Structure and Diversity of Bacterial Communities in the Fermentation of Da-Jiang

Annals of Microbiology (2018) 68:505–512 https://doi.org/10.1007/s13213-018-1355-x

ORIGINAL ARTICLE

Structure and diversity of bacterial communities in the fermentation of da-jiang

Pengfei Zhang 1 & Rina Wu1 & Ping Zhang1 & Yiming Liu2 & Dongbing Tao1 & Xiqing Yue1 & Ying Zhang1 & Jing Jiang1 & Junrui Wu1

Received: 15 December 2017 /Accepted: 28 June 2018 /Published online: 8 July 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Abstract Da-jiang is the traditional soybean fermented food which is popular in the world for a long time. In order to improve the quality and nutritional value of da-jiang, structure and diversity of bacterial communities in the fermentation of da-jiang were analyzed. Illumina MiSeq platforms coupled with bioinformatics approach were used in this study. In the first 28 days, the trends of bacterial abundance were similar in different regions which are increasing firstly, decreasing secondly, and rising again. The quantity of bacteria in post-fermentation is lower than pre-fermentation. In the fermentation of da-jiang, Firmicutes and Proteobacteria are the dominant phyla. The dominant genera in da-jiang from different regions are different: Tetragenococcus (58.1–73.0%) is the dominant genus in da-jiang from Xinmin; Leuconostoc (9.2–25.7%) is the dominant genus in da-jiang from Tieling; Acinetobacter (8.7–25.1%) and Leuconostoc (12.4–22.0%) are the dominant genera in da-jiang from Shenyang. Additionally, Weissella, Lactobacillus, Staphylococcus, Erwinia,andPseudomonas also were found in da-jiang. It is identified that Leuconostoc steadily existed in all da-jiang samples. These results demonstrate the diversity of microbes in traditional fermented da-jiang, which will probably provide a data basis for choosing starter culture for da-jiang industrial fermentation.

