Bacterial Community Dynamics of Salted and Fermented Shrimp based on Denaturing Gradient Gel Electrophoresis Kook-Il Han, Yong Hyun Kim, Seon Gu Hwang, Eui-Gil Jung, Bharat Bhusan Patnaik, Yeon Soo Han, Kung-Woo Nam, Wan-Jong Kim, and Man-Deuk Han

The Korean traditional seafood jeotgal is consumed directly or as an additive in other foods to improve flavor or fermentation efficiency. Saeujot, made from salted and fermented tiny shrimp (SFS; Acetes japonicus), is the best-selling jeotgal in Korea. In this study, we reveal the microbial diversity and dynamics in naturally fermented shrimp by denaturing gradient gel electrophoresis (DGGE). The population fingerprints of the predominant microbiota and its succession were generated by DGGE analysis of universal V3 16S rDNA polymerase chain reaction (PCR) amplicons. Overall, 17 strains were identified from sequencing of 30 DGGE bands. The DGGE profiles showed diverse bacterial populations in the sample, throughout the fermentation of SFS. Staphylococcus equorum, Halanaerobium saccharolyticum, Salimicrobium luteum, and Halomonas jeotgali were the dominant bacteria, and their levels steadily increased during the fermentation process. Certain other bacteria, such as Psychrobacter jeotgali and Halomonas alimentaria appeared during the early-fermentation process, while Alkalibacterium putridalgicola, Tetragenococcus muriaticus, and Salinicoccus jeotgali appeared during the late-fermentation process. The members of the order Bacillales were found to be predominant during the fermentation of SFS. Furthermore, S. equorum was identified as the dominant bacterial isolate by the traditional method of culturing under aerobic and facultative anaerobic conditions. We expect that this information will facilitate the design of autochthonous starter cultures for the production of SFS with desired characteristic sensory profiles and shorter ripening times. Abstract:

M: Food Microbiology & Safety

Keywords: bacterial community, DGGE, 16S rDNA sequence analysis, salted and fermented shrimp, Staphylococcus equorum

This is the 1st report on the microbial dynamics and succession during fermentation of salted and fermented shrimp (SFS; Acetes japonicus) using denaturing gradient gel electrophoresis (DGGE). Staphylococcus equorum was found to be the most persistent bacterial isolate during the 3 month fermentation period of SFS. The isolate is made available under Korean collection of type culture (KCTC) strain 13766. Strains of Staphylococcus equorum and other Bacillales could be beneficial for the design of a predetermined mixture of starter culture.

Practical Application:

Introduction Fermentation is a process in which food is preserved using microorganisms (Ross and others 2002). Salted and fermented shrimp (SFS) has long been used in Korean cuisines, and is recognized as a valuable aquatic protein resource. SFS is also used as an additive to improve the taste of foods such as Kimchi (Jung and others 2011). SFS is usually made by the fermentation of highly salted [approximately 25% (w/w)] aquatic animals such as tiny shrimp (Acetes japonicus), and becomes palatable through subsequent preservation. Most traditional fermentation technologies used for making different types of SFS in Korea, include the use of soil caves. The ripening stage of SFS takes about 3 months, including the fermentation process. The sources of spontaneous fermentation of seafood are primarily organisms associated with the environment of the animal being MS 20140941 Submitted 6/1/2014, Accepted 10/6/2014. Authors Han, Y.H. Kim, Hwang, Jung, Nam, W.J. Kim and Han are with Dept. of Life Science and Biotechnology, Soonchunhyang Univ., Asan, Chungnam, 336-745, Republic of Korea. Authors Patnaik, and Han are with Div. of Plant Biotechnology, College of Agriculture and Life Science, Chonnam Natl. Univ., Gwangju, 500-757, Republic of Korea. Direct inquiries to author Han (E-mail: [email protected]).

