Effects of Leuconostoc mesenteroides starter cultures on microbial communities and metabolites during kimchi fermentation

International Journal of Food Microbiology 153 (2012) 378–387

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Effects of Leuconostoc mesenteroides starter cultures on microbial communities and metabolites during kimchi fermentation Ji Young Jung a, Se Hee Lee a, Hyo Jung Lee a, Hye-Young Seo b, Wan-Soo Park b, Che Ok Jeon a,⁎ a b

School of Biological Sciences, Chung-Ang University, Seoul, 156-756, Republic of Korea World Institute of Kimchi, An annex of Korea Food Research Institute, Sungnam-si, Gyeonggi-do, 463-746, Republic of Korea

a r t i c l e

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Article history: Received 6 September 2011 Received in revised form 20 November 2011 Accepted 28 November 2011 Available online 4 December 2011 Keywords: Kimchi fermentation Leuconostoc mesenteroides Microbial community Starter culture Barcoded pyrosequencing 1 H NMR spectroscopy

a b s t r a c t Kimchi fermentation usually relies upon the growth of naturally-occurring various heterofermentative lactic acid bacteria (LAB). This sometimes makes it difficult to produce kimchi with uniform quality. The use of Leuconostoc mesenteroides as a starter has been considered to produce commercial fermented kimchi with uniform and good quality in Korea. In this study, a combination of a barcoded pyrosequencing strategy and a 1H NMR technique was used to investigate the effects of Leu. mesenteroides strain B1 as a starter culture for kimchi fermentation. Baechu (Chinese cabbage) and Chonggak (radish) kimchi with and without Leu. mesenteroides inoculation were prepared, respectively and their characteristics that included pH, cell number, bacterial community, and metabolites were monitored periodically for 40 days. Barcoded pyrosequencing analysis showed that the numbers of bacterial operational taxonomic units (OTU) in starter kimchi decreased more quickly than that in non-starter kimchi. Members of the genera Leuconostoc, Lactobacillus, and Weissella were dominant LAB regardless of the kimchi type or starter inoculation. Among the three genera, Leuconostoc was the most abundant, followed by Lactobacillus and Weissella. The use of Leu. mesenteroides as a starter increased the Leuconostoc proportions and decreased the Lactobacillus proportions in both type of kimchi during kimchi fermentation. However, interestingly, the use of the kimchi starter more highly maintained the Weissella proportions of starter kimchi compared to that in the non-starter kimchi until fermentation was complete. Metabolite analysis using the 1H NMR technique showed that both Baechu and Chonggak kimchi with the starter culture began to consume free sugars earlier and produced a little greater amounts of lactic and acetic acids and mannitol. Metabolite analysis demonstrated that kimchi fermentation using Leu. mesenteroides as a starter was completed earlier with more production of kimchi metabolites compared to that not using a starter, which coincided with the decreases in pH and the increases in bacterial cell number. The PCA strategy using all kimchi components including carbohydrates, amino acids, organic acids, and others also showed that starter kimchi fermented faster with more organic acid and mannitol production. In conclusion, the combination of the barcoded pyrosequencing strategy and the 1H NMR technique was used to effectively monitor microbial succession and metabolite production and allowed for a greater understanding of the relationships between the microbial community and metabolite production in kimchi fermentation. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Kimchi, an emblematic traditional food in Korean culture, is made through fermentation of vegetables such as Chinese cabbage and radish seasoned with various spices including red pepper powder, garlic, ginger, green onion, fermented seafood (jeotgal), and salts (Cho et al., 1999). In recent years, kimchi has become a globally popular food because of its taste as well as its health-promoting effects (Song, 2004; Kim et al., 2007; Choi and Islam, 2009). Many taxonomic studies have ⁎ Corresponding author at: School of Biological Sciences, Chung-Ang University, 84, HeukSeok-Ro, Dongjak-Gu, Seoul, 156-756, Republic of Korea. Tel.: + 82 2 820 5864; fax: + 82 2 821 8132. E-mail address: [email protected] (C.O. Jeon). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.11.030

shown that kimchi preparation without the sterilization of raw materials leads to the growth of various bacteria originated from the raw materials during kimchi fermentation (Cheigh and Park, 1994). Among them, heterofermentative lactic acid bacteria (LAB), including members of the genera Leuconostoc (Leu.), Lactobacillus (Lb.), Weissella (W.), and Lactococcus species, likely play key roles in kimchi fermentation (Kim et al., 2000; Bae et al., 2005; Cho et al., 2006; Kim and Chun, 2005; Chang et al., 2008; Lee et al., 2011). Spontaneous fermentation using various naturally-occurring bacteria usually results in variations in the sensory qualities of kimchi products. This makes it difficult to produce commercial kimchi with uniform quality. Moreover, the influence of ingredients and fermentation conditions on the microbial community has yet to be explored, and a rational approach to the control of the microbial community for

