Microbial Ecology of Watery Kimchi Kyu Hang Kyung, Eduardo Medina Pradas, Song Gun Kim, Yong Jae Lee, Kyong Ho Kim, Jin Joo Choi, Joo Hyong Cho, Chang Ho Chung, Rodolphe Barrangou, and Frederick Breidt Abstract: The biochemistry and microbial ecology of 2 similar types of watery (mul) kimchi, containing sliced and unsliced radish and vegetables (nabak and dongchimi, respectively), were investigated. Samples from kimchi were fermented at 4, 10, and 20 °C were analyzed by plating on differential and selective media, high-performance liquid chromatography, and high-throughput DNA sequencing of 16S rDNA. Nabak kimchi showed similar trends as dongchimi, with increasing lactic and acetic acids and decreasing pH for each temperature, but differences in microbiota were apparent. Interestingly, bacteria from the Proteobacterium phylum, including Enterobacteriaceae, decreased more rapidly during fermentation at 4 °Cinnabak cabbage fermentations compared with dongchimi. Although changes for Proteobacterium and Enterobacteriaceae populations were similar during fermentation at 10 and 20 °C, the homolactic stage of fermentation did not develop for the 4 and 10 °C samples of both nabak and dongchimi during the experiment. These data show the differences in biochemistry and microbial ecology that can result from preparation method and fermentation conditions of the kimchi, which may impact safety (Enterobacteriaceae populations may include pathogenic bacteria) and quality (homolactic fermentation can be undesirable, if too much acid is produced) of the product. In addition, the data also illustrate the need for improved methods for identifying and differentiating closely related lactic acid bacteria species using high-throughput sequencing methods. Keywords: high-throughput sequencing, microbial ecology, watery kimchi Practical Implication: This research may aid the understanding of how processing conditions for fermented vegetable products may affect microbiota and product quality. & Safety Introduction cabbage as the main vegetable ingredient), dongchimi (with whole M: Food Microbiology There are many different kinds (perhaps hundreds) of fermented or quartered radish), and nabak kimchi (with thinly sliced radish). vegetable kimchi, a traditional food of Korea, which can be A variety of other vegetable ingredients may also be included in roughly classified into 2 groups based on whether or not brine mul kimchi as minor constituents. was added to the fermentation (Cheigh and Park 1994). These The changes in microbial populations during baechu kimchi and fermentations are typically prepared with flavoring ingredients in- dongchimi fermentation have been documented by isolate-based cluded and do not require further processing or desalting before and high-throughput DNA sequencing methods (Cheigh and Park consumption. Typically, salt concentrations of 2% to 3% sodium 1994; Fleming and others 1995; Park and others 2009; Jeong and chloride (equilibrated) are used for fermentation. A common in- others 2013; Jung and others 2014). It is evident that the rate of gredient in many types of kimchi is Chinese cabbage (Brassica reduction in pH, biochemistry, and microbial populations are de- compestries) used in traditional chopped baechu or whole cabbage pendent on temperature (Mheen and Kwon 1984; Lee and others (tongbaechu) kimchi. Other common types of kimchi include radish 2005; Cho and others 2006; Park and others 2008). A study of (Raphanus spp.) kimchi varieties, including kakdugi (cubed) and baechu kimchi fermentation isolates using 16S rDNA sequencing yeolmoo (whole small radishes) kimchi, and others. Watery kimchi has shown that Leuconostoc spp. and Weissella spp. predominated (mul kimchi) is fermented with water (or salt brine) added to the at 10 and 15 °C during the initial stage of fermentation, with vegetables, to typically exceed 2 or more times the volume of the Leuconostoc gasicomitatum and Leuconostoc citrium predominating dur- vegetables. Varieties mul kimchi include biak kimchi (with baechu ing the first 4 d of fermentation at 15 °C (Jeong and others 2013). At 10 °Corcolder,Weissella koreensis was found to be the dom- inant species, with fermentation occurring by this organism at MS 20141519 Submitted 9/11/2014, Accepted 2/17/2015. Author Kyung is − ° with Dept. of Food Science, Sejong Univ, 98 Gunja-dong, Gwangjin-gu, Seoul, temperatures as low as 1 C (Jeong and others 2013). 