Dynamics and Diversity of a Microbial Community During the Fermentation of Industrialized Qingcai Paocai, a Traditional Chinese

Annals of Microbiology (2018) 68:111–122 https://doi.org/10.1007/s13213-017-1321-z

ORIGINAL ARTICLE

Dynamics and diversity of a microbial community during the fermentation of industrialized Qingcai paocai, a traditional Chinese fermented vegetable food, as assessed by Illumina MiSeq sequencing, DGGE and qPCR assay

Huipeng Liang1,2 & Liguo Yin3 & Yahao Zhang2 & Cong Chang2 & Wenxue Zhang2

Received: 4 July 2017 /Accepted: 20 December 2017 /Published online: 6 January 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Abstract Paocai is a traditional Chinese fermented food and typically produced via spontaneous fermentation. We have investigated the microbial community utilized for the fermentation of industrialized Qingcai paocai using the combination of Illumina MiSeq sequencing, PCR-mediated denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) assay. Three main phyla, namely Firmicutes, Proteobacteria and Bacteroidetes, were identified by both MiSeq sequencing and PCR-DGGE. The dominant genera observed in the fermentation were Lactobacillus, Pseudomonas, Vibrio and Halomonas.Mostgenera affiliated with Proteobacteria or Bacteroidetes were detected more often during the earlier part of the fermentation, while Lactobacillus (affiliated with Firmicutes) was dominant during the later fermentation stages. Fungal community analysis re- vealed that Debaryomyces, Pichia and Kazachstania were the main fungal genera present in industrialized Qingcai paocai, with Debaryomyces being the most dominant during the fermentation process. The quantities of dominant genera Lactobacillus and Debaryomyces were monitored using qPCR and shown to be 109–1012 and 106–1010 copies/mL, respectively. During the later fermentation process of industrialized Qingcai paocai, Lactobacillus and Debaryomyces were present at 1011 and 108 copies/mL, respectively. These results facilitate further understanding of the unique microbial ecosystem during the fermentation of industrialized Qingcai paocai and guide future improvement of the fermentation process.

Keywords Qingcai paocai . Illumina MiSeq sequencing . PCR-DGGE . qPCR . Fermentation