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fermented (for example, sea water or sea-tidal flats), or the natural microflora of marine salt used to prepare fermented seafood. Many microorganisms have been isolated from SFS (Yoon and others 2005a, 2005b, 2006; Aslam and others 2007a, 2007b; Mah and others 2008; Park and others 2010; Guan and others 2011; Kim and Park 2014). We previously reported isolation of chitinolytic Bacillus and Paenibacillus sp. from SFS using biochemical, phenotypic, and 16S rRNA-based molecular phylogenetics study (Han and others 2014). Another study documented the bacterial communities of traditional and fermented seafoods from Jeju island of Korea using 16S rRNA gene-clone library analyses and culture isolation (Kim and Park 2014). Although the microbial communities associated with SFS have been studied, there is no compelling evidence regarding microbial succession during fermentation and microbial fingerprinting. Earlier studies have characterized microbial communities by classical taxonomic methods and thus been restricted to the isolation of a small number of strains. Accordingly, comprehensive microbial typing of SFS is necessary to design a specific starter culture and adjunct cultures such as been done for other foods such as artisanal cheeses and Korean fermented soybean paste (Randazzo and others 2002; Kim and others 2009). The use of starter cultures for fermented food production is becoming R  C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12707 Further reproduction without permission is prohibited

increasingly necessary to standardize the product’s properties and obtain consistent flavor, texture, color as well as a shorter ripening time (Alegria and others 2009; Tu and others 2010). Molecular phylogenetics have been useful in investigation of the diversity and dynamics of microbial communities involved in the fermentation process (Delong and Pace 2001; McCartney 2002). The most promising approach includes genetic typing of 16S ribosomal RNA gene (16S rRNA) sequences for rapid microbial community analyses (Guan and others 2011; Han and others 2014). Many molecular biology methods documenting the microorganisms from fermented food samples have employed 16S rDNA sequence analysis (Andorra and others 2008). Polymerase chain reaction-denaturing gel electrophoresis (PCR-DGGE) profiles the 16S rRNA populations based on amplification of ribosomal DNA and electrophoresis of the PCR product in polyacrylamide gels

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Figure 1–DGGE fingerprints of bacterial V3 16S rDNA from SFS samples at different stages of fermentation. The DGGE profile was analyzed by denaturing gradient from 10% to 50%. Lane numbers denote time points (weeks) of the fermentation process. Bands indicated by numbers were excised, reamplified and subjected to sequencing. The number of bands in the DGGE profile corresponds to the bacteria isolates in Table 1.

containing an increasing gradient of denaturant (Muyzer and others 1993). PCR-DGGE studies enumerating microbial diversity of different samples generally concentrate on comparing the number and pattern of bands generated. Although, this may not be completely foolproof, subsequent identification of PCR-DGGE profiles by hybridization analysis, cloning, and sequencing of excised bands can improve the information obtained. PCR-DGGE has been employed to monitor microbial community dynamics in culture-independent analyses, and in parallel with traditional culture methods to study complex microbial communities originating from food samples (Masco and others 2005; Beccaceci and others 2006; Florez and Mayo 2006; Chang and others 2008; Tu and others 2010; Feng and others 2012), and other environments including sliced vacuum-packed cooked ham (Hu and others 2009), rice vinegar (Haruta and others 2006), kefir grains (Magalhaes and others 2010; Miguel and others 2010), and cheese (Alegria and others 2009; Dolci and others 2010). This study was conducted to evaluate the microbial flora associated with the SFS fermentation process using PCR-DGGE, and subsequent identification of species from the band patterns. Furthermore, we were able to characterize Staphylococcus equorum as the predominant bacterial isolate by conventional culturing and 16S rDNA sequence identity. The results of this study will be useful in starter culture selection, design, and optimization of SFS for fermentation. To the best of our knowledge, this is the 1st investigation on the bacterial community dynamics and succession during SFS fermentation using DGGE analyses.