Keywords Da-jiang . Bacteria diversity . Leuconostoc . Illumina Miseq

Introduction Da-jiang, a kind of important soybean paste in the northeast of China, has 2000 years of history (Xu 2013;Geetal.2012). Fermentation is one of the oldest and most economical ways It is one of four major traditional fermented soybean products of producing and preserving foods (Hugenholtz 2013). Due to in China. As we all know, the flavor of da-jiang has a strong the improvement of nutritional value and sensory attributes, relationship with microbial metabolism. The usual methods of fermentation became popular in many cultures. At present, making da-jiang are as follows: soybeans are soaked and fermented foods are part of the daily intake (Chaves-López steamed to make 6 cm × 5 cm × 5 cm bricks, then naturally et al. 2014). China has an ancient civilization with a long fermented for 1 to 2 months; next, we mix the fermented history. Chinese has founded many fermented foods, which bricks with brine in a certain ratio, stir daily, and ferment for has unique flavor and nutrition. The typical fermented foods 1 to 2 months; finally, we can get delicious da-jiang. The are da-jiang, sufu, Puer tea, cheese, and vinegar. The produc- whole fermentation of da-jiang usually lasts for 6 months. tion of fermented food is becoming easier and easier along There are approximately three different stages in da-jiang fer- with the development of technology. However, people always mentation. The first period is degradation of components in prefer foods, which are made by traditional methods. raw materials by a series of complex enzymes secreted by microorganisms. In this stage, the most significant thing is the degradation of protein and starch, which can generate * Junrui Wu kinds of small molecule materials that will be helpful to the [email protected] growth of microorganisms in later fermentation. The second 1 College of Food Science, Shenyang Agricultural University, stage is the flavor formation phase. Various biochemical reac- Shenyang 110866, People’s Republic of China tions occur during the formation of flavor, such as Maillard 2 Department of Foreign Language, Shenyang Agricultural University, reaction which react between the reducing sugar and amino Shenyang 110866, People’s Republic of China compounds and generate melanin and flavor substances. The 506 Ann Microbiol (2018) 68:505–512 third stage is flavor re-balance period. Volatile flavoring sub- Materials and methods stances are mainly formed by microorganisms in the environ- ment that secrete enzymes. Ester is one of the typical flavor Sample collection substances, which are generated from alcohols and organic acids under the action of enzymes secreted by salt-tolerant Seventeen da-jiang samples were collected from three differ- yeast and bacteria (Sun 2007;Liu2010). ent regions (Xinmin (T), Tieling (Z), Shenyang (S)) in Natural fermentation is a spontaneous mixed-culture Liaoning Province of China. All samples were prepared by fermented process under appropriate temperature and humid- traditional fermented methods in the same year and collected ity conditions. This traditional food processing method has every 7 days during fermentation. Before collection, da-jiang been reserved in China without disappearance for many cen- in the bowl needs to be mixed. The collected da-jiang was turies. The microbial diversity in fermentation system is influ- loaded in sterile zipper lock bag and immediately stored at enced by the fermented method. As we all know, different − 80 °C waiting for further analysis. This work was approved microorganisms are responsible for different fermented stages by the Shenyang Agricultural University. All work was ap- and play different essential roles (Kim and Chun 2005; Zhang proved by relevant Chinese laws and institutional guidelines. et al. 2011). In recent years, studies on microbial diversity in traditional fermentation can often be seen. Everyone knows Determining pH, total acidity, and amino nitrogen that the traditional fermentation is very complicated. at different fermented stages Therefore, research on microbial diversity can not only help to analyze the relationship between metabolites and microor- SH-2100 pH meter (OHAUS, Jiangsu, China) was used to ganisms but also help to propose a potential approach to mon- measure pH of da-jiang. Total acidity (TA) of da-jiang was itor the traditional fermentation. determined by alkali titration as follows: to determine total The traditional methods for detecting food-related micro- acidity as lactic acid, 25 ml of properly diluted sample was organisms are mainly culture-dependent method and colony titrated with 0.01-N NaOH to pH 8.1. The determination of counts. However, these methods are limited to exploring the AN was as follows: firstly, formol nitrogen (FN) and ammo- variation and structure of microbial communities. Firstly, nium nitrogen were determined according to Chinese National culture-dependent method may be only suitable for part of Standard (CNS, 2002). To determine FN, diluted sample and microbes. Secondly, some microbes cannot be accurately dis- formalin solution were adjusted to pH 8.1, then 20 ml of criminated based on their appearance. Currently, only a few formalin solution was mixed with 25 ml of diluted samples microbes were identified in soybean paste, such as for 3 min, and titrated to pH 8.1 with 0.01-N NaOH. Then, Lactobacillus plantarum, Leuconostoc, Bacillus subtilis,and amino nitrogen (AN) was determined by subtraction of am- Enterococcus faecium. Previous limitations of culture- monium nitrogen from FN according to CNS (Chinese dependent method on research on microbial diversity have National Standard (CNS) 2002). For each analysis, three rep- been largely overcome by advances in molecular biology licates tests were performed. technology, which provide a rapid and high-resolution de- scription of microbial communities. In