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the improvement of kimchi flavor has not been developed. Therefore, the use of a starter culture has been considered for the production of standardized kimchi with uniform high quality. Some LAB including Leu. mesenteroides, Leu. citreum, and Lb. plantarum have been suggested as starter cultures for quality development in vegetable fermentation (Choi et al., 2003; Leal-Sánchez et al., 2003; Giraffa, 2004; Tolonen et al., 2004; Wiander and Ryhänen, 2005; Johanningsmeier et al., 2007; Chang and Chang, 2010). Members of the genus Leu. mesenteroides, which are typical heterofermentative LAB that produce lactate, carbon dioxide, ethanol, and acetate from carbohydrates, have been found to be major LAB during kimchi fermentation (Cho et al., 2006; Eom et al., 2007; Cho et al., 2009; Jung et al., 2011). Moreover, Leu. mesenteroides is known to produce mannitol, a naturally occurring six-carbon diabetic polyol that imparts a refreshing taste with noncarcinogenic properties, at high levels in kimchi through the reduction of fructose (Grobben et al., 2001). Therefore, the use of Leu. mesenteroides as a starter culture has been considered in commercial kimchi fermentation (Eom et al., 2008). Many Korean commercial industries are already using Leu. mesenteroides strains as starter cultures. However, the effects of Leu. mesenteroides as starter cultures on the microbial community and metabolite production of kimchi have yet to be identified. Many early studies on the kimchi microbial community were carried out using conventional culture-based approaches (Park et al., 1990; Shin et al., 1996; Cho et al., 2006). However, culture-based approaches have known limitations in terms of the reproducibility and unculturability of some bacteria. Culture-independent methods such as 16S rRNA gene clone libraries and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA fragments have been frequently used to circumvent the limitations associated with traditional culture-based methods (Lee et al., 2005; Kim and Chun, 2005; Lee et al., 2008), but they themselves also have some limitations in extending information about kimchi microbiology because they involve time-consuming steps such as cloning or DGGE of 16S rRNA gene PCR amplicons. Taking this into account, a pyrosequencing-based 16S rRNA gene survey has emerged as a powerful technique to unveil in detail the microbial community structures in ecological habitats (Huber et al., 2007; Humblot and Guyot, 2009; Zhang et al., 2009; Ercolini et al., 2011) and to exclude the laborious steps involved in the other methods. In addition, multiplex barcoded pyrosequencing strategies allow for the analysis of multiple samples in a single run (Roh et al., 2010; Sakamoto et al., 2011). It is well known that metabolites, such as organic acids (lactic and acetic acids) and other flavoring compounds (mannitol and amino acids), are important components in kimchi tastes and flavors. The metabolite compositions reflect a more direct collective phenotypic view of the kimchi microbial community (Ha et al., 1989). Proton nuclear magnetic resonance ( 1H NMR) is one of the most comprehensive and powerful tools for simultaneously monitoring several metabolites present in one sample (Figueiredo et al., 2006; Lee et al., 2009). The major objective of this study was to investigate the effects of the Leu. mesenteroides strain B1 as a starter culture for kimchi fermentation. Comparisons of microbial successions and metabolites between Leu. mesenteroides-inoculated kimchi (starter kimchi) and naturally fermenting kimchi (non-starter kimchi) are very important aspects for the successful understanding of kimchi fermentation (Choi et al., 2003; Lee and Lee, 2010). Here, we applied a barcoded 454-pyrosequencing strategy and a 1H NMR technique to analyze kimchi microbial successions and metabolites, respectively. 2. Materials and methods 2.1. Starter culture preparation Leuconostoc mesenteroides strain B1 (KACC 16355) used for starter cultures was isolated from the best-selling kimchi (Daesang F&F