Materials and Methods Sample preparation SFS samples with approximately 22% (w/v) salt were prepared in bulk using fresh shrimp, Acetes japonicus (2.4 ± 3 mm in length) according to a traditional manufacturing method (Jung and others 2013). The samples collected from Shinan-suhyup market (Shinan-gun, Jeollanam-do, Korea) were dispensed into 3 plastic containers (25 kg) with the addition of 22% (w/v) sodium chloride. The container was covered with a cap and the SFS was allowed to ripen for 12 wk in Togul (a traditional food pantry soil

Brevundimonas halotolerans MCS 24T

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Alkalibacillus salilacus BH163T

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Staphylococcus equorum subsp. equorum ATCC 43958T 100

Salinicoccus jeotgali S2R53-5T

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Alkalibacterium putridalgicola T129-2-1T Tetragenococcus muriaticus X-1T

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Psychrobacter jeotgali YKJ-103T Halomonas jeotgali KCTC 22487T

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Halomonas alimentaria YKJ-16T Salinivibrio costicola subsp. costicola NCIMB 701T

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Figure 2–Unrooted phylogenetic analyses of the 16S rDNA-V3 sequences from DGGE fingerprints. Figures at the nodes represent the bootstrap replication percentage and were based on 1000 replicates. Vol. 79, Nr. 12, 2014 r Journal of Food Science M2517

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Microflora in fermented salted shrimp . . .

Microflora in fermented salted shrimp . . . cave). The pH and salt concentration of SFS were intermittently measured. The fermentation temperature and humidity conditions in Togul were measured weekly. During fermentation, 9 SFS samples from 3 different batches were collected from the container every 7 d over a period of 84 d for culture-dependent analyses and extraction of total DNA for culture-independent PCR-DGGE analyses.

Extraction of total DNA from SFS Bulk community DNA was isolated from SFS using the bead beating method (Yeates and others 1998). Subsequently, the DNA samples were treated with RNase A (Sigma Chemical Co., St. Louis, Mo., U.S.A.) and purified by ethanol precipitation. Extracted DNA was further purified using an Ultraclean Microbial DNA isolation kit (Mo Bio Laboratories, Solana Beach, Calif., U.S.A.), after which it was quantified using a spectrophotometer (Nanodrop Technologies, Rockland, Del., U.S.A.). The extracted DNA was used as a template for PCR amplification of the V3 region of the 16S rRNA gene.

M: Food Microbiology & Safety

PCR-DGGE analysis The bacterial community DNA was amplified using the GCclamp-338F (5’-CGCCCGCCGCGCGCGCGGGCGGGGC GGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3’) and 518R (5’-ATTACCGCGGCTGCTGG-3’) primers, which correspond to the V3 region of the 16S rDNA. PCR was performed in a total reaction volume of 25 µL containing 1 µL of template DNA, 0.5 µM of each primer, 200 µM of dNTPs, 1.5 mM MgCl2 , 2.5 µL of 10× PCR buffer, and 1.25 U of Taq polymerase (Bioneer, Daejeon, Korea). Touchdown PCR was carried out using specific primer sets to increase the amplification specificity. The amplification was successful using an initial denaturation step of 5 min at 94 °C and a program in which the annealing temperature was decreased by 1 °C from 65 °C during each successive cycle until a touch-down temperature

of 55 °C was reached. Additionally, 10 cycles of PCR were conducted at 55 °C for 1 min, followed by an elongation step of 72 °C for 3 min and final extension at 72 °C for 10 min. PCR was performed using a 9400 thermal cycler (Perkin-Elmer, Foster City, Calif., U.S.A.). Samples were analyzed under UV light after running on agarose gels. The PCR products were subjected to DGGE on a DCode System apparatus (BioRad, Hercules, Calif., U.S.A.) using the method described by Muyzer and Smalla (1998). Briefly, samples were applied to an 8% (w/v) polyacrylamide gel in 1× Tris– acetate EDTA TAE buffer with a denaturing gradient of 10% to 50% urea-formamide, with 100% defined as 7 M urea and 40% (v/v) formamide. Following electrophoresis for 30 min at 20 V, and 16 h at 60 V, the gel was stained with SYBR Green I (Sigma Co., St. Louis, Calif., U.S.A.) for 30 min and analyzed using the Gel Doc system (Bio Rad). Subsequently, DGGE bands were excised with a sterile scalpel and the DNA from each band was eluted overnight at 4 °C in 10 µL of sterile water. Next, 1 µL of each DGGE band was reamplified using the conditions described above. The fragments were subsequently purified using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Sequencing was performed using an ABI PRISM dye terminator kit (Applied Biosystems, Foster City, Calif., U.S.A.) including the 338F and 518R primers. Sequence identity was determined by a BLAST nucleotide search at the GenBank database, while phylogenetic tree was constructed using the neighborjoining method (Saitou and Nei 1987) within the MEGA v5.0 program (Tamura and others 2011).

