Analysis of the Microbial Structure in Daqu for the Main Types of Chinese Baijiu Volume 1 - Issue 1

XingLin Han^1,2, DeLiang Wang², JinYuan Sun¹, WuJiu Zhang² and BaoGuo Sun^1*

• ¹Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University (BTBU), China
• ²China National Research Institute of Food and Fermentation Industries, China

Received: January 21, 2021;   Published: February 07, 2022

*Corresponding author: BaoGuo Sun, Beijing Laboratory for Food Quality and Safety, Beijing Technology & Business University (BTBU), China

DOI: 10.32474/CTBM.2022.02.000146

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Abstract

This investigation undertook a systematic analysis of the microbial structure of six kinds of daqu from different types of Chinese baijiu. From the results, it was noted that the prokaryotic microbial species present in daqu were much more complex than the eukaryotic microorganisms, but the number of eukaryotic microorganisms was greater than that of prokaryotic microbes. The investigation identified 8 phyla, 74 classes, 130 orders, 203 families, and 255 genera for eukaryotic microbes and 11 phyla, 74 classes, 116 orders, 411 families, and 473 genera for prokaryotic microorganisms. The main eukaryotic microbe in daqu was Ascomycota, followed by Basidiomycota; Ascomycota species are mainly moulds and yeasts, and they play a major role in of baijiu fermentation. The main prokaryotic microorganism in daqu was Firmicutes, followed by Actinobacteria, Proteobacteria, Bacteroidetes, Euryarchaeota and Planctomycetaceae. The number of eukaryotic microbes OTUs were ranked in the order Nongxiang> Zhima-xiang>Qing-xiang>Feng-xiang>Chi-xiang>Jiang-xiang, and the abundance values were ranked in the order Qingxiang> Nong-xiang>Jiang-xiang>Zhima-xiang>Chi-xiang. The number of prokaryotic microorganisms OTUs were ranked in the order Qing-xiang>Nong-xiang>Zhima-xiang>Feng-xiang>Chi-xiang>Jiang-xiang, and the abundance values were ranked in the order Qing-xiang>Nong-xiang>Zhima-xiang>Feng-xiang>Chi-xiang>Jiang-xiang.

Keywords: Chinese Baijiu; High-Throughput Sequencing; Lactobacillaceae; Eurotium

Introduction

Chinese baijiu is one of the world’s oldest distilled alcoholic beverages and is very popular in China. It uses daqu as the fermentation starter, which is a saccharifying and fermenting agent that has a significant impact on the flavour of the product [1]. However, the daqu is different when used in baijiu brewing with different technologies. For example, Jiang-xiang baijiu uses a daqu with a higher temperature in the fermentation process, but Qing-xiang baijiu uses a daqu with a lower temperature in the fermentation process [2]. Moreover, the different kinds of daqu use different raw materials. Therefore, the microbial structure should be different for the daqu for the different types of baijiu. The microorganisms found in daqu have been analysed and reported in the literature; for example, in 2007, Yang Daiyong et al. used the selective culture method to carry out a pilot study of the microbial composition in daqu from Maotai [3]. In 2010, Meng Zhen et al. used PCR-DGGE technology to analyse the bacterial community structure of daqu and found that Firmicutes, Lactobacillaceae, Bacillaceae and Thermoactinomycetaceae were the main bacteria [4]. In 2020, Chen meng-en et al. used the selective culture method to identify Pantoea, Bacillus, Leuconostoc, Thermoascus, Aspergillus, and Thermomyces in Tao-rong daqu [5]. In 2020, Sun li-lin et al. used high-throughput sequencing technology to identify Oceanobacillus, Bacillales, Aspergillus, Thermoascus and Thermomyces in Jiang-xiang daqu [6]. However, this previous research perhaps involved only one kind of daqu and perhaps used the traditional culture method, yielding a large error value [7-10]. High throughput sequencing simultaneously sequences several million to billion DNA molecules and allows in-depth, meticulous, and global analysis; hence, it is called deep sequencing. It enables the rapid and convenient determination of the complicated microbial structure of a sample. The analytical ability and precision of PCR-DGGE technology is markedly more advanced than those of 16S rDNA sequence homology technology, which was the first method of analysing complex multi-strain samples and, at present, has not been used for analysing the microbial structure of daqu from different types of Chinese baijiu [11-14]. This study used high-throughput sequencing technology to research and analyse microorganisms in the main type of baijiu of Nong-xiang daqu, Qing-xiang daqu, Jiang-xiang daqu, Zhima-xiang daqu, Chi-xiang daqu and Feng-xiang daqu.

