Culture-independent analysis of the bacterial communities in Chinese fermented vegetables and genomic analysis of lactic acid bacteria

doi:10.21203/rs.3.rs-218303/v1

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Culture-independent analysis of the bacterial communities in Chinese fermented vegetables and genomic analysis of lactic acid bacteria

https://doi.org/10.21203/rs.3.rs-218303/v1

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Six different fermented vegetables were collected from Zhejiang Province, China, to explore the associated bacterial communities using a high-throughput sequencing platform. A total of 24 phyla, 274 families and 569 genera were identified from six samples. Firmicutes and Proteobacteria were the main phyla in all of samples. Meanwhile, Brevibacterium was the major genus in Xiaoshan pickled radish. Lactobacillus-related genera and Vibrio were the major genera in fermented potherb mustard and its brine. Enterobacter and Cobetia were the major genera in fermented radish and its brine. Chromohalobacter was the major genus in the tuber mustard. These results indicated there were clear differences between the bacterial genera present in Xiaoshan pickled radish, fermented potherb mustard, fermented radish, and tuber mustard. This demonstrated the possible influences of raw materials and manufacturing processes. Furthermore, a large number of lactic acid bacteria were isolated and identified by culture-dependent and 16S rRNA gene sequence analysis, which accounted for more than 68% of all the isolates. In addition, whole genome analysis of Lactobacillus suantsaii, Lactobacillus sakei subsp. sakei, and Weissella cibaria showed they had large numbers of genes associated with carbohydrate metabolism. This may explain why these three bacterial strains can grow in fermented vegetable environments.

Applied & Industrial Microbiology

culture-independent analysis

bacterial community

Chinese fermented vegetables

genomic analysis

As early as thousands of years ago, the ancestors of the Chinese people started to ferment vegetables to extend shelf life (Guan et al. 2020; Zhang et al. 2021). To date, most Chinese people still make fermented foods using perishable and seasonal vegetables, such as radish, cabbage, mustard, and cucumber (Liang et al. 2018; Zhai et al. 2018; Liu et al. 2019a; Rao et al. 2020). The purpose of fermenting the vegetables is not only to prolong shelf-life, but also to enhance nutrition and improve organoleptic characteristics. There are various fermented vegetables in China, such as paocai in Southwest China, and serofluid dish in Northwest China (Li et al. 2017; Liu et al. 2019b). Zhejiang Province, which is located in Southeast China, has a unique environment with rich topography and abundant rainfall. Due to this unique climate, local people particularly enjoy the fermented vegetable products produced in the region. Xiaoshan pickled radish, fermented potherb mustard, and meigan cai are distinctive traditionally fermented foods that have been consumed for thousands of years in Zhejiang Province (Guan et al. 2020; Zhang et al. 2019). Despite the consistency of the geography and climate, the flavor, taste, and texture of these fermented vegetables differ. The changes in the texture and flavor of the fermented vegetables are dependent on the microorganisms present during the fermentation process. This is because bacteria contribute to the flavor formation and acidification of the foods (Guan et al. 2020). Therefore, various measures have been employed to elucidate the structure and composition of the bacterial communities in the fermented vegetable products.

Over recent decades, culture-independent methods have been successfully employed to analyze the microbial communities in fermented vegetables. For example, denaturing gradient gel electrophoresis has been used to study the microbial ecology of suancai and serofluid dish in China (Wu et al. 2015; Zhang et al. 2018; Zhou et al. 2018). Real-time quantitative PCR has also been used to detect both bacteria and yeast in Chinese paocai (Xiong et al. 2019). At present, high-throughput sequencing (HTS) is the most common method used to reveal the major bacterial community members in fermented vegetables. HTS can provide more detailed information and deeper insight into the microbial communities compared to conventional molecular method (Chen et al. 2019; Xiao et al. 2020). Nevertheless, HTS is based on community analysis of environmental DNA, and so pure cultures cannot be isolated and their physiological potentiality remains unknown. Therefore, some studies have combined culture-independent and culture-dependent techniques to analyze the bacterial communities and obtain pure cultures (Lu et al. 2020; Wang et al. 2020).

