Analysis of kimchi microflora using denaturing gradient gel electrophoresis

Abstract

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique was used to determine the microfloral composition during the fermentation of kimchi, a traditional Korean fermented vegetable food. The kimchi was fermented at 10 °C or 20 °C for 30 or 20 days, respectively. DGGE of the partially amplified 16S rDNA was performed and the most intense bands sequenced. The application of this culture-independent molecular technique determined that the lactic acid bacteria Weissella confusa, Leuconostoc citreum, Lactobacillus sakei, and Lactobacillus curvatus were the main microorganisms responsible for kimchi fermentation.

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.