Materials and Methods

Experimental Samples

Ten typical daqu samples of Nong-xiang, Qing-xiang, Jiangxiang, Zhima-xiang, Chi-xiang and Feng-xiang were used. Each sample was broken down and mixed uniformly to obtain a mixed sample representing Nong-xiang daqu (ND), Qing-xiang daqu (QD), Jiang-xiang daqu (JD), Zhima-xiang daqu (ZD), Chi-xiang daqu (CD) and Feng-xiang daqu (FD), and microbial analysis was performed.

Testing Methods and Instruments

To analyse the microbial structure of daqu, Illumina MiSeq sequencing technology was used. The samples were mixed and well distributed and were analysed by the China National Research Institute of Food and Fermentation Industries.

Results and Analysis

Microbial Structure from All the Daqu Types (1,5,15-17)

To determine the microbial diversity in daqu samples, UCLUST software was used for the cluster analysis of the orders. Ninetyseven percent of the orders were similar, and UCLUST was used to designate operational taxonomic units (OTUs). OTUs indicate a group of orders that came from the same classified unit. The results regarding the kinds and numbers of microbes from daqu of 6 different types of baijiu are shown in Figure 1. Prokaryotic microorganisms accounted for 73% of the total kinds, but they accounted for only 38% of the total number. High-throughput sequencing revealed 8 phyla, 74 classes, 130 orders, 203 families, and 255 genera of eukaryotic microbes and 11 phyla, 74 classes, 116 orders, 411 families, and 473 genera of prokaryotic microorganisms. (Table 1) shows the eukaryotic microbes whose abundance value was more than 0.5%, which were detected in this experiment. From the statistical analysis results, the main eukaryotic microbe in daqu was Ascomycota, followed by Basidiomycota; therein, Ascomycota are mainly mould and yeast, and they play a major role in baijiu fermentation. Additionally, Wallemia, Trichosporon and Meyerozyma were not reported in the previous literature. Wallemia is mainly spread over the surface of food, and Trichosporon has been reported to be present in the maiqu for yellow rice wine. From the statistical analysis results, the main prokaryotic microorganism in daqu was Firmicutes, followed by Actinobacteria, Proteobacteria, Bacteroidetes, Euryarchaeota and Planctomycetaceae. Consistent with literature reports, Firmicutes was the primary prokaryotic microorganism in daqu, and most of these microorganisms are heat resistant, such as Thermoactinomycetes, Bacillus, and Lactobacillus.

Microbial Structure of Daqu of Different Types of Baijiu (15-21)