In this study, six fermented vegetable products were collected from Zhejiang. Culture-independent HTS and a culture-dependent method were used in combination to investigate the bacterial communities and isolate specific strains from the six fermented vegetable products. Furthermore, the genomes of Lactobacillus suantsaii (L. suantsaii) CBA3634, Lactobacillus sakei subsp. sakei (L.sakei subsp. sakei) CBA3635, and Weissella cibaria (W. cibaria) CBA3636, which were isolated from fermented potherb mustard and its brine, were sequenced using PacBio and Illumina platforms. This work aimed to deepen the understanding of the bacterial communities in the fermented vegetable products of Zhejiang and isolate and elucidate key features of the dominant lactic acid bacteria (LAB) responsible for vegetable fermentation. The results of this study will be helpful for stabilizing and enhancing the characteristics of fermented vegetable products of Zhejiang.

Sample collection

Samples were collected from six traditional fermented vegetables (C1-C6) from Zhejiang Province in 2018. Xiaoshan pickled radish (C1) was obtained from the factory in Hangzhou and the manufacturing process was as follows. The fresh radish was washed, cut evenly into strips, and air dried for three to five days. The radish was then mixed with a 6–7% (6–7 g/100 g) concentration of sodium chloride and stored at room temperature in the dark for approximately one year. Fermented potherb mustard (Brassica juncea var. multiceps; C2) and the brine of fermented potherb mustard (C6) were obtained from the factory in Hangzhou. The potherb mustard was pickled in a 16–18% concentration of sodium chloride and had been stored at room temperature in the dark for approximately one year. Fermented radish (C3) and its brine (C5) were obtained from the factory in Hangzhou. The fresh radish was washed and pickled in an 18–20% (18–20 g/100 g) concentration of sodium chloride. Then the radishes were stored at room temperature in the dark for approximately one year. Tuber mustard (Brassica juncea var. tumida; C4) was obtained from the factory in Hangzhou. Tuber mustard was pickled in a 14–17% concentration of sodium chloride and had been stored at room temperature in the dark for approximately one year.

Culture-independent HTS analysis of the bacterial communities

Total genomic DNA was extracted from the six samples using a FastDNA SPIN kit for soil (MP Biomedicals, Solon, OH, USA) according to the manufacturer’s instructions. The amplicon library was prepared using a two-step PCR approach. In the first step, the hypervariable V3–V4 region of the bacterial 16S rRNA gene was amplified using the primer pair 341F and 805R (Fadrosh et al. 2014). In the second step, the dual index barcodes, i5 and i7, of Illumina Nextera were attached to each sample. The quantity and quality of the constructed library were determined using a Quanti-iT PicoGreen dsDNA Assay kit (Invitrogen, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA). The final library products were sequenced using an Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to Illumina's standard protocol. Bioinformatic analyses were performed using the free online platform of Majorbio Cloud Platform (www.majorbio.com) by Shanghai Majorbio Bio-pharm Technology Co. Ltd. according to the method described by Huang et al. (Huang et al. 2020).

Cultivation of LAB

De Man, Rogosa, and Sharpe (MRS) and Rogosa SL media were used to isolate cultivable LAB from all of the six fermented vegetable samples. The samples were serially diluted and plated on MRS and Rogosa SL agar media. Each plate was incubated in aerobic conditions at 30°C for 48 h. After cultivation, forty-four colonies were randomly picked and transferred several times until pure cultures were obtained. The 16S rRNA gene fragments of each isolate were amplified by colony PCR using the bacterial universal primer set 27F and 1492R. The PCR products were analyzed by agarose gel electrophoresis and sequenced using Sanger sequencing. The 16S rRNA gene sequences were identified using the EzBioCloud database (https://www.ezbiocloud.net/identify).

Genomic analysis of the isolated LAB

DNA extraction

Genomic DNA was extracted using an MG genomic DNA Purification Kit (MGmed, Seoul, Korea) according to the manufacturer’s protocol. The quality and quantity of extracted genomic DNA were estimated using an Agilent 2100 Bioanalyzer and NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, UK).

Library construction and sequencing

Whole genome sequencing of the isolates was performed using a combination of PacBio RS II and Illumina HiSeq sequencing platforms. The Illumina data were used to evaluate the complexity of the genome. For the PacBio sequencing, 20 kb SMRTbell libraries were constructed using a standard method and sequenced using the PacBio RS II system with the P6 DNA polymerase and C4 chemistry. For the Illumina HiSeq sequencing, paired-end libraries were constructed for the isolates using a TruSeq Nano DNA kit (350 bp insert size) following the manufacturer’s recommendations and were sequenced using the Illumina HiSeq system.

