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PCR-DGGE Analysis of the Microbial Communities in Three Different Chinese “Baiyunbian” Liquor Fermentation StartersS
PCR-DGGE Analysis of the Microbial Communities in Three Different Chinese “Baiyunbian” Liquor Fermentation StartersS
Journal of Microbiology and Biotechnology. 2014. Aug, 24(8): 1088-1095
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : January 23, 2014
  • Accepted : May 03, 2014
  • Published : August 30, 2014
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About the Authors
Xiaomao Xiong
Hubei Baiyunbian Liquor Industry Co. Ltd., Songzi, Hubei 434200, P.R. China
Yuanliang Hu
College of Life Sciences, Hubei Normal University, Huangshi, Hubei 435002, P.R. China
Nanfeng Yan
State Key Laboratory of Agricultural Microbiology, and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Yingna Huang
State Key Laboratory of Agricultural Microbiology, and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Nan Peng
State Key Laboratory of Agricultural Microbiology, and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Yunxiang Liang
State Key Laboratory of Agricultural Microbiology, and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Shumiao Zhao
State Key Laboratory of Agricultural Microbiology, and College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
shumiaozhao@mail.hzau.edu.cn

Abstract
A systematic investigation was performed on the bacterial, Bacillus , fungal, and yeast communities of the three types of Daqu (mechanically prepared, manually prepared, and mixed prepared) used in Baiyunbian Company by reconditioning PCR-denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE results showed that the microbes in the three types of Daqu were mainly thermotolerant and thermophilic microbes, and the most dominant bacterial species were Bacillus and Virgibacillus , followed by Lactobacillus and Trichococcus . Furthermore, the dominant fungi were found to be molds, such as Rasamsonia , Penicillium , Aspergillus , and Monascus , and the dominant yeasts were Saccharomyces cerevisiae , Saccharomycopsis fibuligera , Pichia anomala , and Debaryomyces hansenii . In general, the three types of Daqu showed slight differences in microbial communities, and the Shannon indexes (H’) of the manually prepared and mechanically prepared Daqu were similar. The results suggest that mechanically prepared Daqu can replace manually prepared Daqu in liquor production, and this research provides useful information for liquor production and process improvement.
Keywords
Introduction
Chinese liquor is one of the six most well-known distilled spirits in the world, with a production of 12.26 million liters in 2013. Chinese liquor can be categorized into five aroma styles: soy sauce aroma, strong aroma, light aroma, sweet honey, and miscellaneous aroma [29] . The miscellaneous aroma style includes several types that are derived from the other four styles. “Baiyunbian” liquor, a major liquor in China (production of 0.2 million liters/ year), is a typical representative of Maotai-Luzhou-flavor liquors. Its production cycle of 300 days yields a unique aroma style that is a combination of the soy sauce aroma (Maotai-flavor liquor) and the strong aroma (Luzhou-flavor liquor).
Daqu , an ancient fermentation starter, is produced by solid-state fermentation of wheat, barley, and/or peas, with the ingredients being formulated by grinding and mixing, shaping, incubation, and maturation [32 , 36 , 37] . Daqu is prepared through the natural inoculation of bacteria, molds, and yeasts derived from raw materials, air, ground, the human body, and other related materials in nature. Daqu is an important saccharifying and fermentation agent for the production of Chinese liquor, and provides nutrients for microbial growth. The numerous flavor compounds derived from the microbes present in Daqu influences the liquor quality during fermentation [30 , 36 , 37] .
By both culture-dependent and culture-independent methods, a large variety of microbes have been detected in Daqu . These are considered to be the functional microbiota in Daqu responsible for the formation of a range of lytic enzymes, substrates for alcoholic fermentation, and flavor compounds [24 , 28 , 30 , 36] . Daqu substrates vary in their specific microbial compositions. This variation may arise from intrinsic and extrinsic factors, such as the raw material, moisture, temperature, and environment, that influence the microbial community of Daqu [30] . Because the majority of microbes in Daqu can not be cultured under laboratory conditions, the investigation of Daqu microflora by culture-independent methods has attracted increasing interest from researchers. PCR-denaturing gradient gel electrophoresis (DGGE) is a useful tool for investigating the composition of microbial communities, and has been used successfully to study the taxonomy of the microbial communities in fermentation products [4 , 20 , 32 , 33 , 37] . However, to date, only a few studies about the microflora in Daqu have been performed using culture-independent methods.
