Bacterial Communities of Traditional Salted and Fermented Seafoods from Jeju Island of Korea Using 16S rRNA Gene Clone Library Analysis Min-Soo Kim and Eun-Jin Park

Jeotgal, which is widely consumed as a nutritional supplement in Korea, is traditional type of preserved seafood that is prepared by salting and fermenting. Here, we report on the bacterial community structure and diversity of jeotgal obtained from the Korean island of Jeju, which has a subtropical climate. Two samples of Jeotgal were collected from Jeju, made from either damselfish (Chromis notata; jari-dom-jeot, J1 and J2) or silver-stripe round herring (Spratelloides gracilis; ggot-myulchi-jeot, K1 and K2). The physical characteristics (pH and salinity) were assessed and the bacterial communities characterized using 16S rRNA gene-clone library analysis and cultural isolation. No difference was found in the community composition between the J and K fermented seafoods. Both fermented seafoods had relatively high salinity (26% to 33%) and high pH values (pH 6.08 to 6.72). Based on the 16S rRNA gene sequences, the halophilic lactic-acid bacteria Tetragenococcus halophilus and T. muriaticus were observed to be dominant in the J and K fermented seafoods, accompanied by halophilic bacteria including Halanaerobium spp., Halomonas spp., and Chromohalobacter spp. When compared with 7 other types of fermented seafood from a previous study, the communities of the J and K fermented seafoods were separated by the most influential group, the genus Tetragenococcus. The results suggest that these 2 types of traditional salted fermented seafood from Jeju have distinct communities dominated by Tetragenococcus spp., which are derived from the raw ingredients and are dependent on the physical conditions. This may explain how the seafoods that are made in Jeju may differ from other jeotgals. Keywords: bacterial community, jeotgal, salted fermented food, Tetragenococcus

Introduction Fermented seafood is widely consumed across the world. Fermented seafood is prepared from fish or shellfish, and preserved with the addition of salts to prevent spoilage (Rhee and others 2011). In Korea, there are about 150 different kinds of fermented seafood, termed “jeotgal” or “jeot” (Suh and Yoon 1987). Jeotgal is produced with a combination of high salt, additives, and various types of seafood, which depend on the region and climate of the locale (Do and others 1993). Jeju is the largest Korean island and is located off the southernmost tip of Korea. Jeju is geographically located in a subtropical region, and its climate is warmer than that of the Korean peninsula (http://web.kma.go.kr/eng/biz/climate_01.jsp). Corresponding to Jeju’s distinctive climate, subtropical fish species such as the jari-dom (Chromis notata), a type of damselfish, and the ggot-myulchi or silver stripe round herring (Spratelloides gracilis) are frequently found around Jeju coast, and are widely used as ingredients for fermented seafood. Both have been traditionally used as food sources, are popular local foods of Jeju in summer, and jeotgal made from jari-dom and ggot-myulchi serve as a side dish throughout the year in Jeju (Ha and Han 1986; Kim and others 2009a). Microorganisms play a critical role in food fermentation (Guan and others 2011). The microbial community of several types of

fermented seafood has been described using culture-dependent and culture-independent methods (Kim and others 2009b; Roh and others 2010; Guan and others 2011; Chuon and others 2013; Jung and others 2013). The microbial characteristics of fermented seafood are influenced by the fermenting microorganisms present in the raw material (Roh and others 2010; Park and others 2011b). In addition, the differences in microbial characteristics result in unique flavors and affect the longevity of the preservation. However, little is known about the microbial diversity and the role of these representative microorganisms in Jeju-type fermented seafood. To characterize the structure and diversity of the bacterial community of the Jeju traditional fermented seafoods, 4 samples of jeotgal made with Chromis notata (jari-dom-jeot) and Spratelloides gracilis (ggot-myulchi-jeot) were collected and their bacterial communities were analyzed by 16S rRNA gene clone library sequencing and cultural isolation. The bacterial assemblages of these 2 types of the fermented seafood from Jeju were compared with other types of jeotgal. To our knowledge, no investigation has been reported about the bacterial communities of Jeju traditional fermented seafood. Jeju-type jeotgal is expected to have distinctive microbial characteristics due to the unique geography and climate of the island.