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Corporation) in South Korea. The genome of strain B1 has similar general features to those of the Leu. mesenteroides type strain (ATCC 8293) (Makarova et al., 2006; Jung et al., 2011). Strain B1 cells were cultured in MRS broth (Difco, USA) at 30 °C for 24 h to a concentration of ~10 9 cells/ml and were harvested by centrifugation. The harvested cells were washed twice with 0.9% (w/v) saline and resuspended in 0.9% saline to a concentration of 10 10 cells/ml for starter inoculation in kimchi. 2.2. Preparation of kimchi and sampling Two kinds of kimchi, Baechu kimchi and Chonggak kimchi, were prepared using Chinese cabbage (Brassica rapa subsp. pekinensis) and radish (Raphanus sativus var. sativus L.), respectively, according to a traditional manufacturing method (Jung et al., 2011). Briefly, the Chinese cabbages and radishes were soaked in a solution of 10% (w/v) solar salt (Shinan, Korea) for 10 h. The soaked Chinese cabbages and radishes were washed three times with tap water and drained. The salted Chinese cabbages and radishes were then mixed with various seasoning ingredients using the following ratio; cabbage or radish:red pepper powder:Korean leek:garlic:ginger = 93.2:2.1:2.9:1.4:0.4. The prepared Baechu kimchi and Chonggak kimchi were divided into two sets of kimchi samples of the same weights. One paired set of Baechu kimchi and Chonggak kimchi was inoculated with the cultured kimchi starter (Leu. mesenteroides strain B1) to a concentration of 10 7 cells/g-kimchi, while the other paired set of Baechu kimchi and Chonggak kimchi was added with 0.9% (w/v) saline instead of the starter inoculation as a control. The four prepared kimchi samples were dispensed into three polyethylene plastic bags in 5-kg portions for triplicate analysis and stored at 4 °C. Kimchi soups were periodically sampled and their pH values were immediately measured. The kimchi soups were filtered through four layers of sterile coarse gauze (Daehan, Korea) to remove large particles and the filtrates were centrifuged (8000 rpm for 20 min at 4 °C) to harvest microorganisms. The separated pellets and supernatants were stored at −80 °C for microbial community and metabolite analyses, respectively. The kimchi samples were labeled as “BK” and “CK” for Baechu kimchi and Chonggak kimchi, respectively, and “S” was added for starter kimchi. 2.3. Quantitative real-time PCR to determine 16S rRNA gene copy number To estimate the 16S rRNA gene copy number of total bacteria during kimchi fermentation, total genomic DNA from pellets derived from 1.0 ml of kimchi filtrate was extracted in triplicate using a FastDNA Spin kit (MPbio, USA) according to the manufacturer's instructions. Quantitative real-time PCR (qRT-PCR) was performed in triplicate, and a standard curve was generated on the basis of the number of a pCR2.1 vector (Invitrogen, USA) carrying the 16S rRNA gene of Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 T as described previously (Jung et al., 2011). The 16S rRNA gene copy numbers of each sample were calculated as described previously (Ritalahti et al., 2006; Jung et al., 2011). 2.4. 16S rRNA gene amplifications for barcoded pyrosequencing Total genomic DNA from the pellets was extracted using the FastDNA Spin kit as described above and the concentrations of the extracted genomic DNA were measured with an ELISA reader equipped with a Take3 multivolume plate (BioTek, USA). Hypervariable regions (V1 to V3) of the bacterial 16S rRNA genes from the genomic DNA were amplified using primers V1-27F (5′-adaptor B-AC-GAGTTTGATCM TGGCTCAG-3′) and V3-541R (5′-adaptor A-X-AC-WTTACCGCGGCT GCTGG-3′), where X denotes unique7–11 barcode sequences inserted between the 454 Life Sciences adaptor A sequence and a common linker AC (Supplementary Table 1) (Roesch et al., 2007; Chun et al., 2010). All PCR amplifications were carried out in a 50-μl C1000 thermal cycler

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(Bio-Rad, USA) containing 150 ng of pooled genomic DNA (derived from three different bags with the sample fermentation condition, 50 ng from each), 20 pmol of each primer, and a Taq polymerase mixture (Solgent, Korea) using a cycling regime of 94 °C for 5 min (1 cycle); 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 1 min (30 cycles); and 72 °C for 10 min (1 cycle).

2.5. Pyrosequencing and phylogenetic analyses The PCR products were purified using a PCR purification kit (Solgent, Korea) and their concentrations were carefully assessed with an ELISA reader equipped with a Take3 multivolume plate. A composite sample was prepared by pooling equal amounts of PCR amplicons from each sample. Pyrosequencing was performed by Macrogen (Korea) using a 454 GS-FLX titanium system (Roche, Germany). Pyrosequencing data were processed and analyzed using the RDP pyrosequencing pipeline (http://pyro.cme.msu.edu/) (Cole et al., 2009). Briefly, the sequences were assigned to specific samples based on their unique barcodes, and then the barcodes were removed. Marginal regions with quality scores b 25 (error, 0.005) and ambiguous characters were trimmed. Only sequences> 400 bp in length were chosen for further analyses using the Pipeline Initial Process of the RDP. The chimera formation of the processed reads were checked using the Bellerophon chimera detection program (Huber et al., 2004), but no chimeric sequence was detected. The sequences with high qualities were aligned using the fast, secondary-structure aware INFERNAL aligner (Nawrocki and Eddy, 2007). The aligned sequences were clustered into operational taxonomic units (OTU) defined by 97% similarity using the complete-linkage clustering tool. The Shannon–Weaver index (Shannon and Weaver, 1963), Chao1 biodiversity indices (Chao, 1987), and evenness were calculated by the RDP pyrosequencing pipeline. Rarefaction analysis was also performed using the RDP pyrosequencing pipeline. In addition, the processed sequences were taxonomically classified using the RDP na ve Bayesian rRNA Classifier (Wang et al., 2007) based on an 80% confidence threshold. On the other hand, sequences assigned as mitochondrial or chloroplast 16S rRNA gene sequences were excluded, and principal component analysis (PCA) was performed on the basis of the relative abundance of all taxonomical community structures using the package ‘Vegan’ (Oksanen et al., 2011) in the R programming environment (http:// cran.r-project.org).

2.7. Nucleotide sequence accession numbers The pyrosequencing data of the 16S rRNA genes are publicly available in the NCBI Short Read Archive under accession no. SRA044882. 3. Results 3.1. The pH profiles and 16S rRNA gene copy numbers during kimchi fermentation Baechu (Chinese cabbage) kimchi and Chonggak (radish) kimchi, typical Korean kimchi types, were prepared using Chinese cabbage leaves and radish roots as the main raw materials, respectively. Leu. mesenteroides strain B1 was cultivated and used for the starter kimchi preparations to address the effects of the kimchi starter on fermentation rate, microbial community, and metabolite production of kimchi. We monitored the pH values and bacterial cells during kimchi fermentation. The pH profiles of the kimchi supernatants over the 40 day-fermentation process were similar to those of typical kimchi fermentation (Fig. 1) (Bae et al., 2005; Chang et al., 2008; Chang and Chang, 2011; Jung et al., 2011). In the present study, the initial pHs of kimchi samples were about 5.3–5.4 (Fig. 1), which increased slightly to around pH 5.5 during the early kimchi fermentation, supposedly caused by the secretion of vegetable saps containing some basic components. The pH values then decreased rapidly after 10 days. The reduction in pH of the starter kimchi was initiated earlier