The Structure of the Eukaryotic Microbes in Daqu

From the statistical analysis of the number of OTUs and the abundance values of eukaryotic microbes for these six kinds of daqu (Figure 2), the results showed that the number of OTUs and abundance values of eukaryotic microbe in daqu of different types of baijiu were different, and the rank order of the number of OTUs was ND˃˃ZD˃QD˃FD˃CD˃JD, and the rank order of the abundance values was QD˃ND˃JD˃ZD˃FD˃CD. The number of OTUs and abundance values of daqu of Nong-xiang baijiu were high, especially 90 OTUs, which was higher than the others. The abundance value of the daqu of Qing-xiang baijiu was 105. The structure of eukaryotic microorganisms in the different types of daqu is shown in Figure 3. The rank order of the relative abundance of eukaryotic microorganisms in Nong-xiang daqu was Aspergillus, Saccharomyces, Eurotium, and Thermomyces, and the relative content of Aspergillus was more than 50%. More than 90% of the total was Saccharomyces in Jiang-xiang daqu. However, differences in the relative abundance of eukaryotic microorganisms were not obvious in Qing-xiang daqu, and the maximum was Xeromyces, which was lower than 20% (Table 2). The eukaryotic microorganisms with the highest relative abundance in Zhimaxiang daqu were Wickerhamomyces and Candida. The eukaryotic microorganisms with the highest relative abundance in Fengxiang daqu were Eurotium and Wickerhamomyces. Overall, the eukaryotic microorganisms with the highest relative abundance in these six types of daqu were Aspergillus, Saccharomyces and some Thermomyces.

The Structure of The Prokaryotic Microorganisms in Daqu

From the statistical analysis of the number of OTUs and abundance values of prokaryotic microorganisms for these six kinds of daqu (Figure 4), the results showed that the rank order of the number of OTUs and abundance values of prokaryotic microorganisms in the daqu of different types of baijiu was QD˃ND˃ZD˃FD˃CD˃JD for the number of OTUs and QD˃ND˃ZD˃FD˃CD˃JD for the abundance values. The number of OTUs and abundance value of the daqu of qing-xiang baijiu were the highest, with 250 OTUs and an abundance value of 4500. Those of the daqu of jiang-xiang baijiu were the lowest.

The structure of prokaryotic microorganisms in different types of daqu is shown Figure 5. The prokaryotic microorganisms with the highest relative abundance in Qing-xiang daqu were Staphylococcus, Planifilum, Pediococcus, Lactobacillus and Citrobacter, and the relative contents of Staphylococcus and Planifilum were 55.8% and 22.58%, respectively. The prokaryotic microorganisms with the highest relative abundance in Nongxiang daqu were Weissella, Rheinheimera, Lactobacillus, Bacillus and Pediococcus, and the relative content of Weissella was 38.9%. The prokaryotic microorganisms with the highest relative abundance in Jiang-xiang daqu were Pediococcus, Lactobacillus and Weissella, and these three accounted for more than 90% of the total. The prokaryotic microorganisms with the highest relative abundance in Chi-xiang daqu were Lactobacillus, Citrobacter, Rheinheimera and Lactobacillus, and the relative content of Lactobacillus was more than 70%. The prokaryotic microorganisms with the highest relative abundance in Zhima-xiang daqu were Lentibacillus, Thermoactinomyces, Thermonude, Lactobacillus and Lactobacillus. The prokaryotic microorganisms with the highest relative abundance in Feng-xiang daqu were Lactobacillus, Thermoactinomyces, Thermonude, Lactobacillus and Lactobacillus, and the relative content of Lactobacillus was 65.3%. As shown in Figure 5, the prokaryotic microorganisms with the highest relative abundances in Chinese baijiu daqu were Bacillus, Trichococcus, Pediococcus, Lactobacillus, Weissella, Thermoactinomyces and Thermonude. However, the others, such as Lactococcus, Propionibacterium, and polysporous Actinomycetes, showed great variation in these samples.

Conclusions

This paper analysed the microbial structure in six different types of Chinese baijiu daqu using NGS.

a) The prokaryotic microorganisms accounted for 73% of the total kinds of microbes, but they accounted for only 38% of the total number. The main eukaryotic microbe in daqu was Ascomycota, followed by Basidiomycota; therein, Ascomycota are mainly moulds and yeasts, and they play a major role in baijiu fermentation. The main prokaryotic microorganism in daqu was Firmicutes, followed by Actinobacteria, Proteobacteria, Bacteroidetes, Euryarchaeota and Planctomycetaceae.

b) The eukaryotic microbes and prokaryotic microorganisms with the highest relative abundances in these six types of Chinese baijiu daqu are shown in Table 3.

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