Introduction materials (Yan et al. 2008). It is mildly salted, and paocai brine contains lactic acid. Paocai is one of the typical representatives Paocai is a traditional Chinese fermented vegetable that is of traditional fermented foods in China (especially in Sichuan commonly produced by the spontaneous fermentation of raw province) and is commonly served as a side dish or as an appetizer. In recent years, paocai has been recognized as a Electronic supplementary material The online version of this article functional food with several health benefits (Yan et al. (https://doi.org/10.1007/s13213-017-1321-z) contains supplementary 2008). Generally, industrialized paocai is made from only a material, which is available to authorized users. single type of vegetable without the addition of seasonings, while homemade paocai is made from a variety of vegetables * Wenxue Zhang (e.g. cabbage, radish, cucumber and cowpea) with the addition [email protected] of seasonings (e.g. red pepper, garlic, ginger). Industrialized paocai is produced by stacking fresh vegetables with salt and 1 National Engineering Research Center of Seafood, School of Food Science and Technology, Dalian Polytechnic University, then allowing natural fermentation at ambient temperature for Dalian 116034, People’sRepublicofChina at least 3 months. The quality of industrialized paocai is un- 2 College of Light Industry, Textile and Food Engineering, Sichuan stable and not uniform due to the many factors that influence University, Chengdu 610065, People’s Republic of China the fermentation process, such as the type and quality of the 3 College of Life Science and Food Engineering, Yibin University, raw vegetables, salt concentration and production season. The Yibin 644007, People’s Republic of China spontaneous fermentation of vegetables is mainly affected by 112 Ann Microbiol (2018) 68:111–122 the presence of microorganisms at different stages of the fermentation process and is highly dependent on the lactic acid bacteria (LAB) that occur naturally in the raw materials (Tanganuratetal.2009;Liuetal.2011), including Lactobacillus, Pediococcus, Leuconostoc, Weissella, Enterococcus and Lactococcus. To improve both the production technology and the quality of industrialized paocai, it is necessary to explore the structure of the microbial community during the fermentation process of industrialized paocai. The conventional culture method is often time-consuming and intensive, but numerous powerful molecular ecological methods have recently been developed and widely used to analyze the microbial community in the field of food more efficiently and rapidly (Hong et al. 2014; Luo et al. 2014; Iwobi et al. 2015). These methods have over- come the shortcomings of conventional culture methods. PCR-mediated denaturing gradient gel electrophoresis (PCR-DGGE) is a classical molecular ecological technique that has been widely applied to directly reveal and rapidly monitor the microbial community during the fermentation Fig. 1 Schematic flow diagram of the production process of Chinese process (Nie et al. 2013, 2015;Liangetal.2016a). industrialized paocai However, these methods also have many limitations for the monitoring of microbial communities because they produce 2015, industrialized Qingcai paocai samples in different fer- limited amounts of information. With the advent of next- mentation stages were collected from a local Chinese paocai generation sequencing technology, a thorough investigation factory (Sichuan Liji Paocai Condiment Co., Ltd., Meishan, of the complete microfloral community during the fermenta- China), which is located in Sichuan province, China. Four tion process became possible. Illumina MiSeq sequencing has samples for each fermentation time were collected from dif- been used to analyze the diversity of the microbial community ferent locations in the fermentation pit, and each set of four in many foods (Jeong et al. 2013;Lietal.2016a). However, to samples were then thoroughly mixed for subsequent analysis. date, no relevant studies on the microbial diversity of indus- The pH values were measured in 50-mL samples of paocai trialized paocai have been performed. Quantitative PCR brine using a pH meter (PHS-3C; Shanghai Electronics (qPCR) has emerged as a specific and sensitive technique that Science Instrument Co., Ltd., Shanghai, China). To determine allows for the quantification of the target organism in different the total titratable acidity (TTA), we first diluted 25 mL of samples (Paul et al. 2015; Liang et al. 2016b). In the study paocai brine sample with cooled boiled water to a volume of reported here, qPCR was used as a complementary method to 250 mL, then titrated the diluted sample using 0.1 M NaOH to quantify dominant microorganisms during the fermentation of a final pH of 8.2. The TTAwas expressed as follows: C × (V1 - industrialized paocai. V0) × n × F × 1000/V, where C represents the concentration of The primary aim of this study was to characterize the mi- NaOH (in mol/L); V1 represents the volume of the 0.1 M crobial community during the fermentation of industrialized NaOH used for sample determination (in mL); V0 represents paocai using Illumina MiSeq sequencing, PCR-DGGE and the 0.1 M NaOH used for the blank control (boiled distilled qPCR techniques. The results provide a deeper understanding water) determination (in mL); n represents the dilution factor; of the microecosystem and lay a foundation for improvements F represents the factor appropriate for lactic acid (0.009); V in standard manufacturing systems and production processes represents the volume of the diluted sample (in mL). of industrialized paocai. DNA extraction and PCR-DGGE analysis of the microbial community Materials and methods Total DNA was extracted from 10 mL of homogenized paocai Sample collection and physico-chemical analysis brine sample using an E.Z.N.A.