Analyses of microbial communities and metabolites in Korean fermented soybean foods, meju and doenjang, and Bacillus subtilis pan-genome

Abstract

In this study, microbial communities and metabolites in Korean fermented soybean foods, meju and doenjang, were investigated and pan-genome analysis of Bacillus subtilis, known to play important roles during meju and doenjang fermentation, was also performed. In addition, I reviewed recent researches and functional properties of meju and doenjang in chapter 1.

In chapter 2, bacterial communities and biogenic amine contents in ten traditional and three commercial doenjang products were investigated. Analysis of bacterial community using pyrosequencing showed that the genera Bacillus, Pseudomonas, Enterococcus, and Staphylococcus were dominated, although the bacterial communities were different depending on the sample. Lactic acid bacteria (LAB) such as Weissella, Lactobacillus, Leuconostoc, and Tetragenococcus were also detected with relatively high abundance in some doenjang samples. Overall, the levels of biogenic amines in traditional doenjang were much higher than those in commercial doenjang. In particularly, the levels of biogenic amines in samples C (547.46 mg/kg), D (525.46 mg/kg), and F (524.19 mg/kg) exceeded the criterion recommended by FDA (500 mg/kg). A total of 432 strains were isolated from six traditional doenjang products showing three high- and tree low-histamine levels, and they were classified into seven genera and 30 species. Amylase, protease, and lipase activities of representative 13 species were tested on TSA additionally supplemented with 5% NaCl. Among them, strains belonging to B. siamensis and B. subtilis subsp. subtilis showed the highest enzyme activities for amylase, protease, and lipase. The features and safety of 12 strains belonging to B. subtilis, which is the prerequisite to use as a starter culture in Korea, was investigated. B. subtilis 10TDI13 was selected as a doenjang starter based on the results. These results suggest that the strains satisfied all requirement that can be used as appropriate starter candidates for doenjang fermentation with high quality and safety.

In chapter 3, B. subtilis represents the best characterized member of the Gram-positive bacteria. In this study, we analyzed the taxonomic status and molecular phenotypes using the whole genome sequences of 99 B. subtilis and its close relative strains including strain 10TDI13, which was isolated from Korean traditional doenjang, available in GenBank. Although B. subtilis that consists of three subspecies (B. subtilis subsp. subtilis, B. subtilis subsp. spizizenii, and B. subtilis subsp. inaquosorum) is not phenotypically distinguishable and shares very high 16S rRNA gene sequence similarities. However, average nucleotide identity (ANI) values showed that the 99 strains can be split into at least 8 different phylogenetic lineages. The phylogenetic tree based on 308 core gene showed that three (lineage VI, VII, and VIII) of these lineages belonged to B. atrophaeus, B. amyloliquefaciens, and B. velezensis, respectively. In addition, the rest five lineages shared low ANI values with other lineages, indicating that they should be reclassified into a different species from B. subtilis. The pan-genome analysis of 91 genomes belonging to B. subtilis was found unique genes exclusively presented in each lineage, which can be used as maker gene to distinguish each lineage. In conclusion, the pan genome data obtained in this study demonstrated that three B. subtilis subspecies (subtilis, inaquosorum, and spizizenii) are clearly distinguished, supported that these five lineages could be classified as separate species.

In chapter 4, I prepared meju samples using a Korean traditional method and inoculated A. oryzae SNU-HR, an aflatoxin- and CPA-non-producer strain isolated from industrial koji, and then compared their qualities and functionalities. Bacteria such as Bacillus, unclassified Bacillales, Staphylococcus, Leuconostoc, and Weissella, were mainly detected and fungal community of doenjang samples included Aspergillus, unclassified Microascaceae, Eurotium, Gibberella, and Mucor. The community analysis revealed that Bacillus and Aspergillus were predominant in meju regardless of the use of starter. The meju inoculated with A. oryzae SNU-HR showed difference in pH, water contents, bacterial and fungal communities, and metabolites. The growth of facultative anaerobic bacteria in early fermentation period may affected to the pH decrease and the decline of bacteria using organic acids led to that the pH of TMA is lower than TMC. The addition of A. oryzae SNU-HR led to increase the concentration of most amino acids including alanine, asparagine, aspartate, glutamate, glutamine, and glycine. This study suggests that the use of A. oryzae SNU-HR can reduce the fermentation time by producing metabolites within comparably short times and produce safe and high-quality doenjang.