Materials and Methods Sample preparation and physical features

MS 20131458 Submitted 10/12/2013, Accepted 2/4/2014. Author Kim is with Four samples of the 2 salted and fermented seafood products, Dept. of Life and Nanopharmaceutical Sciences and Dept. of Biology, Kyung Hee Univ., 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Author jari-dom-jeot (J1 and J2) and ggot-myulchi-jeot (K1 and K2), Park is with Dept. of Food Bioengineering, Jeju Natl. Univ., Jeju 690-756, Republic were purchased from the local markets in Jeju. The 2 samples of of Korea. Direct inquiries to author Park (E-mail: [email protected]). each type of jeotgal were manufactured by different local manu-

facturers in Jeju. The samples used in this study were fully ripened R  C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12431 Further reproduction without permission is prohibited

Vol. 79, Nr. 5, 2014 r Journal of Food Science M927

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Abstract:

Microbiota in Jeju fermented seafood . . . for more than 6 mo, which are generally consumed. The pH of the samples was measured using an Orion 3-star pH Benchtop pH meter (Thermo Scientific Orion, Rockford, Ill., U.S.A.), and the percentage of salt was measured using a Pocket PAL-03S portable refractometer (ATAGO, Tokyo, Japan). The bacterial community of the collected samples was analyzed immediately after collection.

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DNA extraction, PCR amplification, and 16S rRNA gene clone library Metagenomic DNA was extracted from a 0.25 mL of each sample using the PowerSoil DNA kit (MoBio, Carlsbad, Calif., U.S.A.) according to the manufacturer’s instructions. The 16S rRNA gene sequence was amplified with 2 universal bacterial primer set: forward primer 8F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and reverse primer 1492R (5 -GGYTACCTTGTTACGACTT3 ) (Lane 1991). A 20-μL PCR mix (MaximeTM PCR PreMix Kit [i-Taq], iNtRON BIOTECHNOLOGY, Korea) was prepared with the bacterial primer set and approximately 10 ng of extracted DNA as the template. The PCR conditions were as follows: 94 °C for 5 min, followed by 20 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s, and a final extension step at 72 °C for 10 min. The amplicon was purified using the QIAquick PCR purification (Qiagen, Valencia, Calif., U.S.A.), and clone libraries of the samples were constructed using the RBC T&A Cloning Vector and HIT Competent Cells (Escherichia coli DH5α; RBC Bioscience, Taiwan). Forty to fifty clones per sample were sequenced with M13 forward and reverse primers using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif., U.S.A.) according to the manufacturer’s instructions. The reaction mixtures were analyzed using an automated DNA analyzer (3730xl DNA Analyzer; Applied Biosystems). For the community analysis, the forward and reverse sequences from the clone libraries were assembled using SeqMan (DNASTAR, Madison, Wis., U.S.A.). The clone sequences are deposited at DDBJ/EMBL/NCBI under accession nrs KF783278 to KF783401. 16S rRNA gene sequence analysis The community analysis based on the clone sequences was processed using the QIIME software package 1.5.0 (Caporaso and others 2010b). The clone sequences were removed not to contain the sequences of forward and reverse primers and trimmed to contain common hypervariable regions (V1 to V4) of 16S rRNA gene sequence. Subsequently, the sequences were clustered against the Greengenes core set at 97% sequence similarity using UCLUST software (Edgar 2010). Representative sequences for operational taxonomic units (OTUs) were selected and aligned with the Greengenes core set using PyNAST (Caporaso and others 2010a). Chimeric sequences were removed from the representative sequences using ChimeraSlayer (Haas and others 2011) and a phylogenetic tree was constructed using FastTree (Price and others 2010). Taxonomic assignment of the representative sequences was defined by BLAST searches against the Nucleotide collection (nr/nt) in Genbank (BLASTn; E-value < 10−10 , coverage > 99%, identity > 97%). To characterize the community structures, species richness (Observed OTUs and Chao1 index) and biodiversity indices (Phylogenetic Diversity, Shannon, and Simpson indices) were determined, and compared between the communities based on the UniFrac distances (Lozupone and Knight 2005). Principal component analysis (PCA) was also determined by comparison with the communities of the fermented seafood previously described by Roh and others (2010) based on genus-level M928 Journal of Food Science r Vol. 79, Nr. 5, 2014

abundances using the ade4 package in R package (Dray and Dufour 2007).

