Study on relationship between bacterial diversity and quality of Huangjiu (Chinese Rice Wine) fermentation

Abstract Huangjiu (Chinese rice wine) is brewed in an open environment, where bacteria play an important role during the fermentation process. In this study, bacterial community structure and composition changes in the fermented mash liquid of mechanized Huangjiu, well‐fermented manual Huangjiu (wines of good qualities), and poorly fermented manual Huangjiu (wines of poor qualities: spoilage, high acidity, low alcohol content) in different fermentation stages from Guyuelongshan Shaoxing Huangjiu company were analyzed via metagenomic sequencing. And bacterial metabolic difference was analyzed via gene prediction of metabolic pathway enzymes. The results showed that the bacterial diversity degree was abundant, and the number of bacterial species in every sample was approximately 200–400. Lactic acid bacteria (LAB) dominated the bacterial community of Huangjiu fermentation, and lactobacillus was predominant species in well‐fermented Huangjiu while Lactobacillus brevis had an absolute dominance in spoilage Huangjiu. Further, gene prediction revealed that transformation of malate to pyruvate and lactate anabolism was more active in mash liquid of well‐fermented manual Huangjiu, while acetate accumulation was stronger in mash liquid of poorly fermented manual Huangjiu, which explained acidity excess reason in poorly fermented Huangjiu at gene level.


| INTRODUC TI ON
Huangjiu (Chinese rice wine), a traditional Chinese alcohol beverage, is popular among Chinese customers. Huangjiu is honored as national banquet alcohol beverage due to abundant nutrition and health function (Yongmei et al., 2007;Zhao et al., 2018). Huangjiu is brewed in an open environment, with glutinous rice as raw material and wheat Qu as sacchariferous agent. There are two distinct types of Huangjiu starter culture. One is made of steamed wheat and inoculated pure A. oryzae. Another is made of raw wheat and naturally inoculated microorganisms. Mixtures of these starter cultures are used in the modern brewing process of Huangjiu (Cao et al., 2010;Guan et al., 2012;Zhang et al., 2012). It is widely recognized that Huangjiu fermentation is in essence the process of mixed fermentation of yeast, bacteria, and molds to produce alcohol and other metabolites, thus formation of unique flavor and aroma . Recently, polymerase chain reactiondenaturation gel electrophoresis (PCR-DGGE) was employed for analysis of microbial community structure of wheat Qu and fermented mash liquid of Huangjiu Yu et al., 2018).
However, some disadvantages existed in PCR-DGGE method, such as preferential DNA amplification of some templates, and the presence of faint bands on gel images that may be difficult to be seen by naked eye, which resulted in underestimation of microbial diversity in the Huangjiu samples Milanovic et al., 2017). With the advance of high-throughput sequencing, metagenomic sequencing detecting microbial diversity in the whole environmental system has been increasingly mature.
Because of huge data volume, ability to detect all microbes including uncultured ones, metagenomic sequencing has been extensively used in fecal and food microbiota analysis (Chen et al., 2020;Lyu et al., 2021;Mao et al., 2015;Xie et al., 2013). For example, Liu et al. (Liu et al., 2018) identified Burkholderia and Bacillusas predominant bacterial genus in Gu Tian Qu (71.62%) and Wu Yi Hong Qu (44.73%), respectively, and detected 213 bacterial genus with low abundance via Illumina-based metagenomic sequencing.
The brewing technology of Huangjiu is divided into manual Huangjiu and mechanized Huangjiu (Figure 1). The mechanized Huangjiu is fermented in a large stainless steel tank with yeast and wheat starter as saccharifying and fermenting agents. The distiller's yeast is cultivated by pure breed, and the wheat koji is mixed by natural culture and pure breed. Traditional manual Huangjiu uses naturally cultivated wheat koji and yeast as saccharifying and fermenting agents. In the first fermentation stage, pottery jar is used as fermentation container, and in the second fermentation stage, pottery jar is used as fermentation container. Poor quality sacchariferous agents, inappropriate ratio of raw material and unsuitable control of fermentation temperature may lead to poorly fermented Huangjiu (Total acid is over standard, alcohol content is below standard). At the end of fermentation, the control standard for Huangjiu fermentation mash by Shaoxing Huangjiu production enterprises is as follows: the alcohol content is above 18%vol and the total acid is below 6.5 g/L.
Although little literature was available on microbial composition in spoilage Huangjiu, beer spoilage was mainly due to LAB (Geissler et al., 2016;Schneiderbanger et al., 2018). As bacteria was important in Huangjiu brewing, this study aimed at analyses bacterial community diversity of fermented mash liquid of mechanized Huangjiu, well-fermented manual Huangjiu and poorly fermented manual Huangjiu, and figuring out the explanation for spoilage in poorly fermented manual Huangjiu. The results would provide practical guidance for quality control of Huangjiu industry.

| Sample
The fermented mash of Huangjiu was obtained from Guyuelongshan

| Physicochemical indexes determination
Alcohol content and acidity of Huangjiu samples were determined according to the protocol recommended by national standard of Chinese Huangjiu GB/T 13662-2018 (Standardization administration, 2018).