fact, molecular biology Determination of LAB changes methods have generally supported the traditional results, but theadvancedtechniqueshaveidentifiedmuchhighermicro- The 10-fold-concentrated cells were serially diluted (100 to bial diversity in microecology than previously expected 106) in 0.9% sterile saline solution, and 100 μlofproperly (Navarro et al. 2013). High-throughput sequencing technolo- diluted samples was spread on de Man, Rogosa, and Sharpe gy is a promising method, because it can generate hundreds of (MRS, Merck, pH 5.7) agar plates, then incubated for 1–2days thousands of sequences within a short time which can cover at 37 °C for counting of lactic acid bacteria (LAB). The col- the complex microbial communities as well as low-abundance onies formed (25–250) were calculated as log CFU per milli- microorganisms (Ercolini et al. 2012; Quigley et al. 2012). liter of da-jiang. Thus far, this technology has been used to explore the bacterial composition in fermented food samples, such as botrytized DNA extraction and PCR amplification wines (Bokulich et al. 2012), Chinese traditional sourdough (Liu et al. 2016), and grapes from a Californian region Firstly, 20-mL PBS buffer was added into 8-g sample and (Bokulich et al. 2014). Therefore, in order to provide a theo- vortexed for 5 min. The mixture was centrifuged at 1000 g retical basis for the understanding of the traditional fermenta- for 10 min. The supernatant was collected. Next, 20-ml PBS tion for da-jiang, we performed high-throughput sequencing buffer was added into the precipitate and vortexed for 5 min. technology (Illumina MiSeq platform) to analyze bacterial We centrifuged the mixture at 3000 g for 10 min and collected diversity in 17 da-jiang samples, which are from different the supernatant again. The above supernatant is mixed and fermented stages and different areas. centrifuged at 12,000 g for 10 min. The resulted precipitate Ann Microbiol (2018) 68:505–512 507 was washed with sterilized water and mixed with 700-μlTE using UCLUST. The highest abundance sequence in each buffer. Total DNA of da-jiang is rapidly extracted using the OTU is selected as the representative sequence. All the anal- CTAB method. Thirty microliter SDS and 3-μl protease K yses were performed using the QIIME program. The phylo- were added into the mixture, which then was incubated at genetic affiliation of each OTU representative sequence was 37 °C for 60 min. One hundred-microliter NaCl and 100-μl analyzed by default parameters in the QIIME software against cetyltrimethylammonium ammonium bromide (CTAB) are Silva database (version 115, http://www.arb-silva.de). We also added to the above mixture, which then was incubated at calculated the coverage percentage by Good’s method, bias- 65 °C for 25 min. Equal volumes of phenol–chloroform– corrected Chao1 richness estimator, Simpson indices, and the isoamyl alcohol (25:24:1) were added, mixed, settled for Shannon diversity indices (H′). R software was used to calcu- 1 min, and centrifuged at 12,000 g for 10 min. Equal volumes late the total number of common OTUs in each group. Then of chloroform–isoamyl alcohol (24.1) were rapidly added to Venn diagram (https://en.wikipedia.org/wiki/ Venn_diagram) the resulting supernatant, mixed well, allowed to settle for visually shows the share of the common and unique OTU in 1 min, and then centrifuged at 12,000 g for a further 10 min. da-jiang from different regions. QIIME software was used to Finally 500 μl of isopropyl alcohol was added to the upper obtain the composition and abundance distribution table at the phase and incubated at − 20 °C to precipitate the DNA. After level of the genus and the phyla of each sample. centrifugation at 12,000 g for 10 min and washing the precipitate in 70% ethanol, the resulting DNA was dissolved in TE buffer solution and stored at 20 °C prior to evaluation (Wu et Results and discussion al. 2015). To analyze the bacterial community, V4 region of 16S Biochemical properties of da-jiang fermentation rDNA gene was amplified using universal primer pair 502F: (5′-AYTGGGYDTAAAGNG-3′) and 802R: (5′- pH of da-jiang during fermentation is one of the most impor- TACNVGGGTATCTAATCC-3′) as described by tant indexes as it affects both the relative growth rates of Subramanian et al. (2014). The PCR mixture (final volume, different microorganisms and the accumulation rate of meta- 25 μL) contained 5-μl Q5 reaction buffer (5×), 2-μlDNA, bolic products, which can change both safety and flavor of the 1 μl of each primer, 0.25-μlQ5polymerase(5U/μl), 5-μl resulting da-jiang. In this study, the pH in da-jiang firstly de- Q5 GC high enhancer (5×), and 2.00-μl dNTP (2.5 mM). creased and then reached stability in the whole fermentation The following thermal cycling scheme was used: initial dena- (Table 1). It can be speculated that pH has a strong relationship turation at 98 °C for 2 min and 27 cycles of denaturation at with microbial metabolism. What’s more, pH values of da- 98 °C for 15 s, annealing at 50 °C for 30 s, and an extension at jiang from different regions have a slight difference (Table 72 °C for 30 s, followed by a final extension period at 72 °C 1). TA and AN are two of the most important quality indices for 5 min, then held at 4 °C. The quality of the amplified PCR of da-jiang, which contribute not only nutritional values but products was verified by electrophoresis in a 1% agarose gel also sensory characteristics. In general, TA and AN contents and purified (Axgen, USA). An equal amount of the PCR continuously increased during da-jiang fermentation progress product was combined to be sequenced using the pair-end in this study (Table 1). The increased TA and AN contents method by Illumina Miseq. may be owing to the microbial fermentation of carbohydrates and free amino acids. As shown in the Table 1, regions have a Illumina Miseq sequencing great impact on TA and AN contents.