Isolation and identification of cultural bacterial strains The samples were homogenized and 103 -fold diluted with a sterile saline solution (0.85%, w/v). The diluted suspension was plated on marine agar (MA; Difco, Detroit, Mich., U.S.A.) supplemented with 1%, 2%, 5%, 10%, and 15% of NaCl, and incubated at 30 °C for 3 to 5 d. Three to four isolated colonies of different morphologies were selected per plate and their 16S rRNA gene sequences were amplified using colony PCR, and analyzed by Sanger sequencing. The taxonomic identification of the isolates was determined based on 16S rRNA gene sequence comparison against the Nucleotide collection (nr/nt) in Genbank (BLASTn; E-value < 10−10 , coverage > 99%, identity > 97%). Phylogenetic analysis of Tetragenococcus 16S rRNA gene sequences The 16S rRNA gene sequences of the OTUs assigned to the genus Tetragenococcus were aligned with the sequences of the culture isolates found in salt- and sugar-rich environments (Satomi and others 1997; Lee and others 2005; Juste and others 2012) using the ClustalW2 (http://www.ebi.ac.uk/). Sequences including the V1 to V4 hypervariable regions of the 16S rRNA gene were used to construct a phylogenetic tree based on the neighbor-joining algorithm (Saitou and Nei 1987) using MEGA5 (Tamura and others 2011).

Results and Discussion Characterization of the bacterial communities of 2 fermented seafoods In total, of 183 16S rRNA gene sequences were generated after chimeric sequence removal, average 62 ± 26 (mean ± SD) sequences per jeotgal type (an average 31 ± 14 of sequences per sample) were generated. Alpha- and beta-diversity were characterized based on OTUs that were defined by clustering of the sequences based on 97% similarity. Most of the bacterial sequences in the J and K fermented seafoods belonged to the 2 phyla Firmicutes (average 78.9%) and Proteobacteria (average 13.4%) (Figure 1) and a small number of the sequences that matched the phylum Bacteroidetes (average 2.8%) were identified in samples J2, K1, and K2 samples (Figure 1), which is concordant with the previous studies that described bacterial communities in fermented foods (Roh and others 2010; Park and others 2011a). The microbial assemblages of the samples did not show major differences, except in regard to the phylum Planctomycetes which was solely dominated by 2.2% of the sequences generated from sample K1 and sample J1 solely dominated by the phylum Firmicutes. The majority of the sequences in the samples were assigned into the genus Tetragenococcus (including T. halophilus and T. muriaticus) of the order Lactobacillales (average 42.7%) and the genus Halanaerobium (including H. saccharolyticum) of the family Halanaerobiaceae (average 35.1%) (Figure 1A). The genus Tetragenococcus contains typical halophilic lactic-acid bacteria species found in fermented food products. Interestingly, no sequences were identified from the genera Lactobacillus, Leuconostoc, or Weissella, which are wellknown lactic acid bacteria (LAB) frequently observed in traditional fermented food (Bae and others 2005; Roh and others 2010; Park and others 2011a). As our previous studies in terms of bacterial communities in fermented seafoods (Roh and others 2010; Park

Microbiota in Jeju fermented seafood . . . The genus Halanaerobium are halophilic fermentative bacteria (Ivanova and others 2011), and are generally found in high-salt environments including fermented products (Cayol and others 1995; Tsai and others 1995; Kobayashi and others 2000; Ivanova and others 2011; Abdeljabbar and others 2013). In addition, a proportion of the sequences belonging to the phylum Proteobacteria were assigned to the genera Chromohalobacter (6.5% for sample K1 and 2.5% for J2), Halomonas (2.2% for sample K1, 2.5% for J2, and 2.0% for K2) and Psychrobacter (including P. celer; 2.2% for sample K1, 5.0% for J2, and 2.0% for K2) (Figure 1A). The bacterial groups we identified are characterized as halotolerant bacteria

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and others 2011a, 2011b), difference was found in the composition of bacterial communities, although the same type of the fermented seafoods was introduced in our previous studies. While Roh and others (2010) used the fermented seafoods containing other ingredients such as red pepper and garlic, Park and others (2011b) used the fermented seafoods not containing other ingredients, but just salts and seafoods. The comparison between these studies showed that LAB are usually found in the fermented seafoods containing other ingredients, and this indicates that the detection of LAB is dependent on other ingredients such as red pepper and garlic, not seafoods and salts.