SFS samples and bacterial strain isolation SFS were prepared as described above with the sample preparation method. Approximately, 2 g of 3-mo-old samples were collected in individual sterile bags, and subsequently homogenized (ACE homogenizer, Model AM-1, Nissei Co., Japan) at 10000

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Figure 3–Visualization of taxonomic levels. Pie charts show the proportion of predominant bacteria in salted and fermented shrimp representing orders such as Pseudomonadales, Oceanospiralles, Lactobacillales, Halanaerobiales, and Bacillales. The dynamic distribution of bacterial orders at 0 month (A), 1 month (B), 2 months (C), and 3 months (D). Relative intensities of the DGGE bands correspond to the fraction of the assigned taxon.

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rpm for 5 min with 18 mL of sterile peptone water. The larger particles from the mixture solution were removed by filtration using 4 layers of sterile coarse gauze. Pellets and microorganisms were harvested from the filtrates by centrifugation (Combi-514R, Hanil, Seoul, Korea) at 7600 × g for 20 min at 4 °C. The samples were then plated on agar plates after 10-fold serial dilutions. Marine agar, nutrient agar, lactobacilli MRS agar, R2A agar and TSB agar (Becton Dickinson and Co., Sparks, Md., U.S.A.) were used for the isolation of bacteria. All plates were incubated at 15 °C for 72 h, after which the resulting colonies (25 to 35 per agar medium) were collected based on differences in morphology and color.

16S rDNA sequence analysis Bacterial strains were confirmed by partial sequencing of the 16S rDNA. Amplification of 16S rDNA was conducted using forward primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and reverse primer 1492R (5’- GGTTACCTTGTTACGACTT-3’) (Devereux and Willis 1995). Sequencing of the 16S rDNA fragment was performed using an ABI 3730xl DNA analyzer (Applied Biosystems). The closest relatives of the partial 16S rDNA sequences were obtained using the BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/) and the Ribosomal Database Project at GenBank (Maidak and others 1999). Morphological and phenotypic characterization Among the predominant isolates, S. equorum identified by 16S rRNA sequencing method was cultured on marine broth medium for 24 h at 37 °C and pre-fixed in 2.5% glutaraldehyde for 1 h. The pre-fixed samples were then treated with 1% osmium tetroxide (pH 7.2, 0.1 M phosphate buffer) solution for 90 min, dehydrated in a graded ethanol series (60%, 70%, 80%, 90%, and 100% for 10 min each), and in hexamethyldisilazane. Finally, the samples were examined using a Hitachi S-4700 Field Emission Scanning Electron Microscope (Hitachi High-Technologies Corp., Japan) with an acceleration tension of 10 kV. The bacterial isolates were phenotypically characterized using API 50CH system tests (bioMerieux, Inc, Hazelwood, Mo., U.S.A.) as described by Logan and Berkeley (1984). In addition, samples were analyzed for catalase activity (Smibert and Krieg 1994).

Results Physicochemical parameters We recoded the physicochemical parameters during fermentation of SFS. The temperature and humidity during the 12 wk of SFS fermentation were 16.7 ± 1 to 2 °C and 99%, respectively. The changes in pH monitored were 6.74 at 0 wk and 6.97 at 12 wk. Salt concentrations were 22% and 21% at 0 and 12 wk, respectively. Microbial species detection by PCR-DGGE The application of PCR-DGGE to the samples throughout SFS fermentation enabled a comprehensive profiling of the microbial communities. The DGGE separation patterns of the PCRamplified V3 region of 16S rDNA segments derived from SFS samples during the 12 wk fermentation period revealed the presence of 30 bands (Figure 1). Each band was sequenced and identified by NCBI BLAST search (Table 1). The major bands in the PCR-DGGE profiles were identified as Staphylococcus equorum subsp. equorum (Band no. 1), Halanaerobium saccharolyticus subsp.