Amplicons were extracted from 2% agarose gels, purified Changes in bacterial abundance using the AxyPrep DNA Gel Extraction Kit (Axygen during the fermentation of da-jiang Biosciences, Union City, CA, USA), and quantified using QuantiFluor™-ST (Promega, USA). Purified amplicons were The richness index (Chao1/ACE) indicated the tendency of pooled in equimolar amounts and paired-end sequenced on an bacterial abundance. We can see that bacterial abundance of Illumina MiSeq platform at Personal Biotechnology Co., Ltd., da-jiang in the first 28 days of fermentation was similar. Shanghai, China, according to the standard protocols. Overall, it looks like that the bacterial abundance was decreased during 0–7 days and 14–21 days and increased during Statistical and bioinformatics analyses 7–14 days and 21–28 days. The diversity index (Simpson/ Shannon) in da-jiang was decreased in the post fermentation Raw fastq files were demultiplexed, quality-filtered using the of da-jiang indicating that the diversity of bacterial communi- USEARCH (v5.2.236, http://www.drive5.com/usearch/)in ties in da-jiang was decreased from the beginning to the end of QIIME software (version 1.8.0). Operational taxonomic post fermentation. Many studies indicated that LAB plays an units (OTUs) were clustered with a 97% similarity cutoff important role in fermented foods (Jans et al. 2012;Fugletal. 508 Ann Microbiol (2018) 68:505–512

Table 1 Number of sequences analyzed, observed diversity richness, estimated OTU richness, diversity index, and estimated sample coverage for 16S rRNA libraries of da-jiang samples; pH value, TA and AN content, and lactic acid bacteria counts of da-jiang during fermentation

Sample OTUs chao1 ACE Simpson Shannon pH TA (g/100 ml) AN (g/100 ml) LAB (log CFU/ml)