In past centuries, Daqu samples were manually prepared. This required a large amount of work force and increased the costs. Thus, in recent years, to lower the costs and to improve the production process, increasing amounts of Daqu were mass-produced by machine. Currently, the Baiyunbian Company uses three types of Daqu prepared by machine, humans, or a combination of machine and human (mixed prepared: mixing material by machine and stepping up the starter by humans). To determine the differences between the three types of Daqu , a systematic investigation was performed to determine the composition of the general bacterial, Bacillus , fungal, and yeast communities in Daqu by using reconditioning PCR-DGGE. The results may help to facilitate liquor production and process improvement.
Materials and Methods
- Sample Collection
The three types of high-temperature Daqu (mechanically prepared, manually prepared, and mixed prepared) were obtained from Baiyunbian Liquor Industry Co. Ltd. in Songzi, Hubei, China. Five representative samples were collected for each Daqu by the quartering method. The samples were transferred to sterile bags, sealed, and stored at 4℃ for analysis within 48 h.
- DNA Extraction
Ten grams of each powdered Daqu sample was homogenized in 90 ml of 0.85% (w/v) sterile physiological saline, and subsequently filtered through four layers of sterile cheesecloth. The filtrates were centrifuged at 14,000 × g , 4℃ for 10 min, and then the pellets were subjected to DNA extraction using the commercial PowerSoil DNA Isolation Kit (Mo-Bio, Carlsbad, USA). For elution, the matrix-DNA complex was resuspended in 100 μl of elution buffer and stored at -20℃ before use. The DNA concentration and quantity were tested on a Nanodrop ND-100 spectrophotometer (Thermo, Wilmington, USA). The extracted DNA was then used as a template for PCR.
- PCR Amplification
Primers used in this study are shown in Table 1 . The 50 μl reaction mixture consisted of 5 μl of 10× PCR buffer (Takara, Dalian, China), 4 μl of dNTPs (2.5 mM of each; Takara), 1 μl of each primer (10 μM), 2.5 U of Taq polymerase (Takara), and 50 ng of template DNA. A Bio-Rad T100 Thermal Cycler (Bio-Rad, Hercules, USA) was used for amplification according to Table S1. After the first PCR, a reconditioning PCR was performed as previously described [26] .
Primers used in the study.
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Primers used in the study.
To amplify Bacillus selectively, a three-step nested PCR approach was applied as previously described [4] . For the first PCR (referred to as “selective PCR”), primer sets pBF and pHR were used. The forward primer pBF was designed specifically for Bacillus and related genera, and the reverse primer pHR was a universal primer. The first PCR was performed using the 50 μl reaction system described above by following the PCR procedures listed in Table S2. Subsequently, the first PCR product was diluted (10-fold) and used as a template for a nested PCR targeting the V9 region of the 16S rRNA gene using primers Ec1055F and Ec1392- GC, designed specifically for Bacillus species. Finally, a reconditioning PCR was performed as described above to generate products for DGGE analysis.
- DGGE Analysis
Before DGGE analysis, the five PCR products from each Daqu were mixed together as one sample, and then the three mixed PCR samples were c leaned u sing a P C R Purification K it ( Omega, Norcross, USA). Using a Model 475 Gradient Delivery System (Bio-Rad), gels containing 8% (w/v) polyacrylamide (acrylamide/ bisacrylamide, 37.5/1) were formed in 1.0× Tris-acetate-EDTA (TAE) buffer (pH 8.0) with a 40% to 60% urea-formamide denaturing gradient (100% corresponding to 7 M urea and 40% deionized formamide). The gels formed were allowed to polymerize for 4 h before use. DGGE analysis was performed using the DCode Universal Mutation Detection System (Bio-Rad) by electrophoresing 20 μl of PCR products (25 ng/μl, plus 10 μl loading buffer) at 10 V for 10 min and then at 110 V for 10 h in 1.0× TAE buffer at 60℃. The DNA bands in gels were visualized by silver staining [27] and photographed with a Molecular Imager FX System (Bio-Rad).