2.6. Targeted metabolite profiling and multivariate statistical analysis The profiles of metabolites, including monosaccharides, organic acids, and amino acids, during kimchi fermentation were analyzed in triplicate using 1H NMR spectroscopy as described previously (Lee et al., 2009; Jung et al., 2011). Metabolite identification and quantification of individual metabolites from the 1H NMR spectra acquired by a Varian Inova 600-MHz NMR spectrometer (Varian, USA) were performed using the Profiler module of the Chenomx NMR suite, v. 6.1 (Chenomx, Canada) as described previously (Lee et al., 2009; Jung et al., 2011). All 1H NMR spectra for multivariate statistical analysis were manually phased and baseline corrected using Mestrenova software (ver. 6.0.4, Mestrelab Research SL, Spain). The NMR spectral data were reduced into 0.001 ppm spectral buckets and the region corresponding to water (4.6–4.8 ppm) was removed. The NMR spectra were normalized to the total spectral area and converted to ASCII format using Mestrenova software. The resulting data files were then imported into MATLAB (ver. 6.5, Mathworks, USA), and all spectra were aligned using the Correlation Optimized Warping (COW) method (Tomasi et al., 2004). All spectral data were mean-centered with no scaling. PCA was then performed on the basis of the covariance matrix using MATLAB software.

Fig. 1. Changes in pH and 16S rRNA gene copy number with total bacterial number in Baechu (Chinese cabbage) kimchi (A) and Chonggak (radish) kimchi (B) during fermentation. Measurements of pH and bacterial 16S rRNA gene copy number were performed in triplicate, and error bars represent the standard deviations. The kimchi samples are as follows: BK, non-starter Baechu kimchi; BKS, starter Baechu kimchi; CK, non-starter Chonggak kimchi; CKS, starter Chonggak kimchi.

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than that in non-starter kimchi regardless of the kimchi type, but their pH reduction rates were similar to one other. The pH values finally reached approximately 4.3, although the pH values of the starter kimchi were slightly lower than those of the non-starter kimchi. After a 20-day incubation period, the pH values became relatively stable regardless of the kimchi type and starter inoculation. A qRT-PCR approach based on the 16S rRNA gene copies was used to enumerate the total number of bacteria in four kimchi samples during kimchi fermentation. The exact determination of bacterial cell numbers using qRT-PCR is almost impossible because the copy numbers of chromosomal 16S rRNA gene operons vary with species type (Farrelly et al., 1995; Park et al., 2009); however, qRT-PCR analysis allows for the estimation of the changes in the cell numbers of a microbial community. In this study, the total numbers of bacteria in the kimchi samples were estimated using a standard curve (BK, R 2 = 0.985; CK, R 2 = 0.986) generated from the cloned 16S rRNA gene of Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 T. The increase in bacterial abundance inversely correlated with the decreases in pH in all kimchi samples (Fig. 1). Even though there were higher initial cell numbers in the starter kimchi (2–3 × 108 copies/ml) compared to the non-starter kimchi (~108 copies/ml), the final 16S rRNA gene copies reached almost similar values (~10 10 copies/ml) after 18 days of kimchi fermentation, regardless of the kimchi type and starter inoculation (Fig. 1).

3.2. Analysis of microbial succession during kimchi fermentation using pyrosequencing A massively parallel barcoded pyrosequencing approach was applied for the analysis of microbial community succession during kimchi fermentation. A total of 90,426 reads resulted from a single

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run with forty kimchi samples (ten kimchi samples from each kimchi fermentation) using barcoded pyrosequencing. A total of 67,423 high quality reads (74.56% of the total reads) with an average sequence length of ~ 492 bp and an average of >1685 sequences for each sample were obtained after removing low quality sequences that could not be adequately classified (Table 1). Statistical microbial diversities for each sample were computed using the RDP pyrosequencing pipeline based on the 16S rRNA gene sequences. The relative bacterial diversity and OTU value declined in all kimchi samples as fermentation progressed (Table 1). Especially for rarefaction curves, plots of OTU versus the number of sequences that can estimate the true maximum OTU value at any phylogenetic level clearly indicated that the numbers of species (OTU) detected in kimchi decreased dramatically as fermentation progressed (Fig. 2). During the early phase of kimchi fermentation, an average of >1685 sequences for each sample was not sufficient to describe the entire diversities of the kimchi microbial community, but the read numbers became saturated, that is, the slopes neared the zero value as kimchi fermentation progressed. In addition, the rarefaction curves also showed that the OTU numbers of the kimchi samples decreased faster with starter inoculation during the early fermentation time (0 and 6 days), but the numbers of the final OTUs were similar and stable regardless of the kimchi type and starter inoculation (Fig. 2). The Shannon–Weaver index indicating the combinational estimates of OTU richness (total number of OTUs) and species evenness also supported the finding that the microbial diversities of the starter kimchi (BKS and CKS) decreased earlier in the process than did those in the non-starter kimchi (BK and CK), and their microbial diversities were similar and stable after 15 days of fermentation, regardless of the kimchi type and the use of a starter (Table 1). Even the starter Chonggak kimchi (CKS) showed a higher Shannon–Weaver index (high diversity) than the nonstarter Chonggak kimchi (CK) at the late fermentation phase.