® DNA kit (Omega Bio-Tek Inc., Norcross, GA), according to the manufacturer’sproto- In this study the vegetable used to make industrialized paocai cols. The DNA preparations were stored at − 20 °C until was Qingcai (Brassica chinensis var. chinensis). A schematic further use. PCR cycling was performed in a MyCycler™ flow sheet of the production process is shown in Fig. 1.In Thermal cycler (Bio-Rad, Hercules, CA). The reaction Ann Microbiol (2018) 68:111–122 113 volume (50 μL) contained 2 × PCR Mix (TIANGEN Biotech, qPCR analysis of dominant microorganisms Beijing, China), 20 pmol primers, template DNA and distilled water. The 16S ribosome RNA (rRNA) and 18S rRNA gene Quantitative PCR was performed in a Lightcycler® Nano were amplified using primers reported in previous studies System (Roche, Basel, Switzerland) to quantify the dominant (Muyzer et al. 1993;Harutaetal.2006); these former primers microorganisms during the fermentation of industrialized contained a GC clamp at the 5′ end. Qingcai paocai. The primers F-lac (5′-GCA GCA GTA DGGE was performed in 8% polyacrylamide gels, with a GGG AAT CTT CCA-3′), R-lac (5′-GCA TTY CAC CGC linear gradient of 30–55% and 20–40% denaturant for both TAC ACA TG-3′), DerbaF1 (5′-CTT TCG CCC TGT GGT the bacterial and fungal community using the D-Code™ GTT TG-3′) and DerbaR1 (5′-GCG TCA AAA AAA GAA Universal Mutation Detection System (Bio-Rad). The electro- CAA CAC C-3′) were used to amplify the 16S rRNA and 18S phoresis of 16S rRNA and 18S rRNA genes was performed in rRNA genes of Lactobacillus and Debaryomyces (Castillo 1 × TAE buffer at a constant voltage of 200 V for 4 h and et al. 2006; Meng et al. 2017). The amplification reaction 160 V for 5 h, respectively, at 60 °C. The gel was then stained volumes (20 μL) contained SYBR® Green Realtime PCR with SYBR Green I (1:10,000 v/v) and visualized using a Bio- Master Mix (Transgen, Beijing, China), 2 pmol of each prim- Rad gel imaging and documentation system (Gel Doc™ XR). er, template DNA and distilled water. The amplification pro- The DNA in major bands was eluted and amplified using the gram consisted of an initial denaturation at 95 °C for 10 min, primers mentioned above without a GC clamp. The PCR followed by 45 cycles of 95 °C for 15 s, 60 °C for 30 s and products were purified and sent to a company for cloning 72 °C for 15 s, with a collection of fluorescence signal at the and sequencing (Sangon, Shanghai, China). The sequence end of each cycle. information was acquired by aligning the results with se- Melting curve analysis was conducted after amplification quences obtained from GenBank using BLAST (www. via gradual heating from 60 °C to 95 °C at 0.1 °C/s under ncbi.nlm.nih.gov/BLAST/) and Classifier (http://rdp.cme. continuous fluorescence monitoring. The standard curves de- msu.edu/index.jsp). scribing the relationship between the threshold cycle (Cq)and copy number (q) were constructed using a tenfold serial dilu- Illumina MiSeq sequencing analysis of microbial tion of plasmid and evaluated via the correlation coefficient communities (R2). All samples were performed in duplicate, and the average values of the obtained results were presented. Bacterial 16S rRNA genes were amplified using the primers 341F (5′-CCT ACG GGN GGC WGC AG-3′) and 805R (5′- Data analysis GAC TAC HVG GGTATC TAATCC-3′), and fungal internal transcribed spacer (ITS) genes were amplified using the Bands patterns of the PCR-DGGE profiles were analyzed via primers ITS1 (5′-CTT GGT CAT TTA GAG GAA GTA Quantity One (Bio-Rad). The bands, Shannon index and A-3′) and ITS2 (5′-GCT GCG TTC TTC ATC GAT GC-3′) Simpson index were determined based on the relative quantity (Liang et al. 2015;Lietal.2016b). The forward primers of the DGGE bands (Liang et al. 2016a). The coverage, contained the partial Illumina adapter and a six-nucleotide Shannon index and Pielou index were included in the alpha sample-specific barcode. The PCR mixture (50 μL) contained diversity analysis by using the rarefaction.single command PCR buffer, each dNTP at 10 mM, 50 μM primers, 5 U Mothur (Schloss et al. 2009), based on the Illumina Miseq Plantium Taq and genomic DNA. The PCR products were OTU sequencing data. To visualize the similarity of microbi- analyzed by electrophoresis and purified using a PCR ota during the fermentation, principal component analysis Purification kit (Sangon). Composite barcoded amplicons (PCA) was then performed using Canoco for Windows v4.5 were pooled with equal amounts of each sample and se- software (Wageningen University and Research Center, quenced using the Illumina MiSeq 2×300 system (Illumina Wageningen, the Netherlands) based on the abundance of mi- Inc., San Diego, CA). crobial community at the genus level obtained by PCR-DGGE Raw sequences were sorted based on their unique and Illumina Miseq sequencing data. barcodes. Quality-checked sequences were controlled and analyzed using the QIIME pipeline. The sequences that Nucleotide sequence accession numbers contained more than one ambiguous base call (N) or those that the UCHIME algorithm identified as a putative chimera The sequence data reported in this study have been submitted were eliminated. The aligned sequences were clustered into to the Sequence Read Archive (SRA) NCBI (http://www.ncbi. operational taxonomic units (OTU) at a 97% similarity thresh- nlm.nih.gov/) under the BioProject ID PRJNA328338. The old using UCLUST v1.1.579. The phylogenetic affiliation of nucleotide sequences generated in DGGE have been each sequence was analyzed using RDP Classifier (version deposited at DDBJ under accession numbers LC128538 to 2.10) software at a confidence level of 80%. LC128575 and LC227663-LC227677. 114 Ann Microbiol (2018) 68:111–122