Genome assembly and annotation

The sequencing reads obtained from the PacBio sequencer were de novo assembled using the hierarchical genome assembly process. After de novo assembly, the Illumina sequencing reads were integrated into the draft assemblies to obtain accurate genome sequences using the Pilon tool. The circular form of the contigs was confirmed using the UGENE software package (Komstantin et al. 2012).

Genome annotation was performed using Pathosystems Resource Integration Center database (PATRIC) and NCBI Prokaryotic Genome Annotation Pipeline (PGAP). The circular genome map was constructed using the CGview Server tool (http://cgview.ca/) based on the CDS annotated by Prokka (Anna et al. 2020). The predicted protein coding sequence (CDS) was assigned a K number using BlastKOALA and classified into a KEGG pathway using the Reconstruct Module tool in KEGG Mapper (Kanehisa et al. 2016). The Prophage Hunter tool was used to predict prophage activity within the genome. The 16S rRNA gene from the genomes were used to construct a phylogenetic tree using MEGA7 according to the neighbor-joining method. The topology of the phylogenetic tree was evaluated with 1,000 bootstrap replications and values >70% were indicated in the tree.

HTS analysis of bacterial community structure

The number of effective sequence reads obtained from the six fermented vegetable samples ranged from 21,388 to 68,573. Based on a similarity threshold of 0.97, a total of 1,131 operational taxonomic units (OTUs) were identified from all the samples. Among them, 67 OTUs were present in all of the samples. In total, 130, 98, 147, 114, 64, and 176 OTUs were unique to the C1, C2, C3, C4, C5, and C6 samples, respectively (Fig. 1A). Species richness estimators (Table 1) were used to reflect the microbial phylotype richness levels. The results revealed that the C3 sample had the highest microbial diversity among the six samples, and rank abundance curves (Fig. 1B) also illustrated this result.

A total of 24 phyla, 274 families, and 569 genera were identified in the six samples. At the phylum level, the major phyla were Firmicutes and Proteobacteria. Firmicutes was the most dominant phylum in the C1 and C2 samples, accounting for 34.48% and 81.97% of the total phyla, respectively. Further, Proteobacteria, the second most dominant phylum, accounted for 33.37% and 15.51% of the total phyla in the C1 and C2 samples, respectively. On the contrary, Proteobacteria was the most dominant phylum in C3, C4, C5, and C6, accounting for 68.56%, 56.73%, 67.92%, and 79.14% of the total phyla, respectively. Firmicutes was the second most dominant phylum in C3, C4, C5, and C6, accounting for 15.82%, 25.27%, 25.81%, and 8.29% of the total phyla, respectively (Fig. 2A). At the family level, Halomonadaceae was most abundant in the C4 (53.83%) and C5 (27.47%) samples. Lactobacillaceae was the most dominant family in C2 (73.73%) and Brevibacteriaceae was the most dominant family in C1. Enterobacteriaceae was the most dominant family in C3, and Beijerinckiaceae was the most dominant family in C6 (Fig. 2B).

At the genus level, Brevibacterium (17.39%) was the most abundant in the C1 sample, followed by Leuconostoc (13.94%) (Zheng et al., 2020; Kim et al., 2021), Lactobacillus-related genera (11.50%), Pseudomonas (6.29%), and Halomonas (6.19%). In C2, Lactobacillus-related genera (73.72%) was the major genus observed, and this was followed by Weissella (7.46%) and Methylobacterium (4.72%). Enterobacter, Ralstonia, Lactobacillus-related genera, and Lactococcus were the dominant genera in C3, accounting for 6.5%, 5.31%, 5.29%, and 5.15% of the total genera. In C4, Chromohalobacter, Halomonas, Halanaerobium, and Lactobacillus-related genera were the dominant genera, accounting for 27.68%, 21.08%, 16.13%, and 13.82% of the total genera. In addition, Cobetia (27.10%), Lactobacillus-related genera (10.36%), Enterobacter (6.84%), Pseudoalteromonas (5.55%), and Lactococcus (5.42%) were highly abundant in the C5 sample. Vibrio (12.37%), Methylobacterium (12.33%), Pseudoalteromonas (10.78%), Pseudomonas (8.82%), Sphingomonas (6.52%), and Enterobacter (5.95%) were the major genera in the C6 sample (Fig. 2C).