S1 362 279 327.996 0.886 4.202 5.87 0.693 0.465 7.84 S2 388 289 347.557 0.901 4.272 5.83 0.699 0.517 8.07 S3 247 163 187.391 0.825 3.384 5.56 0.718 0.654 7.31 S4 258 174 195.841 0.809 3.305 5.16 0.755 0.631 7.37 S5 239 174 174.000 0.818 3.395 5.17 0.754 0.629 7.26 T1 299 219 262.694 0.566 2.567 5.32 0.837 0.641 7.43 T2 382 292 372.029 0.667 3.001 5.11 0.861 0.587 7.96 T3 339 227 298.825 0.496 2.262 4.99 0.891 0.612 7.47 T4 420 324 374.198 0.881 4.463 4.92 0.932 0.557 8.13 T5 337 245 273.919 0.558 2.569 4.89 0.953 0.687 7.55 T6 368 259 321.546 0.569 2.566 4.86 0.976 0.678 7.78 Z1 386 295 343.505 0.795 3.562 6.66 0.342 0.434 8.21 Z2 405 315 419.670 0.806 3.719 6.58 0.360 0.552 8.57 Z3 396 255 327.741 0.759 3.306 6.60 0.351 0.601 8.69 Z4 438 301 353.208 0.812 3.659 6.48 0.378 0.693 8.63 Z5 424 307 391.993 0.754 3.438 6.50 0.373 0.665 8.21 Z6 368 276 357.178 0.778 3.314 6.52 0.370 0.671 7.85

2017). The results confirmed that the amount of LAB has a 2016), and Brazilian kefir grains (Leite et al. 2012). slight difference in different regions (Table 1). It can be in- Therefore, Firmicutes is the dominant bacterial phylum in ferred that the area has an influence on the composition and traditional fermented foods. content of microorganisms in da-jiang. Different regions can During the post fermentation stage of da-jiang, Firmicutes produce different flavors of da-jiang. and Proteobacteria had a larger fluctuation range. In contrast, the content of Bacteroides, Actinobacteria,andThermi was all Bacterial communities of da-jiang quite a little and relatively stable in da-jiang during the post- fermentation stage. The data indicated that Firmicutes and Venn diagram revealed that 335 OTUs (47.6%) were common Proteobacteria were the dominant bacterial phylum in the in all samples. The common OTUs in sample S and sample T; post-fermentation of da-jiang from three regions in the north- sample T and sample Z; and sample S and sample T are 377 east of China. (53.6%), 494 (70.2%), and 386 (54.8%), respectively. The In addition, 100 known bacterial genera and 37 unknown specific OTUs were 49 (7.0%), 31 (4.4%), and 37 (5.3%) in genera were identified in da-jiang. On the whole, sample S, sample T, and sample Z, respectively (Fig. 1). All Leuconostoc, Enterococcus, and Acinetobacter had a large da-jiang samples were dominated by the phylum Firmicutes, Proteobacteria, Bacteroides, Actinobacteria,andThermi (Table 2). The Firmicutes and Proteobacteria occupied the two most dominant phylum in da-jiang. The content of Proteobacteria in da-jiang from Shenyang area was reached the maximum (64.90–78.80%) in post-fermentation stage. This result was consistent with the study on the predominant bacteria in sauce koji (Hui et al. 2017). The content of Firmicutes in da-jiang from Xinmin and Tieling was reached the maximum (78.30–95.70% and 83.60–92.00%, respectively) in post-fermentation stage. This result was consistent with other studies about microbial composition of traditional fermented foods, such as homemade yoghurts (Xu et al. 2015), tarag (Sun et al. 2014), Korean traditional food Doenjang and kochujang (Nam et al. 2012; Kim et al. Fig. 1 Distribution of bacteria OTUs Ann Microbiol (2018) 68:505–512 509

Table 2 The distribution of bacteria of da-jiang in phyla Sample Firmicutes Proteobacteria Bacteroidetes Actinobacteria [Thermi]

S1 20.30% 75.40% 3.20% 1.00% 0.00% S2 30.70% 64.90% 3.30% 1.10% 0.00% S3 16.90% 78.80% 4.10% 0.20% 0.00% S4 18.50% 76.10% 5.30% 0.10% 0.00% S5 23.30% 74.40% 2.10% 0.10% 0.00% T1 92.80% 5.70% 0.00% 1.30% 0.30% T2 94.10% 4.30% 0.10% 1.50% 0.10% T3 95.70% 3.40% 0.10% 0.70% 0.10% T4 78.30% 14.90% 0.10% 6.40% 0.20% T5 92.70% 5.60% 0.00% 1.50% 0.10% T6 86.40% 12.10% 0.30% 1.00% 0.20% Z1 87.20% 10.60% 0.00% 1.90% 0.40% Z2 83.60% 13.30% 1.20% 1.90% 0.00% Z3 91.20% 6.40% 0.20% 2.00% 0.00% Z4 92.00% 5.70% 0.00% 2.30% 0.00% Z5 91.30% 6.20% 0.10% 2.40% 0.00% Z6 91.00% 7.70% 0.00% 1.20% 0.00% proportion in every da-jiang, but the most abundant genus 2002; Kim et al. 2010), and soy sauce (Roling and Verseveld differed significantly in da-jiang from different regions 1996; Hanagata et al. 2003) with high salt content. (Fig. 2). The content of Leuconostoc (12.4–22.0%) was the Tetragenococcus halophilus has a potential to improve the most in sample S, followed by Acinetobacter (8.7–25.1%) and organoleptic properties. Tetragenococcus halophilus strain Pseudomonas (5.2%–11.5%). Tetragenococcus (2.0–73.0%) Th221 was reported to possess immunomodulatory activity were the most in sample T (Table 3). Tetragenococcus by promoting T helper type 1 immunity (Masuda et al. halophilus was also found in the fermentation of some other 2008). The followed bacteria in sample T were Enterococcus traditional fermented foods, such as soy sauce, fish sauces (9.1–30.1%) and Leuconostoc (4.0–19.0%). Sample Z (Nishimura et al. 2009; Udomsil et al. 2011) and fermented showed that Leuconostoc (9.2–25.7%) were the most, follow- fish products (Devi et al. 2015), soybean pastes (Onda et al. ed by Erwinia, Weissella,andLactobacillus,andthe