Results

Physico-chemical property changes during fermentation

The changes in pH and TTA during the fermentation of industrialized Qingcai paocai are shown in Fig. 2. The pH value was 5.71 at the beginning of fermentation and then decreased and steadied at around 3.7. The TTA values increased during the fermentation process, reaching a maximum at 34 days (Fig. 2), following which time it remained almost stable until the end of fermentation.

Fig. 2 Changes in pH and total titratable acidity (TTA)ofsamples Bacterial community diversity based on PCR-DGGE collected during the industrialized Qingcai paocai fermentation process. and Illumina MiSeq sequencing All analyses were conducted in duplicate, and average values are presented The bacterial PCR-DGGE profile is presented in Fig. 3a, showing 37 bands. The number of bands in different samples quality sequences. A total of 12,031 different OTUs were ranged from 12 to 28 (see Table 1). A total of 198,952 reads obtained based on 3% dissimilarity in 16S rRNA sequences, were obtained from a single run with eight bacterial PCR and rarefaction analysis indicated that all bacterial communi- amplicons using barcoded Illumina MiSeq sequencing, and ties were well represented since the rarefaction curves were 183,756 high-quality sequences with an average read length approaching their respective saturation plateaus. All diversity of about 421 bp were obtained after the removal of low- indices of the bacterial community are shown in Table 1.The

Fig. 3 The PCR-mediated denaturing gradient gel electrophoresis and fungal community DGGE analysis was 30–55% and 20–40%, (DGGE) profile of bacterial and fungal microbiota from microbial DNA respectively, and lane designation corresponds to the fermentation time extracted from industrialized Qingcai paocai samples collected at in days (d). The bands indicated by the arrows and numbers were excised different time points during the fermentation process. a Bacterial and sequenced, and the alignment results are listed in Table 2 microbiota, b fungal microbiota. The denaturing gradient of bacterial Ann Microbiol (2018) 68:111–122 115

changes in the Shannon and Simpson indices based on PCR- DGGE and Illumina MiSeq sequencing analysis were parallel; however, the Shannon index based on Illumina MiSeq sequencing analysis was higher than that based on PCR- DGGE analysis, while the Simpson index showed the oppo- site result. The Shannon index based on both methods initially