Isolation and molecular identification of LAB strains

Several colonies on MRS or Rogosa SL plates were isolated and purified from the four samples (C1, C2, C5, and C6). Forty-four isolates were identified via 16S rRNA gene sequence analysis. From C1, eight Staphylococcus (six Staphylococcus piscifermentans and two Staphylococcus nepalensis) and one Terribacillus (Terribacillus goriensis) were obtained. Five Lactobacillus (three Lactobacillus alimentarius, one Lactobacillus suantsaii, and one Lactobacillus versmoldensis) and two Bacillus (one Bacillus altitudinis and one Bacillus gossypii) were obtained from the C2 sample. Eleven Lactobacillus (five Lactobacillus curvatus, five Lactobacillus sakei subsp. sakei, and one Lactobacillus pentosus) were obtained from the C5 sample. From C6, three Lactobacillus (three Lactobacillus sakei subsp. sakei), eleven Leuconostoc (four Leuconostoc mesenteroides subsp. mesenteroides, five Leuconostoc mesenteroides subsp. jonggajibkimchii, and two Leuconostoc carnosum), two Weissella (one Weissella soli and one Weissella cibaria) and one Enterococcus (Enterococcus casseliflavus) were obtained. Among them, 30 strains were LAB strains, which accounted for more than 68% of all isolates. Furthermore, the representative LAB strains from the fermented foods, L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, and W. cibaria CBA3636, were selected for whole genome analysis (Table S1).

Whole genome analysis of L. suantsaii, L. sakei subsp. sakei, and W.cibaria

The genomes of the three strains, L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, and W. cibaria CBA3636, were sequenced using PacBio sequencer and HiSeq platforms. The L. suantsaii CBA3634 genome consisted of a circular chromosome and five plasmids. The length of the genome was 2,661,801 bp with a GC content of 50.49% (Fig. 3A, Table 2). The genome contains 18 rRNA genes, 70 tRNA genes, and 2,362 CDSs. Of the CDSs, 56.0% (1,322 of 2,362) were assigned protein functions and classified into 38 KEGG categories. The top three assigned KEGG categories were “global and overview maps,” “carbohydrate metabolism,” and “amino acid metabolism” (Fig. 3D). One active and two ambiguous prophage regions were predicted in the CBA3634 genome. The active prophage closely matched with the Lactobacillus phage LBR48 and the ambiguous prophages closely matched with the Lactobacillus phage LBR48 and the Streptococcus phage phiJH1301-2.

The complete genome of L. sakei subsp. sakei CBA3635 was composed of a single circular chromosome of 2,006,110 bp with a GC content of 41.13%, and one circular plasmid of 56,165 bp with a GC content of 41.11%. There were 2,002 CDSs, 21 rRNA operons, and 66 tRNAs in the genome (Fig. 3B and Table 2). The 1,130 CDSs of L. sakei subsp. sakei CBA3635 were assigned protein functions and classified into 37 KEGG categories as shown in Fig. 3D. With regard to the KEGG categories of strain CBA3635, “global and overview maps” (528 genes), “carbohydrate metabolism” (174 genes), and “translation” (81 genes) were assigned at high proportions. Two active and two ambiguous prophage regions were predicted in the CBA3635 genome. The active prophages closely matched with Lactobacillus phage PL-1 and Staphylococcus phage IME1361_01. The ambiguous prophages closely matched with Lactobacillus prophage Lj965 and Staphylococcus phage SPbeta-like.

The complete genome of W. cibaria CBA3636 included one circular chromosome and two circular plasmids. The total length was 2,423,709 bp with a GC content of 44.99 % (Fig. 3C and Table 2). The genome contained 2,165 CDSs, 28 rRNA genes, and 89 tRNAs. Of the CDSs, 56.9% (1,232 of 2,165) were assigned protein functions and were divided into 38 KEGG categories. Among them, the highest proportion of genes was assigned to the category of “global and overview maps” (597 genes), followed by “carbohydrate metabolism” (152 genes) and “membrane transport” (87 genes; Fig. 3D). Only three ambiguous prophage regions were predicted in the CBA3636 genome and these were closely related to Weissella phage WCP30, Bacillus phage Anthony, and Streptococcus phage phi-SsUD.1.

The maximum likelihood phylogenetic tree was constructed based on the 16S rRNA sequences of L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, W. cibaria CBA3636, and the reference strains (Fig.4). The phylogenetic tree showed that the three strains are located within two large clades. In addition, L. suantsaii CBA3634 was closely related to the reference strain Lactobacillus suantsaii L88^T, and L. sakei subsp. sakei CBA3635 was closely related to the reference strains Lactobacillus sakei subsp. sakei JCM 1157^T and Lactobacillus sakei subsp. carnosus DSM 15831^T.