To identify the species represented by DGGE bands, the bands were excised from the gels, washed twice with ultrapure water and then stored in 50 μl of ultrapure water at 4℃ for 16-24 h to elute the DNA. Subsequently, 5 μl of DNA elution was used to reamplify the DNA using the reaction mixture and conditions as described above, except that the primer without the GC clamp was used. The PCR products were cleaned using a PCR Purification Kit (Omega) and then sequenced (Invitrogen, Shanghai, China). Assembled sequences were aligned and compared with the 16S, 18S, or 26S rRNA sequences available in GenBank ( http://www.ncbi.nlm.nih.gov/BLAST/ ) to identify their closest phylogenetic relatives. The identities of the relatives were determined on the basis of the highest score.
- Analysis of DGGE Gel Images
The Shannon diversity index (H’) was calculated using the software BIO-DAP (Fundy National Park, Canada), based on the quantity and relative intensity of each band, which were obtained by the software Quantity One (ver. 4.6.2; Discovery Series, Bio-Rad) [15] .
Results and Discussion
- Bacillus Species Are Dominant in the Bacterial Community
The microbes found in Daqu play a key role in the production of alcohol and the formation of flavor. Fig. 1 shows the results of the DGGE analysis of the general bacterial, Bacillus , fungal, and yeast communities found in the three types of Daqu . Most of the microbes detected were the thermotolerant and thermophilic microbes ( Tables 2 - 5 ), and the three types of Daqu showed only slight differences in microbial communities ( Fig. 1 ).
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DGGE profiles of bacterial (A), Bacillus (B), fungal (C), and yeast (D) communities of three types of Daqu. The denaturant gradient ranges are from 40% to 60%. The major bands are numbered. MD, HD, and MID correspond to mechanically prepared, manually prepared, and mixed prepared Daqu, respectively.
Summary of identification of bands inFig. 1A (bacteria).
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aBands are numbered according to Fig. 1A. bIdentity represents the sequence identity (%) compared with that in the GenBank database.
Summary of identification of bands inFig. 1B (Bacillus).
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aBands are numbered according to Fig. 1B. bIdentity represents the sequence identity (%) compared with that in the GenBank database.
Summary of identification of bands inFig. 1C (fungi).
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aBands are numbered according to Fig. 1C. bIdentity represents the sequence identity (%) compared with that in the GenBank database.
Summary of identification of bands inFig. 1D (yeast).
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aBands are numbered according to Fig. 1D. bIdentity represents the sequence identity (%) compared with that in the GenBank database.
For bacterial analysis, 25 bands in the DGGE profiles were eluted from gels and sequenced ( Fig. 1 A). The sequence identity of all the bands was ≥94% as compared with that in the GenBank database, and most of them were uncultured bacteria ( Fig. 1 A; Table 2 ). The most dominant bacterial species were Bacillus (11, 17, 20, and 22) and Virgibacillus (2, 12, 14, 15, and 18). Several previous studies also reported that Bacillus species were dominant in Fen- Daqu [24 , 33 , 37] .
During the processing of the three types of Daqu tested here, the highest temperature at the center of the Daqu pile was about 60℃. Bacillus species have a better survivability than other bacteria under low moisture (13-14%) and high temperature (60-62℃) conditions because of their ability to produce spores. Thus, Bacillus species gradually become the dominant bacteria with an increasing ripening time [24 , 28] . The current study showed a low variation of Bacillus in the three types of Daqu ( Fig. 1 B). More Bacillales species, including Gracilibacillus , Halobacillus , Virgibacillus , and Staphylococcus , as well as seven species of Bacillus , were detected ( Table 3 ).
As the most dominant bacteria in Baiyunbian Daqu , Bacillus species may play an important role in the production of Chinese liquor. Bacillus species are important sources of proteases, amylases, lipases, cellulases, pectinases, glucanases, and other enzymes. These enzymes are important for the hydrolysis of the macromolecular components in cereal grains, leading to liquefaction and saccharification of starch. Furthermore, some metabolites from Bacillus species contain aromatic components such as diacetyl that facilitate subsequent reactions necessary for flavor production [28] . Bacillus species can also generate volatile compounds such as C 4 compounds, pyrazines, volatile acids, and aromatic and phenolic compounds in the liquor. These provide an improved sensory effect and various health benefits [5 , 34] . However, the functionality of the Bacillus species in Baiyunbian Daqu requires further investigation.