Figure 1–The composition of the bacterial communities and PCoA analysis based on UniFrac distances. The composition of the bacterial communities in the K and J fermented seafood were determined by BLASTn searches against the Genbank nucleotide collection (nr/nt) database (A). The communities were compared based on unweighted (left) and weighted (right) UniFrac distances (B). Vol. 79, Nr. 5, 2014 r Journal of Food Science M929

Microbiota in Jeju fermented seafood . . . Table 1–Physical conditions and alpha-diversity of the bacterial communities of the J and K fermented seafoods. Sample ID

pH

Salinity (%)

Observed OTUs

Chao1

Shannon

Simpson

Phylogenetic diversity

J1 J2 K1 K2

6.65 6.72 6.16 6.08

33 31 26 28

6 19 16 17

11 72 34 53

1.1 3.6 2.8 3.3

0.36 0.87 0.72 0.84

1 2 2 1

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found in a wide range of aquatic and terrestrial environments with high salinity including fermented seafood (Yoon and others 2003; Jung and others 2005; Yoon and others 2005; Arahal and Ventosa 2006; Kim and others 2010). The average salinities of the K and J fermented seafood were 26.8% and 33.2%, respectively (Table 1), which suggests that the species of bacteria capable of thriving under high salt conditions are dominant in the K and J fermented seafoods. The communities of the J and K fermented seafoods were compared using the principal coordinate analysis (PCoA) based on the unweighted and weighted UniFrac distances. While the communities in the K1 and K2 samples were clustered together, the community of J1 sample was separate from that of the J2 sample (Figure 1B). In addition, the community of the J2 sample was close to those of the K1 and K2 samples (Figure 1B), suggesting that the community of the J1 sample was different from those of the J2, K1, and K2 samples at the OTU-level. The dominant J1 population only belonged to the Firmicutes phylum without any other bacterial groups observed in the communities of the J1 sample. Of the phylum Firmicutes, the genus Halanaerobium was more abundant in the J1 sample community than in the other samples, which reflected that high salinity of the J1 sample may have impact on the dominance of the genus Halanaerobium in the community of J1 sample. It was recently reported that members of the genus Halanaerobium are largely observed during the late stage of fermentation of shrimp fermented seafood, and that their abundance is correlated with the metabolic characteristics at the end of fermentation (Jung and others 2013). This indicates that the J1 sample was at a later stage of fermentation, and the J2, K1, and K2 samples were also in the mid-late or late stages of fermentation.

Culture and isolation of bacteria from 2 fermented seafoods We also investigated the bacterial communities of the J and K fermented seafoods using culture-dependent approach (Table S1). We cultured on MA supplemented with various amounts of NaCl, and isolated 61 bacterial strains. Based on the taxonomic assignment of the 16S rRNA gene sequences, 39 isolates were identified as species of the genus Staphylococcus, including S. nepalensis and S. equorum, which were previously found in fermented sausage and cheese (Place and others 2003; Leroy and others 2009) and fish sauce (Fukami and others 2004). Sixteen isolates were identified as species of the genus Tetragenococcus, including T. halophilus and T. muriaticus, which were dominant members of the bacterial communities in the J and K fermented seafoods determined by 16S rRNA gene clone libraries. Two species of the genus Halomonas and one species of the genus Psychrobacter were isolated, which were also observed in the communities of the J and K fermented seafoods determined by 16S rRNA gene clone libraries. This indicates that the bacterial communities of the J and K fermented seafoods determined by culture-independent approach

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confirmed using culture-dependent approach by the presence of Tetragenococcus spp. and Halomonas spp. as the members of the populations.