Fig. 2 The distribution of bacteria of da-jiang in genus 510 Ann Microbiol (2018) 68:505–512

Table 3 Relative abundance (%) of 18 major species from each da-jiang sample

TaxonnameS1S2S3S4S5T1T2T3T4T5T6Z1Z2Z3Z4Z5Z6

Tetragenococcus 0.2 0.4 0.2 0.2 0.2 67.7 58.1 73.0 2.0 68.9 67.5 0.5 0.5 0.6 26.9 0.7 29.4 Leuconostoc 12.4 22.0 13.8 14.8 18.2 7.0 7.9 4.8 19.0 7.0 4.0 25.7 23.6 23.0 13.6 21.5 9.2 Enterococcus 2.8 3.1 2.0 2.2 3.0 10.7 16.5 9.1 30.1 9.8 9.3 2.2 2.4 3.6 2.4 3.5 2.5 Acinetobacter 11.0 8.7 24.6 24.4 25.1 0.8 0.8 0.5 1.1 0.6 3.5 0.4 4.8 0.4 0.3 0.4 1.1 Weissella 0.0 0.2 0.0 0.1 0.1 0.2 0.3 0.2 0.4 0.2 0.2 3.3 3.6 4.5 2.3 3.3 2.2 Pseudomonas 7.1 5.2 11.5 6.4 6.7 0.4 0.4 0.3 0.3 0.4 0.6 0.2 0.3 0.2 0.2 0.2 0.2 Erwinia 2.4 1.7 2.2 2.3 1.9 0.0 0.1 0.0 0.1 0.1 0.3 5.3 3.8 3.0 2.5 2.6 3.5 Sphingobacterium 3.2 3.3 4.0 5.3 2.1 0.0 0.0 0.0 0.1 0.0 0.2 0.0 1.1 0.2 0.0 0.0 0.0 Ignatzschineria 9.5 15.6 0.2 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Lactobacillus 0.0 0.0 0.0 0.0 0.0 0.2 0.2 0.2 0.7 0.1 0.2 3.8 3.6 3.5 2.6 3.3 2.8 Staphylococcus 0.7 0.6 0.0 0.0 0.0 1.1 1.2 1.1 3.9 0.8 0.7 0.6 0.7 1.1 0.9 1.0 0.8 Serratia 0.4 0.3 1.2 1.2 0.9 0.0 0.0 0.0 0.0 0.0 0.1 2.0 1.5 1.3 1.0 1.3 1.1 Brevibacterium 0.1 0.1 0.0 0.0 0.0 0.2 0.2 0.1 0.6 0.2 0.2 1.5 1.6 1.7 2.0 2.0 1.0 Corynebacterium 0.7 0.5 0.2 0.1 0.1 0.2 0.4 0.2 1.0 0.3 0.2 0.0 0.0 0.0 0.0 0.0 0.0 Bacillus 0.9 0.9 0.0 0.0 0.0 0.3 0.6 0.7 1.1 0.4 0.3 0.2 0.1 0.2 0.1 0.2 0.1 Stenotrophomonas 0.9 1.1 0.1 0.1 0.1 0.3 0.2 0.1 0.2 0.3 0.0 0.0 0.1 0.0 0.0 0.1 0.0 Comamonas 1.0 1.0 0.2 0.3 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 Yaniella 0.0 0.0 0.0 0.0 0.0 0.5 0.4 0.2 2.0 0.6 0.4 0.0 0.0 0.0 0.0 0.0 0.0 fluctuation range of them was 2.5–5.3%, 2.2–4.5%, and 2.6– out a deeper study of the strain. Some strains of Acinetobacter 3.8%, respectively. Wu et al. (2013) identified species of bac- can be used for the biodegradation of pollutants, such as bi- teria in Northeast da-jiang through PCR-DGGE and conclud- phenyl and chlorinated biphenyl amino acid, phenol, crude ed that L. plantarum, Leuconostoc gasicomitatum, E. faecium, oil, and acetonitrile. What’s more, the metabolites of and T. halophilus were the superiority bacteria strains. Acinetobacter also have a high economic value, such as bio Candidatus solibacter, Leucobacter, Rhodococcus, emulsifier, polysaccharide, and lipase. A study showed Flavobacterium, Chryseobacterium, Phormidium, Acinetobacter AVLB2 can increase the biodegradation ability Natronobacillus, Virgibacillus, Sporosarcina, Aerococcus, of contaminant (4-NA) and also can promote plant growth Clostridium, Mycoplana, Ochrobactrum, Phyllobacterium, (Sivagnanam and Vangnai 2016). In recent years, Achromobacter, Comamonas, Comamonas, Kushneria, and Acinetobacter has been collected from vegetable samples Deinococcus were detected only in a specific fermentation (Karumathil et al. 2016). We also found that Acinetobacter period, and they have a smaller impact on the fermentation in our study (unpublished) may be a common genus in vege- of da-jiang. table or fermented products. Leuconostoc can produce lactic acid, acetic acid, ethanol, As a traditional fermented soybean product, the flavor of and other aromatic compounds (Cogan and Jordan 1994). It da-jiang has a strong relationship with microbial metabolism. can promote the value of probiotics in the stomach, inhibit the A variety of microorganisms work together to form a unique growth of pathogenic bacteria in intestinal tract, and play an flavor of da-jiang; so, it is important to note that the microbial important protective role in the quality of fermented vegetable community of da-jiang consists of not only predominant spe- products (Han et al. 2002; Johanningsmeier et al. 2004; cies but also many other rarer species. The microbial flora in Tamminen et al. 2004; Eom et al. 2007). Enterococci are part naturally fermented foods is complex. The production is not of the normal intestinal flora of humans and animals and are stable and poor security, which resulted that we cannot ensure recognized as probiotic bacteria (Fuller 1989). Previous stud- the quality problems often occurred. So, the study of microbial ies showed that E. faecium produce bacteriocins and are com- diversity in fermented food is necessary to solve the above monly found in traditional fermented foods, such as Japanese problems. The test intuitively showed the bacterial diversity miso (Onda et al. 2002). However, as reported by some au- and flora change in the traditional da-jiang fermentation. thors, E. faecium also have the potential side effects, such as What’s more, the microbial difference among samples of dif- carries on antibiotic resistance genes or virulence determinants ferent areas also affects the product characteristics. Although (Klare et al. 2003). In addition, these genes are often harbored there were some common bacterial genera in da-jiang from on plasmids that could be potentially exchanged. Therefore, if different regions, there were also some obvious differences. In we want to achieve purebred fermentation, we need to carry addition, many external environmental conditions and human Ann Microbiol (2018) 68:505–512 511 factors also affect microbial metabolism. Therefore, there are Informed consent Informed consent is not required in this study. certain differences in the product characteristics of da-jiang in different areas. It can reveal that the microbial community structure in different fermented periods is helpful to excavate References rich microbial resources, and the identified dominant bacteria of different fermentation stages provide