Fig. 2. Changes in relative bacterial diversities shown through rarefaction analysis using bacterial 16S rRNA gene sequences during kimchi fermentation. (A) Non-starter Baechu kimchi. (B) Starter Baechu kimchi. (C) Non-starter Chonggak kimchi. (D) Starter Chonggak kimchi. The numbers indicate the kimchi fermentation day. Operational taxonomic units (OTUs) are calculated by the RDP pipeline with a 97% OTU cutoff of the 16S rRNA gene sequences.

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Table 1 Summary of the barcoded pyrosequencing data sets and statistical analysis of the kimchi fermentation samples. Sample (day) BK

BKS

CK

CKS

0 6 9 12 15 18 21 27 33 40 0 6 9 12 15 18 21 27 33 40 0 6 9 12 15 18 21 27 33 40 0 6 9 12 15 18 21 27 33 40

No. of reads

No. of high quality reads

Average read length (bp)

OTUsa

Shannon–Weaver indexa (H′)

Chao1a

Evennessa (E)

No. of phylaa

No. of generaa

753 1850 2501 2073 2669 1797 1746 2474 2575 748 2325 2695 1978 3165 1683 1851 2670 2883 2777 2499 2550 2403 2056 3494 2869 1415 1076 1925 1340 3235 2665 2646 2849 2627 1513 1469 1868 2705 2474 1508

563 1402 1900 1572 1996 1360 1318 1829 1970 560 1673 2185 1624 2586 1253 1396 2089 2216 2198 1937 1877 1861 1586 2608 2138 1055 782 1408 1000 2350 1907 2174 2314 2113 1140 1094 1404 2071 1842 1072

473 481 485 495 496 497 495 495 495 496 478 487 491 494 496 497 494 495 496 495 474 483 489 496 498 496 496 496 496 498 478 488 491 495 497 495 495 495 495 495

71 106 114 74 50 40 41 47 35 13 145 124 85 93 61 43 49 57 46 40 163 162 109 98 62 33 31 45 36 49 146 129 102 79 47 41 50 50 55 42

2.98 3.05 2.73 2.13 1.64 1.53 1.59 1.51 1.48 1.30 3.22 2.56 2.01 1.78 1.72 1.56 1.66 1.62 1.46 1.54 3.21 3.14 2.66 2.14 1.78 1.57 1.65 1.62 1.53 1.49 3.04 2.42 1.98 1.81 2.01 1.74 1.82 1.72 1.72 1.69

117.75 137.50 175.25 101.07 77.14 53.91 68.14 57.00 57.00 16.75 199.03 181.42 137.00 130.05 127.00 71.50 72.33 76.46 57.67 47.33 318.22 279.04 205.25 128.27 78.87 39.11 40.43 83.00 113.00 62.91 215.09 213.00 169.11 153.10 85.00 49.75 56.50 65.30 89.50 54.00

0.70 0.65 0.58 0.49 0.42 0.41 0.43 0.39 0.42 0.51 0.65 0.53 0.45 0.39 0.42 0.42 0.43 0.40 0.38 0.42 0.63 0.62 0.57 0.47 0.43 0.45 0.48 0.43 0.43 0.38 0.61 0.50 0.43 0.41 0.52 0.47 0.46 0.44 0.43 0.45

4 5 5 6 4 4 4 4 4 1 7 6 5 5 5 5 4 4 5 5 6 5 6 5 4 2 3 3 3 3 6 5 6 5 4 4 5 4 4 4

33 46 38 26 16 14 17 14 9 4 66 55 37 36 16 15 17 17 14 10 68 66 48 37 14 8 9 10 8 12 64 56 45 25 11 8 13 11 14 9

Abbreviations: OTUs, operational taxonomic units. a Diversity indices of the microbial communities and numbers of phyla and genera were calculated using the RDP pyrosequencing pipeline based on the 16S rRNA gene sequences.

The 16S rRNA gene sequences were classified using RDP classifier to investigate the bacterial community successions during kimchi fermentation. As a result, considerable proportions of the 16S rRNA gene sequences were classified as chloroplast 16S rRNA gene sequences (streptophyta) (Fig. 3), which were supposedly produced by PCR amplification of the chloroplast 16S rRNA genes that originated from raw materials including Chinese cabbage or radish. The proportions of the chloroplast 16S rRNA genes were highest during the early phase of kimchi fermentation; the proportions of the chloroplast 16S rRNA genes at the first day were approximately 40–50%. However, the proportions of the chloroplast 16S rRNA gene sequences decreased dramatically as kimchi fermentation progressed due to the rapid increases in bacterial abundance; the proportions of the chloroplast 16S rRNA gene sequences after 15 days of incubation were less than 0.6% (Fig. 3). Fig. 3 shows that kimchi fermentation was governed by three lactic acid bacterial genera, Leuconostoc, Lactobacillus, and Weissella. Among them, the genus Leuconostoc was most abundant in kimchi fermentation, followed by Lactobacillus and Weissella regardless of the kimchi type and starter inoculation. The abundance of Leuconostoc increased during the early stages of kimchi fermentation, while the abundance of Lactobacillus increased rapidly in all the kimchi samples after 9 days of fermentation. Fig. 3 also clearly shows that the use of Leu. mesenteroides strain B1 as a starter increased the Leuconostoc proportions and decreased the Lactobacillus