Diversity of bacterial communities in J and K fermented seafoods We determined the species richness of the J and K fermented seafoods as predicted by the observed OTUs and the Chao1 index, and we assessed the biodiversity by using the Shannon and Simpson indices. We identified between 6 and 19 OTUs, and the Chao1 index values ranged from 11 to 73 in the J and K fermented seafoods (Table 1). The observed differences in the species richness and in biodiversity between the K and J samples were not statistically significant. This indicates that the J and K fermented seafoods have similar community structure in bacterial populations. We compared the bacterial diversity of the J and K fermented seafoods with that of 7 kinds of fermented seafood produced from fish or shellfish, which were characterized using a high-throughput sequencing technique (Roh and others 2010). The species richness among the 7 products based on the number of OTUs and Chao1 index were in the range from 8 to 56, and the biodiversity based on the Shannon and Simpson indices were in the range from 1.22 to 1.97 and 0.30 to 0.53, respectively. These were comparable to those of the J and K fermented seafoods reported in this study. Two recent studies have shown a similar community composition and diversity in Korean fermented soybean paste, although either PCR-DGGE fingerprinting or 454-pyrosequencing was used for microbial analysis (Kim and others 2009c; Nam and others 2012). Our results demonstrate that the traditional Korean fermented seafood, jeotgal, has a simple community structure and diversity, and that the clone-based sequencing used in this study is useful approach for the analysis of small and simple microbial communities. Comparison of bacterial communities in various kinds of fermented seafood We compared the communities of the J and K fermented seafoods with those of 7 additional types of fermented seafood (Roh and others 2010) using multivariate PCA (Figure 2). The 7 additional fermented seafoods formed 3 clusters separating shellfish fermented food (JAB2B), oyster fermented food (JAB4B), and the other fermented seafoods (tiny shrimp [JAB1B], cuttlefish [JAB3B], roe of pollack [JAB5B], tripe of pollack [JAB6B], and crab [JAB7B]) clustered as reported in the original study (Roh and others 2010). The communities of the J (J1 and J2) and K (K1 and K2) fermented seafoods in this study were separated from the 7 fermented foods, although the J1 community was relatively close to them. While the dominance of LAB of the order Lactobacillales, such as Lactobacillus spp. and Weissella spp., were dominant in the 7 additional fermented seafood types, Tetragenococcus spp. and halophilic or halotolerant bacteria such as Halanaerobium spp., Halomonas spp., and Chromohalobacter spp. were dominant in the

Microbiota in Jeju fermented seafood . . . Phylogenetic analysis of the sequences assigned into the genus Tetragenococcus Most of the 7 OTUs assigned to the genus Tetragenococcus, including T. halophilus, were dominated in the communities of J and K fermented seafoods (average 42.7%; Figure 1). We aligned the 16S rRNA gene sequences assigned to the Tetragenococcus OTUs with sequences from members of the genus Tetragenococcus and generated a neighbor-joining phylogenetic tree to better determine their phylogenetic positions (Figure 3). Most sequences from all the OTUs were closely related to T. halophilus subsp. flandriensis and T. halophilus subsp. halophilus. The sequences of the remaining OTU were closely related to T. muriaticus. This result indicates that the OTUs were correctly assigned to the genus Tetragenococcus and

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J and K fermented seafoods. The J and K fermented seafoods had extremely high salt concentrations (26.8% and 33.2% w/v, respectively) similar to 2 of the 7 additional fermented seafoods (tiny shrimp [JAB1B], 32.8%; shellfish [JAB2B], 36.0%). However, the pH values of the J and K seafoods were reduced less in fermentation than those of the 7 additional fermented seafoods were (Table 1). Tetragenococcus halophilus and T. muriaticus are capable of growing under high pH conditions (Holzapfel and others 2006) and have previously been found in salted anchovy and fermented herring (Villar and others 1985; Kobayashi and others 2000). This suggests that the establishment of bacterial communities in the J and K fermented seafoods was largely influenced by pH conditions and the raw ingredients.