a good foundation for Bokulich NA, Joseph CML, Allen G, Benson AK, Mills DA (2012) Nextgeneration sequencing reveals significant bacterial diversity of the sauce industrialization which product sauce by artificial botrytized wine. PLoS One 7:e36357 inoculation. It is possible to achieve the modernization of Bokulich NA, Swadener M, Sakamoto K, Mills DA, Bisson LF (2014) traditional sauce and large-scale production. It also provided Sulfur dioxide treatment alters wine microbial diversity and fermen- quality, safety, and excellent flavor sauce to meet the market tation progression in a dose-dependent fashion. Am J Enol Viticult 66(1):73–79 demand. Also, the study provided the theoretical basis for Chaves-López C, Serio A, Grande-Tovar CD, Cuervo-Mulet R, Delgado- further research and development in naturally fermented da- Ospina J, Paparella A (2014) Traditional fermented foods and bev- jiang. erages from a microbiological and nutritional perspective:the Colombian heritage. Compr Rev Food Sci F 13:1031–1048 Chinese National Standard (CNS). (2002). Soy sauce CNS423, N5006 Cogan TM, Jordan KN (1994) Metabolism of Leuconostoc bacteria. J Conclusion Dairy Sci 77(9):2704–2717 Devi KR, Deka M, Jeyaram K (2015) Bacterial dynamics during yearlong spontaneous fermentation for production of ngari, a dry fermented In conclusion, the current study showed that high-throughput fish product of Northeast India. Int J Food Microbiol 199:62–71 sequencing approach can be successfully applied for analyz- Eom HJ, Seo DM, Han NS (2007) Selection of psychrotrophic ing microbial community in fermented foods. In this study, we Leuconostoc spp. producing highly active dextransucrase from lactate fermented vegetables. Int J Food Microbiol 117(1):61–67 can conclude the dominant bacteria phyla of da-jiang were Ercolini D, Filippis DF, Storia L, Lacono AM (2012) BRemake^ by Firmicutes and Proteobacteria in post-fermentation stages highthroughput sequencing of the microbiota involved in the pro- through Illumina MiSeq sequencing. The common dominant duction of water buffalo mozzarella cheese. Appl Environ Microbiol bacterial genera of da-jiang from three regions were 78(22):8142–8145 Fugl A, Berhe T, Kiran A, Hussain S, Laursen MF, Bahl MI, Hailu Y, Leuconostoc, Enterococcus,andAcinetobacter. Although Sørensen KI, Guya ME, Ipsen R, Hansen EB (2017) there were some common features in da-jiang from different Characterisation of lactic acid bacteria in spontaneously fermented regions, there were also some obvious differences: the domi- camel milk and selection of strains for fermentation of camel milk. nant bacteria genus of da-jiang from Shenyang also included Int Dairy J 73:19–24 – Ignatzschineria, Pseudomonas, Sphingobacterium,and Fuller R (1989) Probiotics in man and animals. J Appl Bacteriol 66:365 378 Erwinia; the dominant bacteria genus of da-jiang from Ge JP, Chai YY, Chen L, Ping WX (2012) The dynamics of bacteria Xinmin also included Staphylococcus and Weissella; community diversity during the fermentation process of traditional Erwinia, Lactobacillus, Serratia, and Brevibacterium were soybean paste. Acta Ecol Sin 32:2532–2538 the dominant bacteria genus of da-jiang from Tieling. Han NS, Jung YS, Eom HJ, Koh YH, Roby JF, Seo JH (2002) Simultaneous biocatalytic synthesisof panose during lactate fermen- Through collation and analysis, we concluded that the chang- tation in kimchi. J Microbiol Biotechnol 12(1):46–52 es of bacterial abundance in da-jiang from three regions are Hanagata H, Shida O, Takagi H (2003) Takagi.Taxonomic homogeneity basically the same in the early and middle fermented stages. of a salt-tolerant lactic acid bacteria isolated from shoyu mash. J Gen – At the end of fermentation, microbial community species in Appl Microbiol 49(2):95 100 Hugenholtz J (2013) Traditional biotechnology for new foods and bever- da-jiang eventually reduced. ages. Curr Opin Biotechnol 24:155–159 Hui WY, Hou QC, Cao CX, Xu HY, Zhen Y, Kwok LY, Sun TS, Zhang Funding information This study was funded by the Natural Science HP, Zhang WY (2017) Identification of microbial profile of koji Foundation of China (Grant Nos. 31470538, 31471713), Program for using single molecule, real-time sequencing technology. J Food Liaoning Excellent Talents in University (LJQ2015103, LR2015059), Sci 82(5):1193–1199 China Postdoctoral Science Foundation Funded Project (Grant No. Jans C, Bugnard J, Njape PMK, Lacroix C, Meile L (2012) Lactic acid 2014M560395), and Cultivation Plan for Youth Agricultural Science bacteria diversity of African raw and fermented camel milk products and Technology Innovative Talents of Liaoning Province (Grant No. reveals a highly competitive, potentially health-threatening predom- 2014048). inant microflora. LWT-Food Sci Techno 47:371–379 Johanningsmeier SD, Fleming HP, Breidt R (2004) Malolacticactivity of lactic acid bacteria during sauerkraut fermentation. J Food Sci 69(8): Compliance with ethical standards 222–227 Karumathil DP, Yin HB, Kollanoor-Johny A, Venkitanarayanan K (2016) Conflict of interest The authors declare that they have no conflict of Prevalence of multidrug-resistant bacteria on fresh vegetables col- interest. lected from farmers’ markets in Connecticut. J Food Prot 79(8): 1446–1451 Research involving human participants and/or animals This article Kim JH, Ahn HJ, Yook HS, Park HJ, Byun MW (2010) Biogenic amines does not contain any studies with human participants or animals per- contents in commercial Korean traditional fremented soybean paste. formed by any of the authors. Korean J Food Sci Biotechnol 33(6):682–685 512 Ann Microbiol (2018) 68:505–512