Genome sequence accession numbers

The genomes of L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, and W. cibaria CBA3636 were deposited in NCBI with the accession numbers CP059603-CP059608, CP059697-CP059698, and CP059699-CP059701, respectively.

Bacteria play a vital role in food fermentation. Bacterial community structure, particularly LAB community structure, is closely associated with the texture, flavor, nutrients and quality of fermented products. However, the understanding of bacterial composition is still limited to several major species responsible for traditional fermented vegetable products (Liu et al. 2017). In this study, the bacterial diversities associated with six fermented vegetables from Zhejiang were investigated. HTS analysis identified 24 phyla, 274 families, and 569 genera based on the OTUs identified in the six samples. The major phyla were Firmicutes and Proteobacteria, the relative abundance of which ranged from 67.85% to 97.48%. This indicated a firm correlation between the two types of bacteria and vegetable fermentation. In fact, most previous reports have also found that fermented vegetables mainly contain Firmicutes and Proteobacteria at the phylum level (Liang et al. 2018; He et al. 2020).

At the genus level, however, the abundance of different genera varied greatly among the different fermented vegetable products. This result was in accordance with the results of previous studies. For example, Lactobacillus-related genera and Pediococcus were found to be the major genera in the suancai samples (Liu et al. 2019a), whereas Bacillus and Bacteroides were the main genera in shuidouchi samples (Chen et al. 2019). The results of the present study showed that Brevibacterium was the most abundant genus in Xiaoshan pickled radish. Lactobacillus-related genera and Vibrio were the most abundant genera in fermented potherb mustard and its brine. Enterobacter and Cobetia were the most abundant in fermented radish and its brine. Chromohalobacter was the most abundant in tuber mustard. These differences in bacterial community may result from the different types of raw materials used during fermentation. Additionally, besides the effects of the raw vegetable used, manufacturing processes or environmental conditions such as temperature, humidity and salinity can also significantly affect bacterial communities and the final quality of the fermented vegetable product (Xu et al. 2020).

Comparing the results obtained from the culture-dependent and culture-independent methods, many microorganisms such as Lactobacillus-related genera, Leuconostoc, and Weissella were encountered using both methods. In particular, Lactobacillus-related genera was found to be abundant using both approaches, which effectively confirmed the existence and composition of these microbes and communities in the fermented vegetable products. However, for the C4 samples, no colonies were obtained on the MRS medium. Some halophilic bacteria, such as Chromohalobacter, Halomonas, and Halanaerobium, were dominant in C4 samples and these halophilic bacteria need to be grown in a salty medium. Thus, the development of a more specific medium for target strain growth and reproduction will be critical for future study.

Whole genome analysis of strains CBA3634, CBA3635, and CBA3636 showed that these three LAB had a large number of genes associated with carbohydrate metabolism. Carbohydrate metabolism includes subcategories related to glucose, fructose, mannose, galactose, ascorbate, aldarate, starch, sucrose, amino sugar, nucleotide sugar, pyruvate, glyoxylate, and dicarboxylate, among others. These results indicate that L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, and W. cibaria CBA3636 are capable of metabolizing various carbohydrates. In addition, a tertiary level analysis of the carbohydrate metabolism in the genomes of the three strains indicated that their genes are involved in utilizing a variety of carbon sources such as starch, sucrose, pentose, galactose, fructose, and mannose. According to previous reports (Tashakor et al. 2017), L. sakei can indeed use different carbon sources, such as sucrose, maltose, fructose, and arabinose. Furthermore, it has been shown that Weissella can use glucose, D-maltose, D-ribose, mannitose, and trehalose. This ability to use multiple carbon sources could explain why L. sakei subsp. sakei CBA3635 and W. cibaria CBA3636 are able to grow in the fermented vegetable environment, as it may enhance their survival, competitiveness, and persistence.

Active or ambiguous prophages were detected in the L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, and W. cibaria CBA3636 genomes. In general, the presence of prophages in the genome has been considered to have a negative effect on the host, but recently it has been shown that prophages can provide several benefits to the host. Prophages can act in bacterial cellular processes such as antibiotic resistance, stress response, and virulence, and also mediate defense against superinfection (Song et al. 2019). The prophages were found in the genomes of LAB including Lactobacillus, Weissella, and Leuconostoc. Prophages present in LAB genomes can help the host to survive in their habitats (Durmaz et al. 2008; Wang et al. 2010; Liu et al. 2009; Joseph et al. 2016; Panthee et al. 2019).

