fermentation, Bacterial diversity, Handmade, Machine-made * Correspondence: [email protected] School of Liquor and Food Engineering, Guizhou University, Guiyang 552107, China © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Zuo et al. Annals of Microbiology (2020) 70:57 Page 2 of 10 Introduction (Fan et al. 2011; Fan et al. 2007; Wang et al., 2017a, b, c, Traditional Chinese liquor (Baijiu), alongside brandy, d; Wu and Xu 2013), which are critical for the flavor whiskey, vodka, gin, and rum, has been classed as one of and quality of the resultant Baijiu. The bacterial commu- the six world-renowned distilled spirits (Liu and Sun nities of HT Daqu of Maotai-flavor liquor have been 2018). It is usually divided into five categories, soy sauce extensively investigated in several studies (Wang et al. aroma, strong aroma, fragrant, sweet honey, and others, 2008; Liang et al. 2015; Wang et al., 2017a, b, c, d; Tang based on typical aromatic characteristics. Among these, et al. 2019 ), but most of the studies focused on the fin- the soy sauce aroma (also known as Maotai-flavor) ished Daqu product. During the entire fermentation accounts for 3% of Chinese Baijiu production and con- process of HT Daqu, the composition and abundance of tributes to 15 and 40% of Chinese Baijiu sales and profit, bacterial communities must undergo complex changes. respectively, with an annual output of more than $150 Although the bacterial communities in HT Daqu of billion. Maotai-flavor Baijiu is the most typical and high- roasted sesame-like flavor liquor have been studied est quality Baijiu because of the complex processes during HT Daqu fermentation (Su et al. 2015), bacteria involved in its production (Xiu et al. 2012). Famous for involved in the HT Daqu of Maotai-flavor liquor have its highly complex, sweet, and refreshing flavor, Maotai- not been studied, and the two kinds of HT Daqu are flavor Baijiu is also the most popular distilled Baijiu in very different. The characteristics of bacterial communi- China (Wu et al. 2012). Its characteristics are mainly ties in the fermentation processes of machine-made HT derived from high-temperature Daqu—a traditional Daqu and HT Daqu of Maotai-flavor liquor are still un- fermentation starter (Wang et al., 2017a, b, c, d). known. In particular, the bacterial community structures Daqu, the saccharification-fermentation starter of trad- involved in the fermentation processes of mechanical itional Chinese Baijiu, is mainly composed of diverse mi- and manual Daqu production have never been com- crobes, functional enzymes, and flavor compounds pared. This has limited the development of the Maotai- (Tang et al. 2019). As the primary microbial and enzyme flavor Baijiu industry, particularly with regard to the source for Chinese Baijiu fermentation, it can determine mechanized production of Maotai-flavor Baijiu. In this the quality of the final Baijiu products. Daqu can be clas- study, we analyzed and compared the microbial commu- sified into three types based on the fermentation nity structure and the vital microorganisms in the man- temperature: low-temperature (LT, 40–50 °C), medium- ual and mechanized HT Daqu fermentation processes temperature (MT, 50–60 °C), and high-temperature (HT, using Illumina MiSeq analysis. 60–65 °C) (Li et al. 2016). Daqu, especially HT Daqu, has a unique and dynamic combination of microbiota, Materials and methods and the dominant microbial flora in HT Daqu is bac- Sample collection teria, rather than fungi, as these microbes are better HT Daqu samples were provided by a famous Maotai- adapted to high temperatures (Zheng and Han. 2016). flavor Baijiu distillery in Guizhou province, China (27° The traditional preparation process of HT Daqu 81’N; 106° 41′ E). Two batches of Daqu in fermentation contains three main stages, as shown in Fig. 1: (i) water workshops were selected to collect samples (workshops infiltration of wheat and grinding, (ii) shaping into H and M). Workshop H used manual molding of Daqu bricks, and (iii) fermentation (approximately 40 days). bricks, whereas Workshop M used mechanical molding. The shaping stage, also called the stepping starter stage, The same raw materials and fermentation conditions for involves pressing the raw material into brick shapes to Daqu production were used in the two batches of Daqu preserve the microorganisms and their metabolites, and in the fermentation workshops. Based the on preliminary it is a critical process in the production of HT Daqu. In data, the 0th (bricks placed in Daqu room), 7th (first this stage, two brick-molding methods can be used, turning of Daqu bricks), 14th (second turning of Daqu namely, manual or mechanical. The mechanical molding bricks), and 40th (end of fermentation) days are essential methods can decrease the influence of operators, labor for the fermentation process; therefore, samples were costs, and production time compared with manual collected on days 0, 7, 14, and 40 of fermentation. As processes. Although there are many advantages with shown in Table 1, Daqu samples produced by hand were mechanical molding compared with traditional manual labeled H0 (fermented for 0 days), H7 (fermented for 7 methods, there is always the question of whether the mi- days), H14 (fermented for 14 days), and H40 (fermented crobial structure of Daqu produced by the mechanical for 0 days). Similarly, Daqu produced by machinery was process is comparable to that produced by traditional named M0, M7, M14, and M40. At each sampling event, manual methods. nine samples were collected from the upper, middle, and During Maotai-flavor Baijiu fermentation, the func- lower parts of the Daqu room, specifically, near the tional bacteria and their metabolites in HT Daqu door, in the middle of the Daqu room, and near the win- contribute to the production of aromatic compounds dow. To represent a sample of the whole fermentation Zuo et al. Annals of Microbiology (2020) 70:57 Page 3 of 10 Fig. 1 Process diagram for the production of high-temperature Daqu. Wheat is mixed with 10% (w/w) water and squeezed, and then broken into three or four pieces to adequately release the starch. Then, 40% (w/w) water is added to the wheat, and the mixture is shaped by hand or machine. After the mixture are shaped to bricks weighing 4 to 5 kg each, the bricks are placed in a Daqu room, namely a fermentation chamber, stacked into 4 to 5 layers and fermented for 40 days. Microorganisms, mostly bacteria, are provided by the wheat, water, and air. During the fermentation, workers cover the bricks with rice straw to maintain heat for microbial growth and flavor compound formation. Daqu is turned over to lower the temperature, involving the first turning of Daqu bricks (the 7th day of fermentation) and second turning of Daqu bricks (the 14th day of fermentation). Daqu needs to mature during storage for about 6 months before use room, the nine samples were then thoroughly mixed to 55 °C for 30 s, and extension at 72 °C for 45 s, with a provide a representative sample for each time point.