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 Jejus 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: ejpark@jejunu.ac.kr).
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 manufacturers 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-L 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 DH5; 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 manufacturers 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 < 1010 , 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 < 1010 , 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 1The 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).
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Microbiota in Jeju fermented seafood . . .

Table 1Physical 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 2The 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.
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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

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Figure 3Phylogeny 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.

References
Abdeljabbar H, Cayol JL, Ben Hania W, Boudabous A, Sadfi N, Fardeau ML. 2013. Halanaerobium sehlinense sp. nov., an extremely halophilic, fermentative, strictly anaerobic bacterium
from sediments of the hypersaline lake Sehline Sebkha. Int J Syst Evol Microbiol 63(Pt
6):206974.
Adewumi GA, Oguntoyinbo FA, Keisam S, Romi W, Jeyaram K. 2012. Combination of cultureindependent and culture-dependent molecular methods for the determination of bacterial
community of iru, a fermented Parkia biglobosa seeds. Front Microbiol 3:436.
An C, Takahashi H, Kimura B, Kuda T. 2010. Comparison of PCR-DGGE and PCR-SSCP
analysis for bacterial flora of Japanese traditional fermented fish products, aji-narezushi and
iwashi-nukazuke. J Sci Food Agri 90(11):1796801.
Arahal DR, Ventosa A. 2006. The family Halomonadaceae. Prokaryotes 6:81135.
Bae JW, Rhee SK, Park JR, Chung WH, Nam YD, Lee I, Kim H, Park YH. 2005. Development
and evaluation of genome-probing microarrays for monitoring lactic acid bacteria. Appl
Environ Microbiol 71(12):882535.
Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R. 2010a.
PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics
26(2):2667.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N,
Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE,
Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh
PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R. 2010b. QIIME allows
analysis of high-throughput community sequencing data. Nat Meth 7(5):3356.
Cayol JL, Ollivier B, Patel BK, Ageron E, Grimont PA, Prensier G, Garcia JL. 1995. Haloanaerobium lacusroseus sp. nov., an extremely halophilic fermentative bacterium from the sediments
of a hypersaline lake. Int J Syst Bacteriol 45(4):7907.
Chuon MR, Shiomoto M, Koyanagi T, Sasaki T, Michihata T, Chan S, Mao S, Enomoto T.
2013. Microbial and chemical properties of Cambodian traditional fermented fish products. J
Sci Food Agri (in press), doi: 10.1002/jsfa.6379.
Do S-D, Lee Y-M, Chang H-G. 1993. The study on kinds and utilities of Jeot-Kal (fermented
fish products). Kor J Soc Food Sci 9(3):2229.
Dray S, Dufour AB. 2007. The ade4 package: implementing the duality diagram for ecologists.
J Stat Softw 22(4):120.
Edgar RC. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics
26(19):24601.
Fukami K, Satomi M, Funatsu Y, Kawasaki K, Watabe S. 2004. Characterization and distribution
of Staphylococcus sp. implicated for improvement of fish sauce odour. Fish Sci 70:91623.
Fukui Y, Yoshida M, Shozen K, Funatsu Y, Takano T, Oikawa H, Yano Y, Satomi M. 2012.
Bacterial communities in fish sauce mash using culture-dependent and -independent methods.
J Gen Appl Microbiol 58(4):27381.
Guan L, Cho KH, Lee JH. 2011. Analysis of the cultivable bacterial community in jeotgal,
a Korean salted and fermented seafood, and identification of its dominant bacteria. Food
Microbiol 28(1):10113.

