Analysis of yeast and archaeal population dynamics in kimchi using denaturing gradient gel electrophoresis
Introduction
Fermentation is a well-known and ancient technique that uses microorganisms to process and preserve food (Ross et al., 2002). Kimchi is a Korean food prepared as a result of fermentation and in recent years it has been recognized as a health-promoting functional food (Song, 2004). Since it is interesting to know what microbes are involved by kimchi fermentation, the fermentation process has been studied extensively by microbiologists with respect to its ecology, proteomics, genetics and physiology (Kim and Chun, 2005, Li et al., 2006, Nan et al., 2005). These studies have reported that kimchi is a healthy food, rich in β-carotene, chlorophylls, dietary fiber and various minerals (Park et al., 2003). It supports a wide range of microorganisms, including lactic acid bacteria (LAB), which perform significant roles during fermentation (Bae et al., 2005, Kim and Chun, 2005). Bacterial isolates from kimchi have been investigated with respect to production and characterization of beneficial enzymes such as dextransucrase and alcohol/acetaldehyde dehydrogenase (Eom et al., 2007, Koo et al., 2005), and for biodegradation of toxic compounds such as sodium nitrite and bisphenol (Oh et al., 2004, Yamanaka et al., 2007).
Several halophilic archaea have been isolated from jeotgal, traditional Korean fermented seafood used in kimchi as an ingredient (Roh et al., 2007a, Roh et al., 2007b). Since the average NaCl concentration of kimchi is 3% (Mheen and Kwon, 1984), we reasoned that it could support haloarchaea. Yeasts such as Pichia spp. have been isolated from kimchi at low pH (about pH 4) that have been fermented over a long period of time (Oh and Han, 2003). However, the overall molecular diversity of yeasts and archaea in kimchi has not yet been reported.
Molecular biology techniques are used frequently to explore the diversity and structure of microbial communities, and microorganisms are identified using certain molecular markers such as 16S or 26S rRNA. In particular, the study of microbial ecology has benefited greatly from the introduction of denaturing gradient gel electrophoresis (DGGE), which provided a molecular fingerprinting technique for studying community structure (Yeates et al., 1998). In DGGE analysis, PCR amplicons of the same size but different sequences can be separated (Muyzer and Smalla, 1998) and this technique has been applied widely for studying microbial dynamics in complex environments such as soil (Sharma et al., 2006), sea (Bowman et al., 2003), insects (Reeson et al., 2003), sludge (Xia et al., 2005) and permafrost-affected soils (Ganzert et al., 2007). This method has also been used to investigate yeast diversity in foods such as wine (Cocolin et al., 2000a), sausage (Rantsiou et al., 2005), sourdough (Meroth et al., 2003) and coffee (Masoud et al., 2004).
In the present work, we studied population dynamics in the microbial community (bacteria, archaea and yeast) during fermentation of kimchi. We compared communities from various types of kimchi using culture-independent DGGE, which analyze 16S and 26S rRNA gene markers. For the best of our knowledge, this is the first report to reveal the diversity of archaea and yeast in kimchi analyzed by DGGE.
Section snippets
Kimchi sampling
Kimchi was obtained from the distributors of commercially-available brands. Samples were taken immediately after production in the factory and then stored at 4 °C during the sampling period. Population dynamics were monitored during fermentation using a cabbage kimchi purchased from the ‘C’ corporation (designated P kimchi). P kimchi is the best-selling kimchi brand and the most representative kimchi in Korea. It is processed with a variety of ingredients such as cabbage, red pepper powder,
Change in the microbial community during fermentation of kimchi
The pH change during fermentation of P kimchi is shown in Fig. 1. The DGGE banding patterns for bacteria, archaea and yeasts, were determined for each pH phase of kimchi fermentation (Fig. 2). pH decreased between P3 and P9, ranging between pH 5.3 and 4.1. The DGGE banding patterns of bacteria changed significantly between P3 and P4, but remained unchanged after P4 (Fig. 2A). Bands B1 to B5, which corresponded to two Bacillus and three eukaryotes, were not observed after P4, at which point the
Discussion
In general, the major ingredient of kimchi is Chinese cabbage and does not require the use of a starter culture. It is ripened by lactic fermentation and alcohol fermentation, a process performed primarily by LAB at low temperatures. Its main ingredients are vegetables such as cabbage or radish, and it can include additional ingredients such as onion, garlic, ginger and pepper. Currently, kimchi is in the health food spotlight because it is rich in nutrients and is recognized as a
Acknowledgements
This work was supported by the KRIBB Research Initiative Program, the Environmental Biotechnology National Core Research Center Program (KOSEF: R15-2003-012-02002-0) and the Conservation Technology Research and Development project hosted by the National Research Institute of Cultural Heritage (of the Cultural Heritage Administration). The first author was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund)
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