143-747, South Korea. Author Pradas is with Inst. de la Grasa (CSIC), Av. Padre A microbial ecology study of dongchimi at 5 and 25 °Cusing Garcia Tejero 4, 41012 Seville, Spain. Authors Kim and Lee are with Microbial Re- culture based and denaturing gradient gel electrophoresis methods source Center/KCTC, Korea Research Inst. of Bioscience and Biotechnology, Daejeon, showed discrepancies between the 2 methods, but isolates showed 305-806, Republic of Korea. Author Kim is with Dept. of Microbiology, Pukyong similar species at 5 and 25 °C, with Leuconostoc mesenteroides as Natl. Univ., Pusan 608-737, South Korea. Authors Choi, Cho and Chung are with Dept. of Culinary Science and Food Service Management, Sejong Univ., 98 the dominant organism during the first 3 to 7 d of fermentation Gunja-dong, Gwangjin-gu, Seoul, 143-747, South Korea. Authors Pradas and Bar- (Park and others 2008). A more rapid decline in pH and increase rangou are with Dept. of Food, Bioprocessing and Nutrition Sciences, 400 Dan Allen in lactic acid bacterial populations were seen at 25 °C compared Drive, North Carolina State Univ., Raleigh, NC 27695-7624, U.S.A. Authors to 5 °C. A study of the evolution of microbial populations during Breidt are with USDA-ARS, SAA Food Science Research Unit, 322 Schaub Hall, ° Box 7624, North Carolina State Univ., Raleigh, NC 27695-7624, U.S.A. Direct dongchimi fermentation at 4 C for 90 d using 454 sequencing inquiries to author Breidt (E-mail: [email protected]). technology showed that Leuconostoc species predominated during fermentation (Jeong and others 2013). A variety of Leuconostoc C 2015 Institute of Food Technologists R . This article has been contributed by US Government employees and their work is in the public domain in the USA. r doi: 10.1111/1750-3841.12848 Vol. 80, Nr. 5, 2015 Journal of Food Science M1031 Further reproduction without permission is prohibited Watery kimchi (Nabak and Dongchimi) ecology . species and Weissella were evident during the first 3 d of fermen- Table 1–Experimental design and sequence data. tation; however, 2 species, Le. gasicomitatum and Le. gelidum,were Sample Type Temp (°C) Time (d) No. Readsa the predominant species for the remainder of the 90 d sampling period. A study of the ecology of nabak kimchi using selective C0000 Sliced, Nabak NAb 0 6515 C0407 Sliced 4 7 6628 media showed changes in the number of lactic acid bacteria C0414 Sliced 4 14 NDc (LAB) isolates of Le. spp. and Lactobacilli spp. (Kong and others C0421 Sliced 4 21 4696 2005). Both groups had high numbers (106-7 CFU/mL) after the C0430 Sliced 4 30 4189 initiation of fermentation. Le. spp. were able to grow slowly at C1003 Sliced 10 3 ND 5 °CwhereasLactobacilli did not. The growth rates of these species C1007 Sliced 10 7 6895 ° C1014 Sliced 10 14 7332 were proportional to temperature, increasing at 10 and 20 C, but C1021 Sliced 10 21 8235 their growth rate decreased when acid levels increased. C2001 Sliced 20 1 5782 In this study, a survey of the microbial ecology of mul kimchi C2003 Sliced 20 3 4306 was conducted with sliced and unsliced (nabak and dongchimi)at C2005 Sliced 20 5 6005 ° C2007 Sliced 20 7 ND 10, 20, and 30 C to determine how changes in radish prepara- N0000 Unsliced, Dongchimi NA 0 2396 tion and fermentation temperature affected both the biochemistry N0407 Unsliced 4 7 8502 and microbiota. Although quality factors were not directly inves- N0414 Unsliced 4 14 8748 tigated in this study, our work represents an important initial step N0421 Unsliced 4 21 3305 for determining how processing conditions (slicing, fermentation N0430 Unsliced 4 30 5183 N1003 Unsliced 10 3 4792 temperature) may influence the chemistry and microbiota. N1007 Unsliced 10 7 8827 N1014 Unsliced 10 14 3828 Materials and Methods N1021 Unsliced 10 21 4354 N2001 Unsliced 20 1 6112 Preparation of watery kimchi and sampling N1003 Unsliced 20 3 3320 Laboratory-scale batches of mul kimchi were prepared with N2005 Unsliced 20 5 4283 M: Food Microbiology N2007 Unsliced 20 7 5868 3.6-L glass containers with lids. Ingredients for watery kimchi aNo. Reads, number of DNA sequences used for analysis. used in this study were purchased from a local market in Seoul, b & Safety NA, not applicable, fresh cabbage sample. South Korea, in 2013. Each batch of watery kimchi was prepared cND, not determined. with 1.5 kg of distilled water, 1 kg of radish, 50.36 g of salt, 10 g of green onion, 5 g of garlic, and 3 g of ginger. Radish (17 to 23 cm in length and with a diameter of 8 to 10 cm) was washed with water, eluent, and a flow rate of 0.5 mL/min. Protonated organic acids trimmed, and cut into quarters along the long axis for dongchimi, were calculated based on the pH and acid concentration data using or further sliced into about 0.5- to 1-cm-thick thin pieces for the Henderso–Hasselbalch equation, based on pKa values of 3.86 nabak kimchi. For both dongchimi and nabak kimchi preparations, and 4.76 for lactic and acetic acids, respectively. green onion, garlic, and ginger were added. Prepared materials were placed in jars, which were placed at 4, 10, and 20 °C, as Bacterial 16S rDNA gene amplification and indicated in Table 1. Brine samples (10 mL) were collected and pyrosequencing processed for traditional microbiological methods, then stored at Ten milliliters of mul-kimchi brine, including solid particles, −70 °C before biochemical analysis and microbial DNA sampling. were filtered using 0.2-µm filter paper.