Ha J-H, Han S-W. 1986. Studies on the processing of low salt fermented seafoods. Bull Korean
Fish Soc 19(4):31220.
Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa
D, Highlander SK, Sodergren E, Methe B, DeSantis TZ, Petrosino JF, Knight R, Birren
BW. 2011. Chimeric 16S rRNA sequence formation and detection in Sanger and 454pyrosequenced PCR amplicons. Genome Res 21(3):494504.
Holzapfel WH, Franz CMAP, Ludwig W, Back W, Dicks LMT. 2006. The genera Pediococcus
and Tetragenococcus. In: Stackebrandt E, Holzapfel WH, editors. The prokaryotes. New York:
Spinger. p 22966.
Ivanova N, Sikorski J, Chertkov O, Nolan M, Lucas S, Hammon N, Deshpande S, Cheng JF,
Tapia R, Han C, Goodwin L, Pitluck S, Huntemann M, Liolios K, Pagani I, Mavromatis K,
Ovchinikova G, Pati A, Chen A, Palaniappan K, Land M, Hauser L, Brambilla EM, Kannan
KP, Rohde M, Tindall BJ, Goker M, Detter JC, Woyke T, Bristow J, Eisen JA, Markowitz
V, Hugenholtz P, Kyrpides NC, Klenk HP, Lapidus A. 2011. Complete genome sequence
of the extremely halophilic Halanaerobium praevalens type strain (GSL). Stand Genomic Sci
4(3):31221.
Jung SY, Lee MH, Oh TK, Park YH, Yoon JH. 2005. Psychrobacter cibarius sp. nov., isolated
from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 55(Pt 2):577
82.
Jung JY, Lee SH, Lee HJ, Jeon CO. 2013. Microbial succession and metabolite changes
during fermentation of saeu-jeot: traditional Korean salted seafood. Food Microbiol 34(2):
3608.
Juste A, Lievens B, Frans I, Marsh TL, Klingeberg M, Michiels CW, Willems KA. 2008a.
Genetic and physiological diversity of Tetragenococcus halophilus strains isolated from sugar- and
salt-rich environments. Microbiology 154(Pt 9):260010.
Juste A, Lievens B, Klingeberg M, Michiels CW, Marsh TL, Willems KA. 2008b. Predominance
of Tetragenococcus halophilus as the cause of sugar thick juice degradation. Food Microbiol
25(2):41321.
Juste A, Van Trappen S, Verreth C, Cleenwerck I, De Vos P, Lievens B, Willems KA. 2012. Characterization of Tetragenococcus strains from sugar thick juice reveals a novel species, Tetragenococcus
osmophilus sp. nov., and divides Tetragenococcus halophilus into two subspecies, T. halophilus subsp.
halophilus subsp. nov. and T. halophilus subsp. flandriensis subsp. nov. Int J Syst Evol Microbiol
62(Pt 1):12937.
Kawashima T, Murakami K, Nishimura I, Nakano T, Obata A. 2012. A sulfated polysaccharide,
fucoidan, enhances the immunomodulatory effects of lactic acid bacteria. Int J Mol Med
29(3):44753.
Kim HJ, Yoon MS, Park Y-S, Ha J-H, Heu MS, Kim J-S. 2009a. Food component characteristics of commercial salt-fermented silver-stripe round herring. J Kor Fish Soc 42(2):
11622.
Kim M-S, Park E-J, Jung M-J, Roh SW, Bae JW. 2009b. Analysis of prokaryote communities
in Korean traditional fermented food, jeotgal, using culture-depedent method and isolation
of a novel strain. Kor J Microbiol 45:2631.
Kim TW, Lee JH, Kim SE, Park MH, Chang HC, Kim HY. 2009c. Analysis of microbial
communities in doenjang, a Korean fermented soybean paste, using nested PCR-denaturing
gradient gel electrophoresis. Int J Food Microbiol 131(23):26571.
Kim MS, Roh SW, Bae JW. 2010. Halomonas jeotgali sp. nov., a new moderate
halophilic bacterium isolated from a traditional fermented seafood. J Microbiol 48(3):
40410.
Kobayashi T, Kimura B, Fujii T. 2000. Strictly anaerobic halophiles isolated from canned Swedish
fermented herrings (Surstromming). Int J Food Microbiol 54(12):819.
Lane DJ. 1991. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic
acid techniques in bacterial systematics. New York: Wiley. p 11575.
Lee M, Kim MK, Vancanneyt M, Swings J, Kim SH, Kang MS, Lee ST. 2005. Tetragenococcus
koreensis sp. nov., a novel rhamnolipid-producing bacterium. Int J Syst Evol Microbiol 55(Pt
4):140913.
Leroy S, Lebert I, Chacornac JP, Chavant P, Bernardi T, Talon R. 2009. Genetic diversity and
biofilm formation of Staphylococcus equorum isolated from naturally fermented sausages and
their manufacturing environment. Int J Food Microbiol 134(12):4651.
Lozupone C, Knight R. 2005. UniFrac: a new phylogenetic method for comparing microbial
communities. Appl Environ Microbiol 71(12):822835.
Masuda S, Yamaguchi H, Kurokawa T, Shirakami T, Tsuji RF, Nishimura I. 2008. Immunomodulatory effect of halophilic lactic acid bacterium Tetragenococcus halophilus Th221
from soy sauce moromi grown in high-salt medium. Int J Food Microbiol 121(3):
24552.
Nam YD, Lee SY, Lim SI. 2012. Microbial community analysis of Korean soybean pastes by
next-generation sequencing. Int J Food Microbiol 155(12):3642.
Park EJ, Chun J, Cha CJ, Park WS, Jeon CO, Bae JW. 2011a. Bacterial community analysis
during fermentation of ten representative kinds of kimchi with barcoded pyrosequencing.
Food Microbiol 30(1):197204.
Park EJ, Kim KH, Abell GC, Kim MS, Roh SW, Bae JW. 2011b. Metagenomic analysis of the viral communities in fermented foods. Appl Environ Microbiol 77(4):
128491.
Place RB, Hiestand D, Gallmann HR, Teuber M. 2003. Staphylococcus equorum subsp. linens,
subsp. nov., a starter culture component for surface ripened semi-hard cheeses. Syst Appl
Microbiol 26(1):307.
Price MN, Dehal PS, Arkin AP. 2010. FastTree 2approximately maximum-likelihood trees for
large alignments. PLoS One 5(3):e9490.
Rhee SJ, Lee JE, Lee CH. 2011. Importance of lactic acid bacteria in Asian fermented foods.
Microb Cell Fact 10(Suppl 1):S5.
Roh SW, Kim KH, Nam YD, Chang HW, Park EJ, Bae JW. 2010. Investigation of archaeal
and bacterial diversity in fermented seafood using barcoded pyrosequencing. ISME J 4(1):
116.
Roling WF, van Verseveld HW. 1997. Growth, maintenance and fermentation pattern of the
salt-tolerant lactic acid bacterium Tetragenococcus halophila in anaerobic glucose limited retention
cultures. Antonie van Leeuwenhoek 72(3):23943.
Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing
phylogenetic trees. Mol Biol Evol 4(4):40625.
Satomi M, Kimura B, Mizoi M, Sato T, Fujii T. 1997. Tetragenococcus muriaticus sp. nov., a
new moderately halophilic lactic acid bacterium isolated from fermented squid liver sauce. Int
J Syst Bacteriol 47(3):8326.