proportions in kimchi fermentation in the two kimchi types. Respective taxonomic analysis of the 16S rRNA gene sequences from pyrosequencing data revealed that all 16S rRNA gene sequences from the non-starter kimchi that were classified as Leuconostoc in the RDP pipeline analysis were most closely related to Leu. mesenteroides, indicating that the kimchi raw materials (Chinese cabbage and radish) themselves contained Leu. mesenteroides-like bacteria that have similar properties to those of the starter strain. Non-starter kimchi contained slightly more Weissella during the early kimchi fermentation and the proportion decreased slowly as fermentation progressed. Interestingly, although the Weissella proportions in starter kimchi were lower than in non-starter kimchi during the early fermentation, their proportions were maintained at higher levels than in non-starter kimchi until fermentation was complete (Fig. 3). 3.3. Statistical analysis of microbial succession during kimchi fermentation The microbial community successions of the kimchi samples were also compared by principal component analysis (PCA) based on the relative abundances of taxonomical community structures after the chloroplast 16S rRNA gene sequences were removed. All samples were continuously distributed into the positive and negative regions of the PC2 within the positive regions of the PC1 as fermentation progressed. Each section of data was represented mainly by Leuconostoc

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Fig. 3. Succession of phylogenetic taxonomic composition during kimchi fermentation. (A) Non-starter Baechu kimchi. (B) Starter Baechu kimchi. (C) Non-starter Chonggak kimchi. (D) Starter Chonggak kimchi. Partial 16S rRNA gene sequences >400 bp in length were classified to the genus level using the RDP na ve Bayesian rRNA Classifier based on the RDP 16S rRNA gene database (80% confidence threshold). Horizontal lines indicate 50% of the relative abundances.

and Lactobacillus (Fig. 4). Leuconostoc was the most influential group especially during the early and middle fermentation stages, while Lactobacillus was the next most influential group during the late fermentation stage in all kimchi fermentations. As expected, the starter inoculation (Leu. mesenteroides strain B1) increased the influences of Leuconostoc on the microbial communities and decreased those of Lactobacillus. The influence of Weissella on the microbial communities was very weak and was negligible in all kimchi samples. PCA analysis also supported that the Weissella abundance in the starter kimchi was more highly maintained than that in the non-starter kimchi during late kimchi fermentation, especially in Chonggak kimchi (Fig. 4C and D). The PCA plots presented by microbial succession indicated that the microbial communities were highly influenced by Leuconostoc during the early and middle kimchi fermentation phases, and the influence of Lactobacillus increased after the middle fermentation phases. 3.4. Analysis of metabolites during kimchi fermentation It is well known that kimchi flavors and tastes are principally related to the contents of the kimchi metabolites that include carbohydrates, amino acids, and organic acids, the productions of which are influenced by the microbial community during kimchi fermentation (Ha et al., 1989; Choi et al., 2003). In order to investigate the effect of the starter (Leu. mesenteroides) on kimchi fermentation, the kimchi metabolites were analyzed in triplicate using 1H NMR spectroscopy. Intense peaks in the 0.5- to 8.5-ppm ranges in the 1H NMR spectra were associated with the presence of various kimchi metabolites including free sugars, amino acids, organic acids, and other organic compounds. Glucose and fructose were detected as major free sugars (Fig. 5A and B) and are known to play important roles as carbon sources for

LAB metabolism in the production of various fermentation products. The levels of free sugars in Baechu (Chinese cabbage) kimchi were relatively higher than those in Chonggak (radish) kimchi. The levels of free sugars in Baechu kimchi increased rather slowly during the early stages, supposedly due to the continuous liberation of free sugars from vegetables. After 10 days of fermentation, their concentrations began to decrease. On the other hand, in Chonggak kimchi, the free sugar levels, especially that of glucose, increased after 20 days (Fig. 5A and B), supposedly due to glucose outflow from radish and the reduced fermentation rate. Generally, the free sugar concentrations of starter kimchi decreased earlier with more reduction compared to those of non-starter kimchi. The levels of lactate and acetate, which are major products of heterofermentative LAB during vegetable fermentation, increased inversely with the decrease in free sugars as fermentation progressed (Fig. 5C and D). However, the levels of ethanol, which is also one of the major products of heterofermentative LAB, were negligible. This might be explained by the ethanol evaporation during lyophilization for 1H NMR analysis (data not shown). Mannitol, a six-carbon polyol produced by the reduction of fructose by LAB (McFeeters and Chen, 1986; Yun et al., 1996; Wisselink et al., 2002), increased during fermentation, and the profiles were inversely correlated with the levels of fructose (Fig. 5E). Starter kimchi inoculated with Leu. mesenteroides produced more fermentation metabolites earlier with larger decrease in free sugars, which can be explained by the presence of high proportions of Leuconostoc during the early fermentation phase. On the other hands, analysis of amino acids using 1H NMR spectroscopy showed that the concentrations of most of amino acids, except for methionine, tryptophan, histidine, asparagine, aspartate, glutamine, alanine and arginine, increased gradually as kimchi fermentation progressed, but the differences