) Shannon Simpson showed a decrease from 1 day to 34 days and then an increase n to a basically steady value after 34 days, indicating that the diversity of the bacterial community was stable during the later stage of the fermentation process (Table 1). The Simpson index showed a similar tendency to vary, with the decrease in the Simpson index demonstrating the appearance of dominant bacteria during the later part of the fermentation process. All PCR-DGGE bands fell into three phyla: Firmicutes, Proteobacteria and Bacteroidetes. The similarity of sequences reached ≥ 99% compared to GenBank (Table 2). In Fig. 3aand ) Shannon Simpson Bands (

n Table 2, Lactobacillus species (bands 1, 2, 4, 5, 9 and 11), including L. alimentarius, L. brevis, L. plantarum and L. sakei, were detected during the entire fermentation process of industrialized Qingcai paocai, with L. alimentarius being the dominant Lactobacillus species during the later fermentation stage (Fig. 3a; Table 2). Most genera affiliated to paocai Proteobacteria were detected during the entire fermentation process of industrialized Qingcai paocai, including Qingcai Pseudomonas (bands 3, 7, 13, 18 and 19), Vibrio (bands 6, 12, 17 and 20), Halomonas (bands 29, 31 and 37), Pectobacterium (bands 27, 28, 32 and 33) and Klebsiella (bands 26, 35 and 36) (Fig. 3a). Flavobacterium (band 8) and Myroides (band 10) affiliated to Bacteroidetes were de-

ses of industrialized tected more during the earlier stage of fermentation (Fig. 4a). ) Coverage Shannon Simpson Bands (

n Based on the results of Illumina MiSeq sequencing analysis, 29 phyla were detected; however, > 95% of the annotated reads were assigned to four phyla, namely Firmicutes,

; OTU, operational taxonomic unit Proteobacteria, Bacteroidetes and Actinobacteria. A total of 619 genera were shared by all samples, with the most abundant genera during the fermentation being Lactobacillus, the fermentation proces Pseudomonas, Vibrio,andHalomonas (Fig. 4b). Three main phyla, namely Firmicutes, Proteobacteria and Bacteroidetes, were detected with both methods, and many mutual dominant genera were detected by both methods (Fig. 4a, b). However, the number of phyla or genera detected using Illumina MiSeq sequencing was much higher than that detected using PCR- DGGE. Bacterial community analysis showed that diverse bacterial groups, including Pseudomonas, Halomonas,

) Coverage Shannon Simpson OTUs ( Sphingomonas, Sphingobacterium, Stenotrophomonas, n Erwinia, Vagococcus, among others, which presumably originated from the raw materials, were present as major popula- Illumina MiSeq sequencingBacteriaOTUs ( Fungi PCR-DGGE Bacteria Fungi tions during the early fermentation stage. With extended fermentation, the bacterial community was rapidly dominated by The diversity indices of microbial community in members of the genus Lactobacillus (Fig. 4a, b). This tendency toward increases in the relative abundance of Lactobacillus 13112134 185450 180172 1448 0.92589 1391 0.935 1330 0.938PCR-DGGE, 4.767 PCR-mediated denaturing 1020 gradient 0.944 gel electrophoresis 4.666 1362 0.956 4.270 0.897 1825 0.955 3.988 0.949 0.958 0.902 1.399 719 0.966 0.841 2.176 502 594 0.145 3.455 0.994 515 0.547 3.270 0.996 194 0.874 0.996 207 3.040 0.766 0.996 193 2.823 0.997 3.172 0.313 194 0.997 2.258 0.611 0.998 0.660 0.169 28 0.998 0.589 0.797 23 22 0.038 0.453 23 0.467 0.729 2.896 12 0.188 2.686 2.613 16 0.347 0.922 2.627 19 0.908 0.897 1.552 17 3 0.893 1.970 6 8 0.635 2.390 5 0.806 2.125 1.066 5 0.880 1.493 7 0.852 1.828 0.644 7 1.168 0.719 9 0.816 1.185 0.581 1.433 0.615 1.567 0.682 1.916 0.685 0.755 Fermentation time (days) Table 1 was consistently observed. 116 Ann Microbiol (2018) 68:111–122

Table 2 Microbial species identification after sequencing of 16S rRNA and 18S rRNA genes purified from PCR-mediated denaturing gradient gel electrophoresis profiles obtained from RNA directly extracted from paocai samples

Band IDa Closest sequences/microorganismsb Identify (%) Length (bp) Phylogenetic affiliation Accession no.