saccharolyticum (Band no. 7), Halomonas jeotgali (Band no. 13), and Salimicrobium luteum (Band nos. 21, 22, and 23). The intensity of bands in DGGE profiles reflected the dominance of microbial species. During the early stages of fermentation, band no. 5 and band no. 28 corresponding to Pseudomonas veronii and Salinococcus jeotgali, respectively, showed high intensity. From the 3rd wk of fermentation, strain Alkalibacillus salilacus (Band nos. 26 and 30) showed increasing intensity. Band nos. 2, 9, and 29 showed high intensity after 7 weeks of fermentation, while band no. 27 was dominant after 10 weeks. These findings indicated that Alkalibacterium putridalgicola, Tetragenococcus muriaticus, S. jeotgali, and Vergibacillus kekensis constitutes the dominant bacterial species during late stage of SFS fermentation. An unrooted phylogenetic tree depicting the bacterial community based on 16S rRNA DGGE bands have been shown in Figure 2. Overall, 10 bands in the PCR-DGGE profile (Band nos. 8, 11, 12, 17, 19, 20, 21, 22, 23, and 25) corresponded to S. luteum BY-5T , 3 bands (Band nos. 24, 26, and 30) corresponded to A. salilacus BH163 and 2 bands (Band nos. 15 and 18) related to Salinivibrio costicola subsp. costicola NCIMB 710T . The dynamic distribution of microorganisms during 12 wk of fermentation of SFS under aerobic or partially anaerobic conditions was classified into predominant orders (Figure 3). During the beginning of SFS fermentation, the bacterial community predominately belonged to the order Oceanospirillales, followed by 60% distribution within the orders Bacillales, and other 40% equally distributed among Halanaerobiales and Pseudomonadales (by 1 mo of fermentation). Throughout the remainder of the fermentation process, the diversity of bacteria belonging to the order Bacillales improved relative to other bacterial orders, and was higher at about 50% and 56.6% at 2 and 3 mo of SFS fermentation, respectively. No bacterial genera belonging to Pseudomonadales were found present during the 3 mo of SFS fermentation, while bacterial genera belonging to order Lactobacillales were found during the later stages of SFS fermentation.

Isolation of dominant bacteria by PCR (16S rDNA) A total of about 150 colonies belonging to strict aerobic and facultative anaerobic bacteria grew from the original filtrate and were subjected to molecular-based identification using the 16S rDNA sequences of the isolates. The isolates were found to belong to 7 orders: Bacillales, Caulobacterales, Lactobacillales, Oceanospirillales, Pseudomonadales, Halanaerobiales, Vibrionales, and included 14 species (Staphylococcus equorum, Staphylococcus xylosus, Staphylococcus arlettae, Staphylococcus saprophyticus subsp. saphyticus, Staphylococcus saprophyticus subsp. bovis, Bacillus cereus, Bacillus thuringiensis, Psychrobacter celer, Psychrobacter marincola, Planococcus maritimus, Salinicoccus salsiraiae, Kocuria rhizophila, Kytococcus sedentarius, Rothia amarae) representing 8 genera. Among the isolates from SFS, the genera Staphylococcus represented 69% of all of the isolates, whereas Bacillus and Psychrobacter represented 10% and 6%, respectively. The proportion of species in the genera Rothia, Planococcus, Kytococcus, and Kocuria were 5%, 5%, 3%, and 2% of all isolates, respectively. The dominant facultative anaerobic bacteria in SFS samples were S. equorum (>60 colony) as was observed with the PCR-DGGE profiles. Strains showing a 16S rDNA sequence identity of 99% or higher with S. equorum species were isolated from all media used in this study. Scanning electron microscopy of S. equorum isolates showed the presence of round and cream-colored colonies, with the individual cells being Gram-positive cocci (Table S1; Figure 4). Phenotypically, the isolates were catalase and indole production positive, and showed an alkaline reaction with litmus Vol. 79, Nr. 12, 2014 r Journal of Food Science M2519

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Microflora in fermented salted shrimp . . .

