J. Gen. Appl. Microbiol., 60, 227‒233 (2014) doi 10.2323/jgam.60.227 ©2014 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Full Paper Pyrosequencing-based analysis of the bacterial community during fermentation of Alaska pollock sikhae: traditional Korean seafood (Received July 2, 2014; Accepted August 13, 2014) Hyo Jin Kim,1 Min-Jeong Kim,1 Timothy Lee Turner,2,3 and Myung-Ki Lee1,* 1 Fermentation Research Center, Korea Food Research Institute, Gyeonggi-Do, Seongnam-Si 463‒746, Republic of Korea 2 Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA 3 Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, 1206 West Gregory Dr., Urbana, Illinois 61801, USA fermented foods were designed for long-term storage, to We analyzed the bacterial community of Alaska supplement various nutrients, and to provide featured pollock sikhae, a traditional Korean food made by flavors. Jeotgal and sikhae are both traditional fermented natural fermentation with Alaska pollock, utilizing seafoods made for long-term storage (Rhee et al., 2011). pyrosequencing. We fermented the Alaska pollock While jeotgal contains a relatively high concentration of salt sikhae at two different temperatures (10°C and [generally 20‒30% (w/w)], sikhae uses a low concentration 20°C). Before fermentations, the bacterial commu- of salt [＜7%] (Cha et al., 2004; Jung et al., 2013a, b; Guan nity was varied. After fermentations, however, Lac- et al., 2011; Lee et al., 2014). In contrast to jeotgal, sikhae tobacillus sakei became dominant. The Alaska pol- contains grains that provide abundant carbon sources to lock sikhae sample before fermentations contained microbes. Gajami-sikhae, Alaska pollock sikhae, and squid only 2% L. sakei, but the sample on day 6 of fermen- sikhae are the three most popular Korean sikhae. In addition tation at 10°C comprised 74% L. sakei (90% at to salt and fish (Alaska pollock), Alaska pollock sikhae is 20°C). In addition, we observed a reduction in the composed of cooked grains, white radish, red pepper powder, composition of unpreferred bacterial species for malt, garlic, and ginger. Alaska pollock sikhae is mainly foods after fermentation. The composition of unpre- divided into Kangwondo- and Kyoungsangdo-style recipes. ferred bacterial species was more than 30% of total While Kyoungsangdo-style recipes use dried Alaska pollock, reads in samples before fermentation and decreased undried fish are utilized for the Kangwondo-style recipes. to less than 0.2% after fermentation. This result Despite its importance as a Korean fermented food, there is suggested that the fermentation of Alaska pollock limited research regarding sikhae and our understanding of sikhae can be beneficial for food safety. Alaska pol- the fermentation mechanisms of sikhae is largely incomplete. lock sikhae might be a favorable habitat for L. sakei. Novel bacterial strains were isolated from sikhae, mainly Our study is the first report illustrating the altera- from gajami-sikhae (Kim et al., 2009; Jung et al., 2012a; tion of the bacterial community of Alaska pollock Shin et al., 2011; Park et al., 2013, 2014). Alishewanella sikhae during fermentation utilizing pyrosequenc- jeotgali MS1T was isolated from gajami-sikhae and its ing analysis. genome was fully sequenced (Kim et al., 2009; Jung et al., 2012a). Shin and co-workers isolated Leutobacter celer Key words: Alaska pollock sikhae; bacterial com- NAL101T from gajami-sikhae (Shin et al., 2011). They munity analysis; Lactobacillus sakei; pyrosequenc- suggested that the gajami-sikhae habitat might not be the ing analysis optimal growth condition for the NAL101T strain (Shin et al., 2011). Park and colleagues isolated Lactobacillus planta- rum LG42 from gajami-sikhae and showed its antiobesity Introduction effect on high-fat-diet-induced obese mice (Park et al., 2013, 2014). Recently, we characterized the bacterial community Numerous fermented foods are popular in Korea. Korean of eight gajami-sikhae products and isolated beneficial ＊Corresponding author: Dr. Myung-Ki Lee, Fermentation Research Center, Korea Food Research Institute, Gyeonggi-Do, Seongnam-Si 463‒746, Republic of Korea. E-mail: [email protected] None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. 