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Fig. 4. Score plots of the principal component analysis (PCA) derived from the relative abundance of bacterial communities during kimchi fermentation. (A) Non-starter Baechu kimchi. (B) Starter Baechu kimchi. (C) Non-starter Chonggak kimchi. (D) Starter Chonggak kimchi. Numbers beside the data points on the score plots represent the fermentation time (day) of kimchi. The directions of the straight arrows indicate the relative loading on the first and second principal components. The directions of the curved arrows indicate the routes of data points on the score plots during kimchi fermentation.

between starter kimchi and non-starter kimchi were not significant (Supplementary Table 2). 3.5. Statistical analysis of metabolites during kimchi fermentation To examine the effects of the kimchi starter (Leu. mesenteroides) on the variations of all kimchi metabolites including carbohydrates, amino acids, and organic acids during kimchi fermentation, a PCA strategy was applied to the entire 1H NMR spectrum data set. In the case of Baechu (Chinese cabbage) kimchi, the PCA score plots showed clear differences between the starter kimchi and non-starter kimchi (Fig. 6A). Rightward phase shifts in the plots demonstrate that continuous metabolic changes (fermentation) occurred in Baechu kimchi up to 40 days after initiation. The fast movements of the starter Baechu kimchi following different routes also indicate that the starter Baechu kimchi fermented faster and the metabolite compositions could be a little different from those of non-starter kimchi. On the other hand, the PCA score plot of Chonggak kimchi shows that, although the starter Chonggak kimchi fermented slightly faster than the non-starter Chonggak kimchi, both samples had similar metabolite compositions unlike Baechu kimchi (Fig. 6B). No differences in the PCA score plots after 20 days of fermentation in Chonggak kimchi indicate that Chonggak kimchi fermentation might be nearly complete at 20 days regardless of the starter inoculation. 4. Discussion A combination of a barcoded 454-pyrosequencing strategy and a H NMR technique enables the monitoring of changes in the microbial

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communities and metabolites and the investigation of relationships between the microbial community and metabolite formation in microbial ecological processes. Here, we applied a combination of a barcoded 454-pyrosequencing strategy and a 1H NMR technique to investigate the effects of a starter (Leu. mesenteroides) culture on kimchi fermentation. It has been known that heterofermentative LAB such as Leuconostoc mesenteroides and Leu. citreum predominates under weaker acidic and less anaerobic conditions during early and middle kimchi fermentation stages and homofermentative LAB belonging to Lactobacillus and Weissella species becomes dominant as kimchi fermentation conditions change to more anaerobic and acidic conditions (Cho et al., 2006; Chang and Chang, 2010; Lee and Lee, 2010). Therefore, it is thought that, if a heterofermentative LAB such as Leu. mesenteroides is used as a starter culture in kimchi fermentation, kimchi products become less acidic and more refreshing in taste due to the greater productions of ethanol, CO2, and mannitol, and reduced production of lactic acid compared to the use of Lactobacillus as a starter. Therefore, some companies are using Leu. mesenteroides as a starter culture for the fermentation of commercial kimchi. However, some studies have reported contrasting results showing rapid fermentation completion with greater pH decrease with the use of Leuconostoc species as a starter in cabbage fermentation (Choi et al., 2003; Tolonen et al., 2004; Johanningsmeier et al., 2007). Our study showed that the pH of starter kimchi began to decrease earlier than that in non-starter kimchi, but the final pH values were similar (Fig. 1), which did not coincide with our expectations. Community analysis of kimchi showed that the genera of Leuconostoc, Lactobacillus, and Weissella were the main lactic acid bacteria in kimchi fermentation, and the proportion of Leuconostoc was much higher

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Fig. 5. Quantification of identified primary key metabolites in non-starter and starter kimchi supernatants during kimchi fermentation. Data are given as mean ± standard errors, calculated in triplicate. Quantification was determined using the Chenomx NMR Suite v. 6.1 with 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as the internal standard. A, B, C, D, and E represent fructose, glucose, lactate, acetate, and mannitol, respectively.

than that of Lactobacillus or Weissella, especially during the early kimchi fermentation, while the proportion of Lactobacillus increased rapidly during the middle kimchi fermentation stage (Fig. 3). These results were in accordance with previous reports, which have been often explained by the hypotheses that members of Leuconostoc grow well under less acidic and anaerobic conditions, while members of Lactobacillus and Weissella are more competitive under acidic conditions (Cho et al., 2006; Chang and Chang, 2010; Lee et al., 2005). In addition to these explanations, the massively parallel barcoded pyrosequencing analysis allowed us to suggest another possible explanation for the predominance of Leuconostoc during early kimchi fermentation. The relative abundance of Leuconostoc at the first day of fermentation was very high (8.1–9.1%), indicating that the kimchi raw materials (cabbage or radish) already contained a large amount of Leuconostoc (Fig. 3), while the same kimchi raw materials contained a small amount of Lactobacillus (relative abundance of Lactobacillus at the first day: 0.36–0.59%). This could be another important reason for the low abundance of Lactobacillus during the early kimchi fermentation. The