Figure 2–The ordinated diagram of PCA biplot of the jeotgal samples. The comparison of the bacterial communities between the K and J fermented seafood and 7 kinds of the fermented seafood (Roh and others 2010) using PCA based on genus-level abundances. All genus as variables were indicated by arrows in PCA diagram. Representations: JAB1B, tiny shrimp; JAB2B, shellfish; JAB3B, cuttlefish; JAB4B, oyster; JAB5B, roe of pollack; JAB6B, tripe of pollack; and JAB7B, crab. Vol. 79, Nr. 5, 2014 r Journal of Food Science M931

Microbiota in Jeju fermented seafood . . . that some (J.2 181, J.1 15, K.2 133, K.1 33, K.1 11, and K.2 96 sequences) may be novel species. The members of the genus Tetragenococcus are halophilic and osmophilic lactic acid-producing bacteria (Juste and others 2008a; Juste and others 2012). Strains of T. halophilus have been reported to produce volatile compounds by hydrolyzing fish protein, and play a role in the flavor formation during the fermentation of fish sauce (Udomsil and others 2010). Interestingly, none of the studies reported that Tetragenococcus are the dominant species in the bacterial communities of fermented food products, although Tetragenococcus spp. have been identified in combination with Bacillus subtilis, Lactobacillus spp., and Weissella spp. during food fermentation (An and others 2010; Adewumi and others 2012; Fukui and others 2012; Nam and others 2012; Chuon and others 2013). Considering that the genus Tetragenococcus is the only bacteria of lactic-acid produc-

ing bacteria that can grow under a high-salt conditions (Roling and van Verseveld 1997), these bacteria may have been selected for, and thrived in this highly salted fermented seafood, and may have contributed to the distinctive microbial characteristics of the J and K fermented seafoods. Recently, the genetic diversity of Tetragenococcus in some saltand sugar-rich environments has been determined by PCR-based molecular approaches, yet still only a limited number of species have been isolated (Juste and others 2008b; Fukui and others 2012; Nam and others 2012; Chuon and others 2013). It was recently reported that T. halophilus has an immunomodulatory effect that attenuates allergic symptoms by promoting Th1/Th2 immune balance (Masuda and others 2008; Kawashima and others 2012). However, further studies will be needed in order to fully understand the characteristics of this genus. In this study, we found

M: Food Microbiology & Safety Figure 3–Phylogeny of 16S rRNA gene sequences assigned into the genus Tetragenococcus. The phylogenetic status of the 16S rRNA gene sequences of the OTUs (bold) assigned to the genus Tetragenococcus were determined by generating a phylogenetic tree based on the V1-V4 region of the 16S rRNA genes using the neighbor-joining method. Bar, 0.005 changes per nucleotide position.

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tentative new species of the genus Tetragenococcus in the 2 types of jeotgals studied (jari-dom-jeot and ggot-myulchi-jeot), in addition to the species of Tetragenococcus (T. muriaticus and T. koreensis) that have frequently been isolated from other Korean fermented foods (that is, squid liver sauce and kimchi) (Lee and others 2005). This suggests that jeotgal, including “jari-dom-jeot” and “ggot-myulchi-jeot,” is a newly identified reservoir of the genus Tetragenococcus.

Conclusion We determined the bacterial community structures and diversity of the salted and fermented seafoods, “jari-dom-jeot” and “ggotmyulchi-jeot,” by using 16S rRNA gene clone library analysis and cultural isolation. Our study first characterized the bacterial communities of Jeju traditional fermented seafoods, and showed that Tetragenococcus spp. (T. halophilus and T. muriaticus) and Halanaerobium spp. (H. saccharolyticum) were dominant members of the bacterial community in the “jari-dom-jeot” and “ggot-myulchijeot” fermented seafoods. The bacterial communities of these fermented seafoods may originate from the species frequently found in the raw materials when under high-salt conditions. This study will be helpful to establish basic scientific knowledge about foodborne microorganisms in food fermentation and to further need for selecting starter strains for the development of fermented food manufacture. Furthermore, we have demonstrated that 16S rRNA gene clone library sequencing is an effective method for analyzing simple environmental microbial communities.

Acknowledgment This work was supported by the research grant of Jeju Natl. Univ. in 2011.

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Yoon JH, Yeo SH, Oh TK, Park YH. 2005. Psychrobacter alimentarius sp. nov., isolated from squid jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 55(Pt 1):171–6.

Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s website: Table S1. Identification of the isolates cultured from the J and K fermented seafoods.

Bacterial communities of traditional salted and fermented seafoods from Jeju Island of Korea using 16S rRNA gene clone library analysis.

Jeotgal, which is widely consumed as a nutritional supplement in Korea, is traditional type of preserved seafood that is prepared by salting and ferme...
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