Kim M, Chun J (2005) Bacterial community structure in kimchi, a Sivagnanam S, Vangnai AS (2016) Biodegradation of 4-nitroaniline by Korean fermented vegetable food, as revealed by 16S rRNA gene plant-growth promoting Acinetobacter sp. AVLB2 and toxicological analysis. Int J Food Microbiol 103:91–96 analysis of its biodegradation metabolites. J Hazard Mater 302(25): Kim MJ, Kwak HS, Jung HY, Kim SS (2016) Microbial communities 426–436 related to sensory attributes in Korean fermented soy bean paste Subramanian S, Huq S, Yatsunenko T, Haque R, Mahfuz M, Alam MA, (doenjang). Food Res Int 89(1):724–732 Benezra A, DeStefano J, Meier MF, Muegge BD, Barratt MJ, Klare I, Konstabel C, Badstubner D, Werner G, Witte W (2003) VanArendonk LG, Zhang Q, Province MA, Petri WA Jr, Ahmed Occurrence and spread of antibiotic resistances in Enterococcus T, Gordon JI (2014) Persistent gut microbiota immaturity in mal- faecium. Int J Food Microbiol 88:269–290 nourished Bangladeshi children. Nature 510:417–421 Leite AM, Mayo B, Rachid CT, Peixoto RS, Silva JT, Paschoalin VM, Sun CY (2007) Formation and function of major microbial enzymes in Delgado S (2012) Assessment of the 399 microbial diversity of natural fermented soybean paste. Northeast Agricultural University, Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis. 2–5 – Food Microbiol 31(2):215 221 Sun ZH, Liu WJ, Bao QH, Zhang JC, Hou QC, Kwok LY,Sun TS, Zhang Liu JM (2010) Study on production technology of soybean isoflavone HP (2014) Investigation of bacterial and fungal diversity in tarag – nutritional soybean paste. Harbin University of Commerce, 1 6 using high-throughput sequencing. J Dairy Sci 97(10):6085–6096 Liu T, Li Y, Chen J, Sadiq FA, Zhang G, Li Y (2016) Prevalence and Tamminen M, Joutsjoki T, Sjöblom M, Joutsen M, Palva A, Ryhänen EL, diversity of lactic acid bacteria in Chinese traditional sourdough Joutsjoki V (2004) Screening of lactic acid bacteria from fermented revealed by culture dependent and pyrosequencing approaches. vegetables by carbohydrate prowling and PCR-ELISA. Lett App – LWT-Food Sci Techno 68:91 97 Microbiol 39(5):439–444 Masuda S, Yamaguchi H, Kurokawa T, Shirakami T, Tsuji RF, Nishimura Udomsil N, Rodtong S, Choi YJ, Hua YL, Yongsawatdigul J (2011) Use I (2008) Immunomodulatory effect of halophilic lactic acid bacteri- of Tetragenococcus halophilus as a starter culture for flavor im- um Tetragenococcus halophilus Th221 from soy sauce moromi provement in fish sauce fermentation. J Agr Food Chem 59(8): grown in high-salt medium. Int J Food Microbiol 121(3):245–252 101–108 Nam YD, Park SL, Lim SI (2012) Microbial composition of the Korean traditional food Bkochujang^ analyzed by a massive sequencing Wu JR, Zhang JC, Shi P, Wu RN, Yue XQ, Zhang HP (2013) Bacterial – community involved in traditional fermented soybean paste dajiang technique. J Food Sci 77(4):250 256 – Navarro D, Mateo E, Torija MJ, Mas A (2013) Acetic acid bacteria in made in northeast China. Ann Microbiol 63(4):1417 1421 grape must. Acetic Acid Bacteria 2:19–23 Wu RN, Yu ML, Liu XY, Meng LS, Wang QS, Xue YT, Wu JR, Yue XQ Nishimura I, Igarashi T, Enomoto T, Dake Y, Okuno Y, Obate A (2009) (2015) Changes in flavour and microbial diversity during natural Clinical efficacy of halophilic lactic acid bacterium Tetragenococcus fermentation of suan-cai, a traditional food made in northeast – halophilus Th221 from soy sauce moromi for perennial allergic China. Int J Food Microbiol 211:23 31 rhinitis. Allergol Int 58(2):179–185 Xu HY, Liu WJ, Gesudu QM, Sun ZH, Zhang JC, Guo Z, Zheng Y, Hou Onda T, Yanagida F, Uchimura T, Tsuji M, Ogino S, Shinohara T, QC, Yu J, Qing YT, Kwok LY, Menhe B, Zhang HP (2015) Yokotsuka K (2002) Widespread distribution of the bacteriocin- Assessment of the bacterial and fungal diversity in home-made yo- producing lactic acid cocci in miso-paste products. J Appl ghurts of Xinjiang, China by pyrosequencing. J Sci Food Agric Microbiol 92:695–705 95(10):2007–2015 Quigley L, O'Sullivan O, Beresford T, Ross P, Fitzgerarld G, Cotter P Xu Q (2013) The spread and regional characteristics of East Asian miso (2012) Highthroughput sequencing for detection of subpopulations culture. China Condiment 2:9–14 of bacterial not previously associated with artisanal cheeses. Appl Zhang JC, Liu WJ, Sun ZH, Bao QH, Wang F, Yu J, Chen W, Zhang HP Environ Microbiol 78(16):5717–5723 (2011) Diversity of lactic acid bacteria and yeasts in traditional sour- Roling W, Verseveld HW (1996) Characterization of Tetragenococcus doughs collected from western region in Inner Mongolia of China. halophila populations in Indonesian soy mash (Kecap) fermenta- Food Control 22:767–774 tion. Appl Environ Microbiol 62(4):1203–1207