relative abundance of Lactobacillus in kimchi raw materials was even lower than that of Weissella (5.2–10.3%) (Fig. 3). The relative abundances of Lactobacillus on the 12th day of fermentation was approximately 20%, although the pH of kimchi after 12 days was similar to their initial pH values (Figs. 1 and 3), suggesting that Lactobacillus can grow relatively quickly, similar to Leuconostoc, even during early kimchi fermentation phase. Barcoded pyrosequencing analysis also showed that the relative abundance of Leuconostoc decreased during the middle stage of kimchi fermentation, while the growth of Lactobacillus continued even during the late fermentation phase, which supported the fact that members of the genus Lactobacillus are more acid-resistant than Leuconostoc. The use of Leu. mesenteroides as a kimchi starter increased the Leuconostoc proportions and decreased the Lactobacillus proportions. The abundance of Weissella in the kimchi raw materials was relatively high, but they were never dominant (Fig. 3). However, interestingly, the proportions of Weissella in the starter kimchi were maintained at higher levels than in the non-starter kimchi until late kimchi fermentation phase, which might be due to the reduced

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those of non-starter kimchi (Fig. 6). This indicates that kimchi with Leu. mesenteroides as a starter can be fermented more rapidly and results in different kimchi flavors and tastes compared to kimchi fermented without a starter. The PCA data showed that metabolite compositions of Chonggak kimchi were not significantly different after 20 days, which might indicate that Chonggak kimchi fermentation was nearly complete at 20 days and the 1H NMR technique is a possible tool for determining the stage of kimchi fermentation. Mannitol concentrations increased as fermentation progressed, and their profiles were related to the increase in total cell number (Figs. 1 and 5E), which might suggest that Leuconostoc as well as Lactobacillus produced mannitol during kimchi fermentation (McFeeters and Chen, 1986; Wisselink et al., 2002). Mannitol provides refreshing tastes to foods and also has antioxidant and non-metabolizing sweetening properties. Therefore, it is a good replacement for sugars in diabetic foods (Wisselink et al., 2002). Kimchi with Leu. mesenteroides as a starter produced more mannitol compared to the non-starter kimchi, indicating that the use of Leu. mesenteroides as a starter may help produce kimchi with more nutritional value. In conclusion, this study showed that kimchi fermentation was completed earlier with greater production of kimchi metabolites when Leu. mesenteroides was used as a starter. This may contribute to the production of commercial kimchi with greater uniformity and functional properties. A combination of the use of a barcoded 454-pyrosequencing strategy and a 1H NMR technique was a good approach to effectively monitor microbial succession and their metabolites, which may allow for the better understanding of the relationships between the microbial community and metabolite production in diverse fermented foods including kimchi. Heterofermentative Leu. mesenteroides used in this research is a good example of the development of a kimchi starter. However, more studies to link the relationships among microbial communities, metabolites, and sensory characteristics (kimchi tastes or flavors) are necessary, which will finally give good rationales for the selection of a good starter to produce standardized kimchi with functional properties. Supplementary materials related to this article can be found online at doi:10.1016/j.ijfoodmicro.2011.11.030. Fig. 6. Score plots of the principal component analysis (PCA) performed on the 1H NMR spectra of kimchi metabolites during kimchi fermentation in (A) Baechu (Chinese cabbage) kimchi and (B) Chonggak (radish) kimchi. Closed circles and triangles represent starter kimchi and non-starter kimchi, respectively. Numbers beside the data points on the score plots represent the fermentation time (day) of kimchi. The directions of the curved arrows indicate the routes of data points on the score plots during kimchi fermentation.

abundance of Lactobacillus, a Weissella competitor, by the inoculation of Leuconostoc (Figs. 3 and 4). It is known that the members of the genus Leuconostoc including Leu. mesenteroides are heterofermentative LAB that produce lactic acid, ethanol, acetic acid, and CO2,while members of the genus Lactobacillus consist of homofermentative LAB (that produce only lactic acid from sugars) and heterofermentative LAB. The metabolite analysis using the 1H NMR technique showed that the increases in lactate and acetate, fermentation products of heterofermentative LAB, were well correlated with the decrease in free sugars and increase in total cell number regardless of the increase in relative abundance of Lactobacillus (Figs. 1 and 5). This suggests that Leuconostoc and heterofermentative members of Lactobacillus, not homofermentative Lactobacillus, governed the kimchi fermentation. In fact, when respective 16S rRNA gene sequences that were classified as Lactobacillus in RDP pipeline analysis were analyzed using the RDP classifier, they were most closely related to Lb. sakei, a typical facultatively heterofermentative Lactobacillus (data not shown) (Klein et al., 1996). The PCA using all kimchi metabolites including carbohydrates, amino acids, organic acids, and others from the 1H NMR-based metabolic analysis showed that starter Bachu kimchi fermented faster, and their final metabolite compositions were also a little different from

Acknowledgments This work was supported by the Technology Development Program for Agriculture and Forestry (TDPAF) of the Ministry for Agriculture, Forestry and Fisheries and by the Next-Generation BioGreen 21 Program (No. SSAC2011-PJ008220), Rural Development Administration, Republic of Korea.

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