228 KIM et al. strains (Kim et al., 2014). The study indicated that the lactic adjusted to equimolar concentrations and sequencing was acid bacteria were dominant in the bacterial community of executed on a 454 GS Junior system (Roche, Basel, Switzer- the gajami-sikhae products. land) following the manufacturer’s instructions. Novel strains isolated from various fermented foods had Processing of sequenced data from the microbial community been characterized and their functionality as probiotics had in the Alaska pollock sikhae samples using pyrosequencing. also been investigated (Abbasiliasi et al., 2012; Shin et al., Sequences collected by pyrosequencing were sorted by a 2008; Wu et al., 2009; Chang et al., 2010; Park et al., 2013). unique barcode for each sikhae sample. Among the sorted Although research on an individual strain from a specific sequences, sequences less than the 300 bp read length or habitat is important, understanding the microbial communi- sequences whose average quality score was less than 25 bp ty present in the environment also provides valuable were removed. In addition, sequences were further edited by information. To estimate the microbial community composi- primer sequence trimming utilizing pairwise alignment and tion of the specific environment, numerous tools have been sequencing error correction. We employed the EzTaxon employed. Among them, next-generation sequencing (NGS) extended database for sequence identification by searching technologies such as pyrosequencing have gained attention the highest similarity among the top five hits of the BLASTN for microbial community analysis as tools that can overcome algorithm (Chun et al., 2007). UCHIME software was also issues in the conventional methods, such as limitations of employed for chimeric sequences and non-16S rRNA gene collecting information, fastidious culturability of some sequences that were further removed (Edgar et al., 2011). An microbes, and laborious processes (Lee et al., 2012; Jeong et operational taxonomic unit (OTU) was obtained by CD-HIT al., 2013; Mardis, 2008). In fact, numerous studies analyzing software with a 97% sequence identity cutoff (Li and the microbial community compositions in traditional Korean Godzik, 2006). foods have been performed utilizing pyrosequencing (Jung Statistical analysis to determine the richness and diversity et al., 2011, 2012b, 2013a, b; Nam et al., 2012b; Jeong et al., of species of each Alaska pollock sikhae sample. We 2013; Lee et al., 2014; Nam et al., 2012a; Mardis, 2008). performed statistical analysis using the Chao1 richness Our previous research revealed that Lactobacillus sakei index and Shannon diversity index with MOTHUR software was dominant in the gajami-sikhae microbiota (Kim et al., (Schloss et al., 2009). We obtained the Chao1 richness index 2014). In this study, we conducted pyrosequencing analysis and Shannon diversity index to estimate the richness and of the bacterial community in Alaska pollock sikhae made diversity of species of the different Alaska pollock sikhae with a Kangwondo-style recipe. Alteration of the bacterial samples. Fast UniFrac distance utilizing CLcommunityTM community compositions at two different temperatures software (ChunLab, Inc., Seoul, Korea) was used for princi- consistently indicated that L. sakei became prominently pal coordinate analysis (PCoA) to determine the differences dominant among the varied bacterial community. This result in the richness and diversity of bacterial communities among suggested that L. sakei might have the capability to suppress sikhae samples (Hamady et al., 2010). Pyrosequecing reads other species in the bacterial community or utilize nutrients reported in this study have been deposited in the EMBL of sikhae better than other bacterial species present. Sequence Read Archive database under the project accession number PRJEB6648 (http://www.ebi.ac.uk/ena/data/view/ Materials and Methods PRJEB6648). Sample preparation and fermentation of Alaska pollock Results sikhae. We prepared Alaska pollock sikhae following a Kangwondo-style recipe. Firstly, 5 kg of Alaska pollock Characteristics of the sequenced data for Alaska pollock fillet were completely washed with 3% salt water. The sikhae samples utilizing pyrosequencing analysis washed fish fillet was chopped and mixed with 1.05 kg of Alterations in the bacterial community compositions of cooked millet, 2.72 kg of shredded radish, 800 g of red Alaska pollock sikhae during the fermentation under differ- pepper powder, 10 g of green onion, 8 g of garlic, 5 g of ent temperatures (10°C and 20°C) were monitored by ginger, 1 g of salt, and 21 g of malt. We collected samples for high-throughput pyrosequencing of the partial 16S rRNA the bacterial community analysis and fermented the Alaska gene. The sequence length of the Alaska pollock sikhae pollock sikhae for further analysis under two different samples was distributed between 300 and 530 bp (Fig. 1). temperature conditions (10°C and 20°C). We collected The total reads for each Alaska pollock sample ranged from samples of the two different temperature conditions