Analysis of kimchi microflora using denaturing gradient gel electrophoresis
Introduction
Kimchi is a generic term used to denote a group of fermented vegetable foods in Korea. The flavor of kimchi is dependent on the ingredients, fermentation conditions, and lactic acid bacteria (LAB) involved in the fermentation process (Mheen and Kwon, 1984, Lee et al., 1992, Cheigh and Park, 1994). To demonstrate the role of LAB in kimchi fermentation, it is essential to investigate the dynamics of the whole bacterial community. Many LAB have already been isolated from kimchi and systematic taxonomic studies on these bacteria have also been reported. In the 1990s, Lee et al. published taxonomic studies on the LAB isolated from kimchi using an analysis of the cellular fatty acids (Lee et al., 1996a, Lee et al., 1996b) and carbon source utilization patterns (Lee et al., 1997a, Lee et al., 1997b). However, the results obtained with these different methods of analysis are not directly comparable.
The isolation of DNA and/or RNA from microflora in complex environments has been described (Woese, 1987, Amann et al., 1995, Pace, 1997, Hugenholtz et al., 1998). Due to the known limitations of cultivation methods, many recent studies have used the culture-independent 16S rDNA-based PCR technique to determine the diversity and dynamics of the microflora in foods, such as pozol (Ampe et al., 1999, Omar and Ampe, 2000), cassava (Ampe et al., 2001), malt whisky (Beek and Prist, 2002) and Italian sausage (Cocolin et al., 2000, Cocolin et al., 2001). Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons has also been shown to be a suitable tool for the analysis of microbial communities, as it allows the rapid detection of species and changes in the compositions of microflora (Muyzer and Smalia, 1998, Water et al., 2000). However, little is yet known about the diversity and dynamics of the microbial communities present during kimchi fermentation. Therefore, the current study used a molecular approach, combining PCR-amplification of the V3 region of the 16S rDNA gene and DGGE, to monitor the dynamics of the microbial communities involved in the fermentation of kimchi.
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Preparation of kimchi
Chinese cabbages were trimmed, washed, cut into 5 cm lengths, soaked in a 10% (w/v) salt solution for 3 h at room temperature, and then rinsed with water to reduce the salt concentration to 2%. After draining the excess water from the salted Chinese cabbages, kimchi was prepared with the following ratio of ingredients: Chinese cabbage 100 g, sugar 1 g, green onion 4 g, garlic 2 g, ginger 1 g, red pepper powder 2 g, and fermented anchovy sauce 1.4 g. Fifty 250-ml glass bottles, each containing
pH
The pH of the kimchi decreased during fermentation. At 10 and 20 °C the pH started at 5.4, then decreased to 3.7 and 3.2 after 20 and 14 days of fermentation, respectively, and remained at these levels for the remainder of the process.
Community fingerprinting using DGGE
The fingerprints obtained from the kimchi preparations during the course of the fermentation revealed 12 and 10 visible bands for the kimchi fermented at 10 and 20 °C, respectively (Fig. 1). The fingerprints of the independently duplicated kimchi preparations
Discussion
It is well established that LAB are important microorganisms in the kimchi fermentation process (Mheen and Kwon, 1984, Lee et al., 1992, Cheigh and Park, 1994). There have been previous analyses of the composition and population dynamics of the microbial communities involved in kimchi fermentation. However, all such studies have been based on the random isolation of LAB using LAB culture media, such as MRS agar, and identification using phenotypic characteristics and polyphasic methods.
The
Acknowledgements
This work was partially supported by the 21C Frontier Microbial Genomics and Application Center Program (MG02-0302-006-2-1-0) and KRIBB Research Initiative Program, Korea.
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