Microflora in fermented salted shrimp . . . Table 1–Identification of bands excised from 16S rRNA DGGE fingerprints of SFS during fermentation. Band(s)a

M: Food Microbiology & Safety

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 a

Closest relative

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Staphylococcus equorum subsp. equorum ATCC 43958T Alkalibacterium putridalgicola T129–2–1T Psychrobacter jeotgali YKJ-103T Pseudomonas marincola KMM 3042T Pseudomonas veronii CIP 104663T Brevundimonas halotolerans MCS 24T Halanaerobium saccharolyticum subsp. saccharolyticum DSM 6643T Salimicrobium luteum BY-5T Tetragenococcus muriaticus X-1T Halomonas salina F8–11T Salimicrobium luteum BY-5T Salimicrobium luteum BY-5T Halomonas jeotgali KCTC 22487T Halomonas taeanensis BH 539T Salinivibrio costicola subsp. costicola NCIMB 701T Halomonas salina F8–11T Salimicrobium luteum BY-5T Salinivibrio costicola subsp. costicola NCIMB 701T Salimicrobium luteum BY-5T Salimicrobium luteum BY-5T Salimicrobium luteum BY-5T Salimicrobium luteum BY-5T Salimicrobium luteum BY-5T Alkalibacillus salilacus BH 163T Salimicrobium luteum BY-5T Alkalibacillus salilacus BH 163T Virgibacillus kekensis YIM-kkny16T Halomonas alimentaria YKJ-16T Salinicoccus jeotgali S2R53–5T Alkalibacillus salilacus BH 163T

100 90.6 98.1 100 100 97 100 98.1 100 98.8 97.5 97.5 96.3 99.4 99.4 97.5 98.1 100 97.5 98.8 97.5 97.5 98.1 99.4 96.9 99.4 95.7 99.4 100 100

AB009939 AB294167 AF441201 AB301071 AF064460 M83810 X89069 DQ227305 D88824 AJ295145 DQ227305 DQ227305 EU909458 AY671975 X95527 AJ295145 DQ227305 X95527 DQ227305 DQ227305 DQ227305 DQ227305 DQ227305 AY671976 DQ227305 AY671976 AY121439 AF211860 DQ471329 AY671976

Bands are numbered as indicated on the DGGE gel (shown in Figure 1). of identical nucleotides in the sequence obtained from the DGGE band. Accession number of the sequence of the closest relative found by a BLAST search.

b Percentage c

milk (Table S1). Furthermore, the biochemical characteristics of the isolates indicated that they can utilize a variety of substrates as carbon sources, including glucose, fructose, lactose, xylitol, Nacetylglucosamine, and urea (Table S1). S. equorum strain isolated from SFS samples have been deposited at Korean Collection for Type Cultures (KCTC) with No. 13766.

A

Discussion Saeujeot, a salted and fermented shrimp food in Korean cuisine, is made by adding 22% (w/v) salt to shrimp. SFS is consumed as fermented seafood by itself or as an additive to foods such as Kimchi to improve the taste or fermentation efficiency. Product processing of SFS by fermentation without the sterilization of raw

Figure 4–Scanning electron micrographs of the predominant isolate, Staphylococcus equorum. The isolate is a Gram-positive coccus that grows in grape-like clusters.

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1㎛

Microflora in fermented salted shrimp . . . justified in our DGGE fingerprinting analyses as it has been isolated from a marine solar saltern in Korea (Yoon and others 2007). The dominance of S. luteum in PCR-DGGE analysis was not represented in the culture-based study and conforms to earlier reports (Choi and others 2014).

Conclusions PCR-DGGE analyses showed the bacterial succession during 3–mo fermentation schedule of SFS. The dominant bacteria throughout the fermentation period were identified as Staphylococcus equorum, Halanaerobium saccharolyticum, Salimicrobium luteum, and Halomonas jeotgali. The identification of the most persistent and dominant members of the microbial community, especially Staphylococcus equorum and other Bacillales is critical to effectively design autochthonous starter cultures for the production of SFS with desirable sensory profiles and reducing ripening times. We are currently, evaluating the technical properties of potential starter cultures, and it is expected that the results will contribute to the overall knowledge regarding microflora involved in SFS.

Acknowledgment This work was supported in part by the Soonchunhyang Univ. Research Fund.