Bacteria 1 Lactobacillus brevis strain FJ006 99 195 Lactobacillus KP889231.1 2, 5 Lactobacillus plantarum strain SP-5 99–100 194–196 Lactobacillus KT371518.1 3 Pseudomonas sp. KAR34 100 194 Pseudomonas KR054996.1 4, 9 Lactobacillus alimentarius strain: JCM 1095 100 195–196 Lactobacillus LC063166.1 6, 12, 20 Vibrionaceae bacterium X1 99–100 193–194 Vibrio KJ158197.1 7, 18 Pseudomonas sp. 138-FB 100 194–195 Pseudomonas AB566059.1 8 Flavobacterium sp. isolate M1_I3 99 190 Flavobacterium HG934362.1 10 Uncultured bacterium clone JCD02C 97 190 Myroides JF692526.1 11 Lactobacillus sakei strain S39 100 194 Lactobacillus KT327858.1 13 Pseudomonas fluorescens strain b225 100 179 Pseudomonas EU434518.1 14 Acinetobacter sp. SS48 99 195 Acinetobacter KP258131.1 15 Erwinia sp. INT22–36 99 194 Erwinia KP708594.1 16 Acinetobacter sp. strain DD79 99 196 Acinetobacter LN871436.1 17 Vibrio sp. LC7 100 196 Vibrio KP731564.1 19 Pseudomonas oryzihabitans strain MOSEL-MD11 100 196 Pseudomonas KP890064.1 21, 24 Uncultured bacterium gene clone: 1X68 99–100 196 Gammaproteobacteria LC002927.1 22 Lactococcus piscium strain H5 100 197 Lactococcus KT767784.1 23 Solibacillus silvestris strain SN8 100 196 Solibacillus LN870317.1 25 Sphingomonas faeni strain IHBB 11138 100 171 Sphingomonas KR085870.1 26, 35, 36 Klebsiella sp. MOSEL-SA4 100 194–195 Klebsiella KP890070.1 27, 28, 32 Pectobacterium carotovorum subsp. brasiliense strain Y65 99–100 195–196 Pectobacterium KP187523.1 29 Halomonas sp. JB380 100 194 Halomonas KF669533.1 30 Pantoea sp. S71 98 195 Pantoea KT025920.1 31 Halomonas sp. HL-48 100 194 Halomonas KJ004410.1 33 Pectobacterium atrosepticum strain CbUr23 100 195 Pectobacterium KM371724.1 34 Halomonadaceae bacterium PH27A 100 196 Oceanospirillales AF513467.1 37 Halomonas subglaciescola strain NBRC 14766 99 196 Halomonas NR_113667.1 38 Erwinia piriflorinigrans strain EA7 100 195 Erwinia KR812394.1 Fungi 1, 2, 3, 6, 7 Uncultured Debaryomyces gene isolate: DGGE band VZ3 99–100 401–402 Debaryomyces LC050968.1 4 Kazachstania aerobia strain AS 2.2384 98 405 Kazachstania AY881652.1 5 Uncultured Kazachstania gene clone: ZQC7 98 402 Kazachstania AB986206.1 8, 10, 14 Uncultured Debaryomyces gene isolate: DGGE band VZ5 99–100 402 Debaryomyces LC050970.1 9 Pichia sp. NRRL Y-27259 99 384 Pichia EF550366.1 11 Debaryomyces hansenii strain Nc6HA-1 99 399 Debaryomyces KR336844.1 12, 13 Pichia kluyveri strain NRRL Y-11519 99 387 Pichia EF550389.1 15 Cystofilobasidium macerans strain CBS10757 100 403 Cystofilobasidium KF036666.1

Only highest homology matches are presented a The number of each band corresponds with the profiles shown in Fig. 3 b Sequences were compared with known sequences in the NCBI database

Fungal community diversity based on PCR-DGGE similarity of sequences was ≥ 98% compared to those in and MiSeq sequencing GenBank (Table 2). A total of 459,244 reads was obtained using barcoded Illumina MiSeq sequencing, and 458,475 As shown in Fig. 3b, a total of 15 fungal bands were observed high-quality sequences were obtained after removal of low- and the sequencing results are shown in Table 2.The quality sequences. A total of 3118 different OTUs were Ann Microbiol (2018) 68:111–122 117