Other bacterial species such as Lactobacillus (3) and Trichococcus (2, 15) were also dominant in Daqu ( Fig. 1 A; Table 2 ). Several Lactobacillus species, including L. brevis , L. helveticus , L. panis , and L. fermentum , were observed in Daqu [30] . The Lactobacillus species regulate the diversity in a microbial consortium present in food and dairy manufacturing systems, and produce a variety of enzymes and organic acids to produce flavor compounds [13 , 23] . Trichococcus species are frequently isolated from sewage sludge and the anaerobic bioreactor used to treat organic wastewater. However, little information is available about their role in fermented products [3] .
The main differential bands in the three types of Daqu were related to the uncultured Firmicutes bacterium (4), uncultured bacteria (7, 8, and 9), uncultured Trichococcus (10), and Lentibacillus salis (21) ( Fig. 1 A; Table 2 ). Obtaining functional information about these bacteria is very difficult because most are uncultured. In the Daqu prepared by machine, a new band (21) related to L. salis was identified. L. salis is a moderately halophilic bacterium first reported in Daqu . Lentibacillus species belong to Bacillaceae , whose role in the liquor production may be similar to that of genus Bacillus .
- Molds Are Dominant in the Fungal Community
The saccharification and alcoholic fermentation of Chinese liquor result partially from the combined actions of molds and yeasts that grow more or less in succession throughout the brewing process [30] . As shown in Fig. 1 C and Table 4 , the dominant fungi in the three types of Daqu were Talaromyces emersonii (1), Penicillium oxalicum (2), Aspergillus (3, 4, and 9), and Monascus (11). Previous studies have described the isolation and identification of fungi found in Daqu and in the fermentation of Chinese liquor [24 , 28] , and some functions of fungi in fermented foods were determined.
A study in 2012 proposed that, based on their genetic relationships, T. emersonii , T. byssochlamydoides , Talaromyces eburneus , and Geosmithia argillacea belong to Rasamsonia gen. nov. [6] . These are thermotolerant and thermophilic fungi, and little information is available about their roles in fermented products. However, Penicillium species are considered to be undesirable contaminants, as they can interfere with Daqu quality by inhibiting the growth of other beneficial microbes [36] . Aspergillus and Monascus species are very common in fermentation starters. The role of Aspergillus species in fermented products has been studied extensively. Aspergillus species can produce proteolytic and other lytic enzymes, which are linked to the transformation of insoluble grain compounds into water-soluble peptides, sugars, free amino acids, and other degradation products found in soy sauce, thus affecting liquor flavor [17 , 30] . Monascus species can esterify mixed acids and ethanol to form esters such as ethyl caproate that contribute to the aroma of the Chinese liquor. This species can also produce citric acid, acetic acid, succinic acid, and substances with physiological activity that facilitate the formation of flavor and provide health benefits [14 , 16] .
Other fungi such as Thermoascus crustaceus (5), Rhizomucor pusillus (7), and Absidia blakesleeana (10) were detected in the three starters ( Table 4 ). Thermoascus is commonly found in high-temperature Daqu , but its functions in alcohol fermentation starters remain unknown [25 , 32] . R. pusillus , a type of thermophilic fungi, is commonly present in cereals, oil seeds, meat products, and Daqu , and can affect liquor flavor by rapidly utilizing numerous carbon sources, and producing glycerol, lactic acid, glucanase, phosphatase, acid proteinase, and alcohol dehydrogenase [22 , 35] . A. blakesleeana is the most common species in some kinds of Daqu . It can secrete hydrolyzing enzymes that decompose macromolecular materials and produce some metabolites that affect liquor flavor [30] .
In the current study, the results of the DGGE analysis showed that most of the fungi present were molds, and that yeasts were not the dominant microflora ( Fig. 1 C; Table 4 ). Temperature is an important factor that affects the growth and death of microbes. In general, the heat resistance of yeast is poorer than that of molds. During the preparation process of the three types of Daqu , the center of Daqu reaches 60℃, which is not compatible for the survival of most yeasts. The high temperature attained during the preparation process may explain the reason for the absence of yeast species in Daqu .
