The bacterial communities of Daqu at different storage periods were analyzed with high- throughput sequencing, which uncovered totally 20 phyla, 38 classes, 84 orders, 138 families, 268 genera and 396 species. With the threshold at relative abundance > 0.5% at the phylum level, 6 predominant bacterial phyla were identified from the 3-month-old Daqu, including Firmicutes (62.66%), Proteobacteria (27.15%), Bacteroidetes (4.40%), Cyanobacteria (1.89%), Actinobacteria (1.30%) and Chloroflexi (0.70%). Six predominant phyla were found in the 6-month-old Daqu, including Firmicutes (63.53%), Proteobacteria (15.26%), Bacteroidetes (14.87%), Spirochaetae (3.08%), Fusobacteria (2.06%) and Actinobacteria (0.65%). Four predominant phyla were identified from the 9-month-old Daqu, including Proteobacteria (80.86%), Firmicutes (17.39%), Bacteroidetes (0.63%) and Cyanobacteria (0.61%). With the prolonging of storage period, the abundance levels of Firmicutes and Bacteroidetes both first rose and then declined, while those of Proteobacteria and Cyanobacteria first dropped and then increased. At the relative abundance > 0.5%, both Actinobacteria and Chloroflexi were only detected in the 3-month-old Daqu. As reported, predominant phyla including Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria were detected from Daqu using high-throughput sequencing (Liang et al. 2017), which are very similar to our results, but we also detected Spirochaetae and Fusobacteria.
With the threshold at relative abundance > 1% at the genus level, 10 predominant bacterial genera were recognized from the 3-month-old Daqu, including Lactococcus (27.90%), Enterobacter (23.14%), Lactobacillus (14.81%), Weissella (7.12%), Bacillus (2.32%), Pediococcus (1.97%), Staphylococcus (1.61%), norank c Cyanobacteria (1.48%), Enterococcus (1.48%), and norank f Bacteroidales S24-7 group (1.00%). 8 predominant genera were recognized from the 6-month-old Daqu, including unclassified f Peptostreptococcaceae (24.64%), Escherichia-Shigella (11.80%), Terrisporobacter (6.47%), Clostridium sensu stricto 1 (5.68%), Lactobacillus (4.61%), norank f Bacteroidales S24-7 group (3.65%), Ruminococcaceae UCG-005 (2.88%), and Rikenellaceae RC9 gut group (2.63%). 4 predominant genera were recognized from the 9-month-old Daqu, including Enterobacter (79.80%), Lactobacillus (6.32%), Weissella (4.59%) and Pediococcus (1.59%). At the genus level, the bacterial community compositions of Daqu varied largely among different storage periods. The absolutely dominant genera in the 3-month-old Daqu included Lactococcus and Enterobacter. The absolutely dominant genera in the 6-month-old Daqu included unclassified f Peptostreptococcaceae and Escherichia-Shigella. The absolutely dominant genus in the 9-month-old Daqu was Enterobacter. At the relative abundance > 1%, Terrisporobacter, Clostridium sensu stricto 1 and Ruminococcaceae UCG-005 were found only in the 6-month-old Daqu.
Studies have shown that Coccus and Bacillus are the main force of bacteria in Daqu.The dominant genera identified from the 3- or 9-month-old Daqu mostly can produce lactic acid, but excessive ethyl lactate concentrations will cause inharmonious fragrance in liquors(He et al. 2018; Zheng et al. 2013). The exclusive genera found in the 6-month-old Daqu were Terrisporobacter, Clostridium sensu stricto 1, and Ruminococcaceae UCG-005, which were caproic acid bacteria, metabolites of which are the major fragrant substances of Luzhou-flavor liquors. The Lactobacillus, Lactococcus and Weissella identified from the 3-month-old Daqu were all lactic acid bacteria(Horvath et al. 2009). During the production of white liquors, lactic acid bacteria can ferment carbohydrates, produding abundant lactic acids, but excessive lactic acids will lead to a bitter taste of liquors and decrease the liquor quality. Moreover, the acid environment will produce bioamine(Linares et al. 2011), but the intake of excessive bioamine into the body will cause adverse reactions(Mah et al. 2002). The Clostridium sensu stricto 1 identified from the 6-month-old Daqu belongs to Clostridia, which is a dominant class of caproic acid bacteria during the production of Luzhou-flavor liquors(Hu et al. 2015). Terrisporobacter and Ruminococcaceae are also caproic acid bacteria(Zheng et al. 2013). The Enterobacter found in the 9-month-old Daqu can ferment glucoses to form lactic acid(Li et al. 2015) and is also a bioamine-producing bacterium in Daqu (Curiel et al. 2011). At the same time, the high content of ethyl lactate formed by lactic acid can inhibit the main aroma of liquor(Li et al. 2015).Based on the characteristics of the bacteria mentioned above and the diversity and abundance of bacterial communities in the 6-month-old Daqu, we suggest the storage period of Daqu for fermentation is 6 months. Liu Junhong et al. studied the growing rules of microorganisms in Daqu after different storage periods by using the traditional isolating culture and found the 4- to 6-month-old Daqu masses were suitable for fermentation(Liu et al. 2009). With high-throughput sequencing, Liang et al.(2017)explored the prokaryotic microoganism composition and flavor compositional variation of 0- to 6-month-old Daqu and found the 4- to 6-month-old Daqu was more suitable for fermentation of high-quality liquors. Our findings are basically consistent with these studies.
Moreover, the species identified from Daqu of Luzhou-flavor liquors for the first time are norank c Cyanobacteria, Escherichia-Shigella, Terrisporobacter, norank f Bacteroidales S24-7 group, Ruminococcaceae UCG-005, and Rikenellaceae RC9 gut group. With high-throughput sequencing, Hu et al. detected norank c Cyanobacteria from ensilage corn straws at the genus level(Hu et al. 2018). Dai Yijie et al. found with high-throughput sequencing that the dominant genus in the Daqu of Maotai-flavor liquors was Escherichia-Shigella (Dai et al. 2019). Terrisporobacter was found in the washes of Luzhou-flavor liquors(Li et al. 2018). High-throughput sequencing also identified norank f Bacteroidales S24-7 group in the intestinal tracts of mice(Li et al. 2018). Moreover, high-throughput sequencing identified Ruminococcaceae UCG-005 and Rikenellaceae RC9 gut group in the microbial communities of milch cow claw corners (Bay et al. 2018). Nevertheless, the functions of bacteria in the fermentation of Luzhou-flavor liquors should be further investigated.
Totally 396 bacterial species were identified through high-throughput sequencing, and a resource library of Daqu bacteria from Luzhou-flavor liquors after different storage periods was preliminarily established. The information of bacteria was richer compared with traditional isolating culture. This study enriches the knowledge on the diversity of bacterial communities in the Daqu of Luzhou-flavor liquors and will theoretically underlie the rational judgement of Daqu storage time during practical productions and promote the standardized and scientific Daqu production.