Fig. 4 Taxonomic composition analysis of microbial communities at the bacterial community composition based on Illumina MiSeq sequencing genus level during the industrialized Qingcai paocai fermentation process analysis, c fungal community composition based on PCR-DGGE based on PCR-DGGE and Illumina MiSeq sequencing analysis. a analysis, d fungal community composition based on Illumina MiSeq Bacterial community composition based on PCR-DGGE analysis, b sequencing analysis obtained based on 3% dissimilarity in ITS sequences. The differed depending on whether PCR-DGGE or Illumina diversity indices based on PCR-DGGE and Illumina MiSeq MiSeq sequencing was used. As shown in Fig. 4c and d, the sequencing analysis are shown in Table 1. Changes in these relative abundance of Debaryomyces obtained by PCR- indices based on both methods were similar. The diversity of DGGE was higher than that obtained by Illumina MiSeq se- the fungal community was lower than that of the bacterial quencing during the later stage of fermentation. The dominant community. fungus obtained by both methods during the fermentation Figure 4cshowsthefourmainfungalgeneradetectedby process of the industrialized Qingcai paocai belonged to the PCR-DGGE, i.e. Debaryomyces (bands 1, 2, 3, 6, 7, 8, 10, 11 same genus, Debaryomyces (Fig. 4a, b). and 14), Kazachstania (bands 4 and 5), Pichia (bands 9, 12 and 13) and Cystofilobasidium (band 15). Debaryomyces was Multivariate analysis of the microbial community clearly the dominant fungal genus during the fermentation during fermentation process of industrialized Qingcai paocai (Fig. 4c). According to Illumina MiSeq sequencing analysis, a total of Successions of the microbial community of the industrialized 154 genera were detected, with the dominant fungal genus Qingcai paocai samples were compared using PCA analysis according to this method also being Debaryomyces (Fig. based on the relative abundances of taxonomical community 4d), which matches the results of the PCR-DGGE analysis. structures. PCA analysis based on PCR-DGGE and Illumina Kazachstania and Pichia were also detected using Illumina MiSeq sequencing data basically obtained in similar results MiSeq sequencing (Fig. 4d). The composition of the fungal (Fig. 5a, b). community during the fermentation process of industrialized Among the genera detected by both methods, many micro- Qingcai paocai differed depending on which of these two organisms correlated highly with the early fermentation period methods were used and which genetic region was analyzed. (Fig. 5a, b), indicating that a copious number of microorgan- Unclassified fungi were identified more abundantly by isms adhered to the raw materials of industrialized Qingcai Illumina MiSeq sequencing. The relative abundances of mu- paocai. Figure 5 shows that the main genera of Pseudomonas, tual fungi, i.e. Debaryomyces, Kazachstania,andPichia,also Vibrio and Halomonas were strongly correlated with the early 118 Ann Microbiol (2018) 68:111–122

Fig. 5 Principal component analysis (PCA) of microbial community in directions of the straight arrows indicate the relative loading on the first industrialized Qingcai paocai fermentation based on PCR-DGGE and and second principal components. The directions of the curved arrows Illumina MiSeq sequencing data. a PCA of microbial community based indicate the routes of data points on the score plots during industrialized on PCR-DGGE data, b PCA of microbial community based on Illumina Qingcai paocai fermentation MiSeq sequencing data. Circles represent individual samples. The fermentation process, while Lactobacillus, Debaryomyces distinguish false positive signals due to non-specific amplifi- and Kazachstania correlated strongly with the later fermenta- cation or primer-dimers (Soares et al. 2013). The amplification tion process. The microbial community structure during dif- curves in our study took on a sigmoidal form, and the melting ferent fermentation stages of the industrialized Qingcai paocai curves only had one peak (Electronic Supplementary Material differed. As shown in Fig. 5, the succession of the microbial Fig. 6S), indicating a good specificity of primers. The standard community observed by both methods during the fermenta- curves for Lactobacillus and Debaryomyces were Cq = − tion process of industrialized Qingcai paocai showed a similar 3.40log10(q) + 52.48 and Cq = − 3.32log10(q) + 46.42, tendency toward change. respectively. The R2 of both formulas were 0.99, indicating a good cor- Quantitative analysis of dominant microorganisms relation between Cq and the natural logarithm of q. The am- during fermentation using qPCR plification efficiency of the formulas was 0.97 and 1.00, respectively, demonstrating a good amplification. The quantities The quantities of Lactobacillus and Debaryomyces during the of Lactobacillus and Debaryomyces during the fermentation fermentation of industrialized Qingcai paocai were monitored process of industrialized Qingcai paocai are shown in Table 3. by qPCR with SYBR Green I. DNA melting curves can Lactobacillus and Debaryomyces were present at 109–1012

Table 3 Thequantitiesofthe a dominant microorganisms in the Fermentation time (days) Concentration (copies/mL) fermentation of industrialized Qingcai paocai Lactobacillus Debaryomyces

1 (1.37 ± 0.03) × 109A (6.19 ± 0.76) × 106 a 3 (9.37 ± 1.23) × 1011 B (4.70 ± 0.40) × 108 a 11 (3.63 ± 0.16) × 1010 A (1.20 ± 0.29) × 107 a 21 (2.11 ± 0.18) × 1011 C (1.34 ± 0.02) × 108 a 34 (4.85 ± 0.22) × 1012 D (4.50 ± 0.63) × 1010 b 50 (5.94 ± 0.82) × 1011E (1.19 ± 0.72) × 1010 c 72 (1.59 ± 0.09) × 1011 AC (3.30 ± 0.37) × 109 a 89 (1.08 ± 0.08) × 1011 AC (4.03 ± 0.07) × 108 a