Although yeasts were not the dominant fungi in Daqu , they could gradually become the dominant microbes and have a significant effect on the subsequent fermentation processes used in liquor production. The dominant yeasts found in the three types of Daqu were Saccharomyces cerevisiae (8, 9, and 18), Saccharomycopsis fibuligera (7), Pichia anomala (12 and 13), and Debaryomyces hansenii (15) ( Fig. 1 D; Table 5 ). The yeasts identified in Baiyunbian Daqu are similar to those found in other alcoholic starters. Most of them are present naturally in the fermentation products [12 , 28 , 30 , 37] . S. cerevisiae is usually dominant in alcoholic fermentations because of its competitive growth under strict anaerobic conditions and its tolerance to ethanol. Thus, S. cerevisiae is an efficient microbe for ethanol production [31 , 37] . A previous study reported that S. cerevisiae was the major yeast species active during Fen-liquor fermentation [12] .
Several non- Saccharomyces yeasts, including S. fibuligera , P. anomala , and D. hansenii , were found in Baiyunbian Daqu. S. fibuligera occurs commonly in fermentation starters and typically grows prior to the main alcoholic fermentation, thereby playing an important role during the initial stages [7] . S. fibuligera has a strong saccharification capability and produces various enzymes, particularly glucoamylase and α-amylase, that metabolize the native starch into maltose, dextrin, and glucose [1] . P. anomala is a film-forming yeast and can improve the taste, texture, yield, and safety of agricultural products by outcompeting undesirable fungi [8] . Supplementation with P. anomala , a potent producer of esters, during production can increase the ester content of liquor [8 , 9 , 30] . D. hansenii is a haloduric and lipid-accumulating oleaginous yeast that is found in the process of making Chinese liquor, and is beneficial for fermented foods [21 , 30] .
Overall, only small differences were detected in the composition of the fungal and yeast communities among the three types of Daqu ( Figs. 1 C and 1 D). The main differences were observed in the mixed prepared Daqu ( Fig. 1 D) where the relative amount of Aspergillus intermedium (16 and 17) was increased, and the amounts of S. cerevisiae (2) and Candida silvae (3) were decreased ( Table 5 ).
- Similarity in Shannon Diversity Index
DGGE profiles were analyzed using the Shannon index (H’) to assess the relative intensity and the absence or presence of the bands ( Fig. 1 and Table 6 ). The three types of Daqu had very similar diversity indexes of bacteria, Bacillus , and fungi. For yeast, the diversity indexes of manually prepared Daqu and mechanically prepared Daqu were similar, but higher than that of the mixed prepared Daqu ( Table 6 ).
Shannon index (H’) calculated from the DGGE banding patterns inFig. 1.
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Shannon index (H’) calculated from the DGGE banding patterns in Fig. 1.
Several researchers have investigated the microbial communities and development of microbes in the Daqu preparation and ripening process as well as production in different regions using different culture temperatures [28 , 30 , 32] . Perhaps some other factors inherent in the different preparation methods would affect the microbial composition in Daqu . For example, mixing material by hand and stepping up the starter by foot may lead to microbial transfer from the human body to the starter. In addition, the force and heat produced by machine may affect the growth and death of microbes in Daqu . However, only slight differences in microbial communities were found among the three types of Daqu , and similar Shannon indexes (H’) were observed in manually prepared and mechanically prepared Daqu . During Daqu preparation, the culture was inoculated with 4% old Daqu . In this way, the Daqu has been handed down from one generation to the next. It is possible that the addition of old Daqu , temperature, and other factors played a decisive role in the microbial composition of Daqu .
In conclusion, this is probably the first study to identify the bacterial, Bacillus , fungal, and yeast communities in three types of Daqu by using reconditioning PCR-DGGE with four different groups of primers. The microbes detected were mainly thermotolerant and thermophilic microbes. The dominant bacterial and fungal species were Bacillus and molds. The preparation methods had minimal effects on the microbial composition, and based on these data, the mechanically prepared Daqu could replace manually prepared Daqu in Chinese liquor production. The findings of our study provide useful information for liquor production and fermentation process improvement.
Acknowledgements
This work was financially supported by grant J1103510 from the NSFC, grant 2013BAD10B02 from the Project of National Science & Technology Support Program of China, and grants 2012MBDX013 and 2014JC004 of the Fundamental Research Funds for the Central Universities.
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