Vol. 79, Nr. 5, 2014 r Journal of Food Science M933

M: Food Microbiology
& Safety

Microbiota in Jeju fermented seafood . . .

Microbiota in Jeju fermented seafood . . .

Suh HK, Yoon SS. 1987. A study on the regional characteristics of Korean chotkal. Korean J
Dietary Cult 2:4554.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum
parsimony methods. Mol Biol Evol 28(10):27319.
Tsai CR, Garcia JL, Patel BK, Cayol JL, Baresi L, Mah RA. 1995. Haloanaerobium alcaliphilum
sp. nov., an anaerobic moderate halophile from the sediments of Great Salt Lake, Utah. Int J
Syst Evol Microbiol 45(2):3017.
Udomsil N, Rodtong S, Tanasupawat S, Yongsawatdigul J. 2010. Proteinase-producing halophilic
lactic acid bacteria isolated from fish sauce fermentation and their ability to produce volatile
compounds. Int J Food Microbiol 141(3):18694.
Villar M, de Ruiz Holgado AP, Sanchez JJ, Trucco RE, Oliver G. 1985. Isolation and characterization of Pediococcus halophilus from salted anchovies (Engraulis anchoita). Appl Environ
Microbiol 49(3):6646.
Yoon JH, Kang KH, Park YH. 2003. Psychrobacter jeotgali sp. nov., isolated from jeotgal, a
traditional Korean fermented seafood. Int J Syst Evol Microbiol 53(Pt 2):44954.

M: Food Microbiology
& Safety
M934 Journal of Food Science r Vol. 79, Nr. 5, 2014

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):1716.

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