a Values followed by different uppercase letters are significantly different at p ≤ 0.05 among the quantities of Lactobacillus during the fermentation process. Values followed by different lowercase letters are significantly different at p ≤ 0.05 among the quantities of Debaryomyces during the fermentation process Ann Microbiol (2018) 68:111–122 119 and 106–1010 copies/mL, respectively, showing that the quan- attached to raw materials belonged to the Proteobacteria. tity of Lactobacillus was higher than that of Debaryomyces Hence, they were strongly correlated with the early fermenta- (Table 3). tion process (Fig. 4a, b). Haque et al. (2015) investigated the endophytic bacterial diversity in Korean kimchi (made of Chinese cabbage leaves) and found Proteobacteria to be one Discussion of the four major phylogenetic groups present in the fermentation process. Therefore, this could account for the abundant In China, fermented foods, such as Chinese liquor, vinegar, genera affiliated to Proteobacteria detected during the earlier soy sauce and sauerkraut, are quite popular. Although many fermentation stage. Members of the versatile Pseudomonas studies have reported that a variety of microbial species con- genus can adapt to a wide range of habitats, and this adapt- tribute to the fermentation of foods (Nie et al. 2013; Nie et al. ability of Pseudomonas could account for their constant pres- 2015; Li et al., 2016a), little is known about the microbial ence throughout a wide-ranging environment (Mena and communities that are active during the fermentation process of Gerba 2009). It is therefore reasonable to assume that industrialized Qingcai paocai. Pseudomonas was detected abundantly during the earlier fer- The changes in pH and TTA have always been used as mentation. However, most genera affiliated to Proteobacteria fundamental variables to determine the ripening time of were restrained by the falling pH and increasing TTA during fermented vegetables (Cheigh et al. 1994). In the present fermentation (Fig. 3a, b), which would account for their initial study, the pH value decreased to about 3.77 and the TTAvalue gradual decline in abundance. When adapted to the higher increased to about 5.5, which is similar to values reported for acidic condition, some of them would grow but not strongly. homemade paocai in China (Liu et al. 2015; Zhang et al. However, a small number of Vibrio sp. was also observed in 2016). However, it only took 3–5 days for homemade paocai samples collected on day 89. These may have survived in to achieve these pH and TTA values, while it can take up to 3 industrialized Qingcai paocai due to their characteristics of months to reach comparable values in industrialized paocai, biofilm formation and halotolerance. Moreover, both viable possibly due to the larger amount of raw materials and the and dead cells are taken into account in a culture- increased volume of the fermentation container. A general independent approach, which could also account for the pres- guideline is that paocai is ripe at a pH of < 4.0 and a TTA of ence of Vibrio sp. on day 89. In contrast, the abundance of >3g/L(Zhangetal.2016). During the fermentation process Lactobacillus with high resistance to acidity increased first the microbial groups shift sequentially from less acidic and and then decreased during fermentation. Lactobacillus, which salt tolerant groups to more acidic and salt tolerant ones that is typically regarded as a dominant LAB in vegetable fermen- are better adapted to the acidic and salty environmental con- tation, can produce abundant lactic acid (Endo et al. 2008; dition (Fierer and Jackson 2006; Plengvidhya et al. 2007). Xiong et al. 2013) and inhibit pathogens such as Salmonella Therefore, with the change in pH and TTA, the initial change spp. and Escherichia coli (Dec et al. 2016). In this study, was a decrease in the band number and the Shannon index of Lactobacillus was the most dominant genus during the later the microbial community in industrialized Qingcai paocai. fermentation stage (Fig. 3a, b), which is in accordance with However, when the microbial communities had adapted to results reported in previous studies on Chinese homemade the acidic and salt environmental condition, both the band paocai (Xiong et al. 2012;Yuetal.2012). However, the dom- number and the Shannon index showed an increasing trend inant species observed in previous studies on Chinese home- until the end of the fermentation process (Table 1). made paocai differ from those found in our study. Both PCR-DGGE and Illumina MiSeq sequencing were Lactobacillus plantarum has been commonly reported as the used to explore the characteristics of the microbial communi- predominant strain in homemade paocai, as well as L. casei, ties during industrialized Qingcai paocai fermentation. In L. acetotolerans and L. brevis (Xiong et al. 2012;Yuetal. terms of the bacterial community structure, both methods de- 2012; Cao et al. 2017). In the present study, L. alimentarius, tected three main phyla, namely Firmicutes, Proteobacteria, closely followed by L. plantarum were the dominant species. and Bacteroidetes, although their relative abundance varied The types of raw materials and production methods are poten- between techniques. Firmicutes and Proteobacteria were tially the main factors leading to the different dominant spe- dominant during the fermentation process. In a previous study cies. The different research methods could also account for we focused on the microbiota in matured industrialized paocai these differences because traditional plate culture has a specif- from a different factory than that used in the present study and ic selectivity. Weissella and Leuconostoc were also detected also found that Firmicutes and Proteobacteria were the dom- using Illumina MiSeq sequencing, but at a lower abundance,, inant phyla in all samples (Liang et al. 2016a). A number of but not using PCR-DGGE, indicating a more comprehensive genera affiliated to Proteobacteria, such as Pseudomonas, analysis using Illumina MiSeq sequencing than using PCR- Vibrio, Halomonas and Pectobacterium, were abundant be- DGGE. During the fermentation of Chinese homemade fore 21 day (Fig. 3a, b), indicating that most microorganisms paocai, Weissella and Leuconostoc were commonly reported 120 Ann Microbiol (2018) 68:111–122 as initiator (Xiong et al. 2012). Many studies have demonstrat- During the early stage of the fermentation, the quantities ed that the LAB of the genera Lactobacillus, Leuconostoc,and of Lactobacillus and Debaryomyces increased when the Weissella predominate during kimchi fermentation (Jeong pH remained unchanged. Subsequently, the quantities of et al. 2013; Jung et al. 2014). Jung et al. (2011) analyzed Lactobacillus and Debaryomyces decreased with decreas- changes in bacterial populations during fermentation using ing pH value. When adapted to the acidic condition, they metagenomic approaches; phylogenetic analysis indicated increased again and achieved a peak value at 34 days at that the kimchi microbiome was dominated by three genera: which point the pH had decreased to a stable but low Leuconostoc, Lactobacillus,andWeissella.Amongthese, value. After 34 days, the quantity of Lactobacillus in the Leuconostoc or Weissella were dominant bacteria during kim- fermentation of industrialized Qingcai paocai was deter- chi fermentation, while Lactobacillus was most abundant dur- minedtobe1011 copies/mL (Table 3). At the end of ing the fermentation of industrialized Qingcai paocai. industrialized Qingcai paocai fermentation process, the Leuconostoc and Lactobacillus were detected in salted quantity of Debaryomyces was 108 copies/mL (Table 3). Chinese cabbage by PCR-DGGE, while Weissella was not In conclusion, this study was the first to report microbial (Hong et al. 2014). In addition, some studies have reported community succession during industrialized Qingcai that Weissella mainly originated from kimchi ingredients paocai fermentation by combined Illumina MiSeq se- (Kim et al. 2004;Hongetal.2014). This is potentially the quencing and PCR-DGGE. Three main phyla, namely reason why only a low abundance Weissella was detected in Firmicutes, Proteobacteria and Bacteroidetes,wereiden- industrialized Qingcai paoai since it was produced without the tified by both methods. Most genera affiliated to addition of ingredients. Proteobacteria or Bacteroidetes,suchasPseudomonas, According to the analysis of fungal community using Vibrio, Pectobacterium, Klebsiella, Flavobacterium and both PCR-DGGE and Illumina MiSeq sequencing, Myroides, were detected more often during the earlier stage Debaryomyces, Pichia and Kazachstania were the main of fermentation. Lactobacillus, including L. alimentarius, and mutual fungi during the fermentation of industrialized L. brevis, L. plantarum and L. sakei affiliated to Qingcai paocai. Debaryomyces was a dominant fungus Firmicutes, was detected and became dominant during during the whole fermentation process of industrialized the later stage of fermentation. The dominant genera de- Qingcai paocai, which was also reported in our previous tected by both approaches were similar. The results of fun- study (Liang et al. 2016a). However, the abundance of gal community analysis using both PCR-DGGE and Debaryomyces during the fermentation process determined Illumina MiSeq sequencing showed that the genera using Illumina MiSeq sequencing was lower than that Debaryomyces, Pichia,andKazachstania were the main using PCR-DGGE. This difference may be due to the fact and mutual fungi during the fermentation of industrialized that most of the uncultured species affiliated to Qingcai paocai. Among these, the genus Debaryomyces Debaryomyces were classified as unclassified fungi in the was the dominant fungi during the fermentation process. Illumina MiSeq sequencing analysis. In the PCR-DGGE The quantities of the dominant genera Lactobacillus and analysis, many bands were also identified as uncultured Debaryomyces weremonitoredbyqPCR,andtheresults Debaryomyces. Debaryomyces hansenii has been reported showed concentrations of 109–1012 and 106–1010 to produce aroma compounds (Cano-García et al. 2014). copies/mL, respectively. The quantities of Lactobacillus Potentially, this is also conducive for the development of and Debaryomyces during the later stages of fermentation flavor during the fermentation of industrialized Qingcai of industrialized Qingcai paocai were determined to be in paocai. A previous study detected Pichia as the dominant 1011 and 108 copies/mL, respectively. This is the first re- fungi in homemade paocai and showed that the members of port on the composition of the microbial community dur- this genus could produce various enzymes during fermen- ing the industrialized Qingcai paocai fermentation process tation (Liang et al. 2016b). To date, there have been very that uses a combined Illumina MiSeq sequencing, PCR- few reported studies on the fungal community in paocai. DGGE and qPCR assay. Our results provide information Quantitative PCR has been used to investigate LAB at which will facilitate improvements in the industrialized the genus or species level in various food matrices paocai fermentation process. Further studies on the corre- (Achilleos and Berthier, 2013;Kimetal.2015;Luetal. lation between paocai flavor properties and the major mi- 2015). Little research has been reported on the use of crobes that contribute to the fermentation are necessary. qPCR to quantify the copy numbers of Lactobacillus and Debaryomyces in the industrialized Qingcai paocai fermentation. Table 3 shows that the quantities of Acknowledgements The study was financially supported by the National Science and Technology Project (NO. 2012BAD31B04) of the Ministry Lactobacillus and Debaryomyces were low at 1 day of of Science and Technology of the People’s Republic of China, and the fermentation, indicating that they accounted for only few Science and Technology Planning Project (NO. 2016NZ0007) of the of the microorganisms attached to the raw materials. Sichuan Science and Technology Department. Ann Microbiol (2018) 68:111–122 121

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