| DNA extraction
MP Fast DNA SPIN Kit for Soil kit (MP Biomedical) was used to extract genomic DNA from the sample. The extracted DNA was determined by NanoDrop nucleic acid quantitative assay. DNA was re-extracted from the sample with concentration less than 5 ng/μl.
The extracted DNA was stored at −20℃ for metagenomic sequencing. Three replicates were performed.

| Metagenomic sequencing
The metagenomic DNA libraries were constructed with 2 μg ge-

| Meatgenomic data analysis
High-quality data after sequencing quality control was aligned to microbial genomes available on NCBI (National Center for Biotechnology Information) and Gen bank, thus identifying the microbial species and calculating its abundance (N. Qin et al., 2014).
Three replicates were performed.

| De novo assembly of illumina highquality reads
De bruijn-graph-based assembler SOAPdenovo was utilized to assemble short reads with parameter k-mers, varying from 39 to 59. N50 was calculated for contigs of different k-mers and only the contigs of largest N50 assembly were attributed to a sample (Qin et al., 2014). In order to utilize as many reads as possible, the unassembled reads from the same batch of fermentation were merged for a second assembly. All these contigs were used for gene prediction.

| Gene prediction and contig origin determination
To avoid rice or wheat gene pollution, SOAPdenovo assembled contigs were aligned to cereal reference genome. Contigs with coverage greater than 90% and similarity greater than 95% were considered to be contaminated and removed. The remaining contig was mapped to bacterial genome database and subject to gene prediction via MetaGeneMark software. Non-redundant gene encoding proteins were aligned to KEGG database, and a protein was assigned if E value was smaller than 1 × e −10 , therefore its gene function could be predicted (Qin et al., 2012).

| Physicochemical indexes analysis of different fermented mash liquid of Huangjiu
Alcohol content and acidity of different Huangjiu fermentation mash were described in  . However, traditional manual Huangjiu fermented relatively slowly, and the alcohol content reached 13%vol on the seventh day. The acidity of Huangjiu should be controlled below 6.5 g/L to maintain good quality. The results showed that acidity of both mechanized Huangjiu and well-fermented manual Huangjiu reached the standard, and spoilage Huangjiu reached the acidity standard at the end of major fermentation (14d), but the acidity elevated obviously and exceeded standard in the postfermentation.

| Bacterial composition analysis
Bacterial composition detected via Illumina-based metagenomic sequencing was shown in Figure 4. LAB, especially lactobacillus dominated the bacterial community of different fermented mash liquid of Huangjiu samples, suggesting that LAB was the predominant bacterial species in Huangjiu fermentation, which was consistent with the PCR-DGGE results (Yu et al., 2018). However, PCR-DGGE could hardly detect bacteria species with very low abundance, while 121 lactic acid bacteria were detected via highthroughput sequencing in the present study (Table 2), providing more detailed information of LAB in Huangjiu fermentation.
Bacterial species except LAB were classified into "others," among which Saccharopolyspora, Bacillus, Pantoea possessed higher proportion. As LAB has strong acid-producing capacity, proper proliferation of them in Huangjiu fermentation reduces pH value of fermentation mash and therefore inhibits growth of other infectious microbes. PCA analysis based on LAB species contribution F I G U R E 6 Metabolic network of bacterial community in Huangjiu fermentation. The gray and black rectangle represent enzyme encoding by higher abundance of gene in fermented mash liquid of well-fermented manual Huangjiu and poorly fermented manual Huangjiu (spoilage, high acidity, low alcohol), respectively (difference above 5 times) showed that there was little difference among the mechanized Huangjiu samples, but there was a significant difference among the well-fermented manual Huangjiu samples (including spoilage Huangjiu) (Figure 5), which indicated that the quality of the mechanized Huangjiu was more easily controlled.
In the mechanized fermented liqueur mash, Lactobacillus plantarum accounted for a large proportion, while the L. plantarum could secrete bacteriocins with antibacterial activity, which gave it an advantage in fermentation (Navarro et al., 2000). The proportion of LAB was particularly high in fermented mash liquid of spoilage Huangjiu, and to the rancidity of Huangjiu, which had a great impact on the quality of Huangjiu. Therefore, the quantity of L. brevis during the fermentation should be listed as one of the objects to monitor the fermentation process in order to take timely measures to reduce the loss of spoilage.

| Gene function prediction
Metagenomic sequencing has an obvious advantage, that is, enormous sequence data size. Not only can researchers calculate the relative abundance of species in microbial community, but predict relevant gene function according to sequence alignment with gene sequence in known genome. Figure 6 described

CO N FLI C T S O F I NTE R E S T
There are no conflicts of interest in this manuscript.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing. TA B L E 3 EC number and its corresponding enzyme shown in metabolic network with above 5 times difference between wellfermented and spoilage Huangjiu samples

D ECL A R ATI O N S
Compliance with ethical standards.

CO D E AVA I L A B I LIT Y
Software applications are available.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data in this manuscript are based on the scientific process and are completely true.