Some strains of L. sakei are known to produce bacteriocins, such as sakacin A, sakacin G, sakacin P, sakacin Q, and sakacin X (Kim et al. 2020). The bioinformatic analysis performed in the present study showed that the L. sakei subsp. sakei CBA3635 genome contained a sakacin P response regulator gene. However, the sakacin P structural gene sppA was not found in the genome. Mretr et al. also found some of L. sakei strains that did not produce sakacin P, but contained sakacin P-related synthetic genes (Mretr et al. 2005). However, the role of the sakacin P-related genes in non-producers is not known. These results indicate that L. sakei subsp. sakei CBA3635 cannot produce sakacin. In addition, Weissella species have been known to synthesize extracellular exopolysaccharides (EPS) (Ye et al. 2018). EPS-producing LAB have been known to have an ability to tolerate gastrointestinal conditions, reduce pathogenic biofilm formation, and help in adhesion to epithelial cells than non-EPS-producing LAB. The bioinformatic analysis performed in the present study showed that W. cibaria CBA3636 genome contained the exopolysaccharide biosynthesis protein, which is an EPS-related gene. Nucleotide sugar biosynthesis is essential for EPS. Through further exploration of the KEGG database, it was found that W. cibaria CBA3636 had a high number of genes (29 genes) related to amino sugar and nucleotide sugar biosynthesis. This shows that W. cibaria CBA3636 is a potential EPS-producing bacterial strain.

Using HTS, the bacterial communities of six fermented vegetable products from Zhejiang Province in China, were found to be highly diverse. Firmicutes and Proteobacteria were the main phyla found in all of the fermented vegetable products. However, Brevibacterium was the major genus in Xiaoshan pickled radish; Lactobacillus-related genera and Vibrio were dominant in fermented potherb mustard and its brine; Enterobacter and Cobetia were the major genera in fermented radish and its brine; and Chromohalobacter was the major genus in tuber mustard. These results indicated that different types of raw materials and manufacturing processes led to differences in the bacterial genera present. A culture-dependent method was used to isolate the cultivable LAB strains. In total, 44 isolates were successfully obtained and identified by 16S rRNA gene sequencing. Among them, 30 strains were LAB strains, which accounted for more than 68% of all of the isolates. Whole genome analysis of L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, and W. cibaria CBA3636 showed that these three bacteria had a large number of genes associated with carbohydrate metabolism. This illustrated that L. suantsaii CBA3634, L. sakei subsp. sakei CBA3635, and W. cibaria CBA3636 can grow in fermented vegetable environments.

Acknowledgments and Funds

This work was supported by the National Natural Science Foundation of China (NO. 31671865); the Key Research and Development Program of Zhejiang Province (NO. 2020c04008), China; and the World Institute of Kimchi funded by the Ministry of Science and ICT (KE2001-2), Republic of Korea.

Author contributions

Daqun Liu and Seong Woon Roh designed experiments. Jianming Zhang and Hye Seon Song analyzed experimental results and wrote the manuscript. Chengcheng Zhang, Yeon Bee Kim, Seong Woon Roh carried out experiments.

Compliance with ethical standards

Conflict of interest The authors declared that they had no competing interests.

Ethical approval Not applicable.

Consent for publication All authors approved the manuscript.

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Table 1. The diversity indices of bacterial communities in the six samples

Sample	ACE	Chao1	Coverage (%)
C1	567.79	549.75	0.99
C2	449.97	436.89	0.99
C3	653.07	643.01	0.99
C4	349.38	341.33	0.99
C5	475.71	467.44	0.99
C6	590.61	573.40	0.99

Table 2. General genome features of Lactobacillus suantsaii CBA3634, Lactobacillus sakei subsp. sakei CBA3635, and Weissella cibaria CBA3636

Feature	CBA3634	CBA3635	CBA3636
Genome size	2,661,801	2,006,110	2,423,709
GC content (%)	50.49	41.13	44.99
rRNAs	18	21	28
tRNAs	70	66	89
Predicted genes	2,362	2,002	2,165

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Culture-independent analysis of the bacterial communities in Chinese fermented vegetables and genomic analysis of lactic acid bacteria

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