Author Contributions Kook-Il Han performed the experiments, analyzed the data, and wrote the paper. Yong Hyun Kim designed and performed the experiments. Seon Gu Hwang performed the experiments. Eui-Gil Jung performed the experiments. Bharat Bhusan Patnaik wrote the paper and analyzed the data. Yeon Soo Han provided technical details. Kung-Woo Nam analyzed the data. Wan-Jong Kim provided technical details. Man-Deuk Han designed the experiments, analyzed the data, provided technical details, and wrote the paper.

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materials leads to the growth of various microorganisms, derived from the raw materials, during fermentation. Therefore, many microbial communities are represented during SFS fermentation. Togul, Korean traditional food pantry soil cave is a good place for fermentation and storage of SFS. To produce shrimp products, SFSs were fermented in Togul for 12 wk at 16.7 ± 1 to 2 °C under 99% humidity. Traditionally, the quantification and identification of microflora in fermented foods rely on culture-dependent methods, which include the basic culture of the sample, counting, and identification of colonies (Lee and others 2014). Due to biased results and unrepresentative data generated under cultivation conditions (Roh and others 2010), culture-independent approaches including PCR-DGGE and 16S rRNA gene clone library analysis, and pyrosequencing have been devised for a comprehensive profile of the microbial populations in processed and fermented food environments (An and others 2010; Matsui and others 2013). In this study, the PCR-DGGE method was used to identify the microbial diversity and dynamics in naturally fermented SFS. This is an important step that will facilitate selection of starter cultures, particularly strains that are well adapted to the desirable characteristics used for this bacteria-fermented SFS. This is an advance over the evaluation of microbial communities in jeotgal by culturedependent methods (Cha and others 1983; Ahn and others 1990; Jung and Park 2004) and 16S rRNA gene sequence analysis (Kim and others 2009a, 2009; Guan and others 2011)-based phylogenetic analysis. A total of 17 strains were identified by DGGE sequencing of 30 distinct bands corresponding to V3 16S rRNA fragments. The dominant facultative anaerobic bacteria found in naturally fermented SFS samples were S. equorum and S. luteum, with the latter being identified in 10 of 30 bands from PCR-DGGE samples. Among the bacterial isolates identified by 16S rDNA sequence analysis after culture of SFS samples, S. equorum constituted the highest number of isolates. The results were in agreement to a report that shows Staphylococcus sp. to be dominant in Saeujeot, with more than 90% of strains showing similarity to S. equorum (Guan and others 2011). The presence of S. equorum throughout the 3–mo fermentation schedule of SFS indicates that the isolate may harbor a dominant niche in tiny shrimps. The dominance of Staphylococcus sp. in fish and other sea organisms may be common as these strains are halotolerant and facultative anaerobes. S. equorum is a member of the coagulase-negative staphylococcus group that is frequently reported in fermented sausages and surface-ripened cheeses (Hoppe-Seyler and others 2004; Talon and others 2008; Guan and others 2011). The 2nd most dominant bacteria were Halanaerobium saccharolyticus and Halomonas jeotgali. Halomonas sp. was recently isolated and conform to reported analyses of microflora from fermented foods (Jung and Park 2004; Kim and others 2009a, 2009b). Halanaerobium growth has been reported during the late period of Saeujeot fermentation and is considered a potential indicator of putrefaction or over-fermentation of seafood. In this study, Virgibacillus kekensis was dominant in SFS samples collected during late-fermentation period, followed by Alkalibacterium putridalgicola, Tetragenococcus muriaticus, and Salinococcus jeotgali. Species of the genus Virgibacillus have been shown to be prominent in myeolchi-jeot and environments with high-salt conditions (Yongsawatdigul and others 2007; Guan and others 2011). The halophilic lactic acid bacteria T. muriaticus have been reported from 2 samples of jeotgal, made from damselfish (Chromis notata) or silver-stripe round herring (Spratelloides gracilis), obtained from subtropical Jeju island of Korea (Kim and Park 2014). Also, the most abundant representation of S. luteum strain BY-5T is well

Microflora in fermented salted shrimp . . .

M: Food Microbiology & Safety

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Supporting Information Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. Table S1. Characteristics of Staphylococcus equorum isolated from salted and fermented shrimp.