Analyses of microbial communities and metabolites in Korean fermented soybean foods, meju and doenjang, and Bacillus subtilis pan-genome
한국의 전통 콩 발효식품인 메주와 된장의 미생물 군집 및 대사체 분석과 Bacillus subtilis 판지놈 분석 연구

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 최상호.-
dc.description.abstractIn 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.
dc.description.tableofcontentsChapter I. Literature Review 1
I-1. Meju 2
I-2. Doenjang 6
I-3. Health benefits of doenjang 10
References 17

Chapter II. A survey of traditional- and commercial-doenjang products focusing on bacterial communities and biogenic amines 22
II-1. Introduction 23
II-2. Materials and Methods 26
II-2-1. Doenjang sample collection and measurement of pH and titratable acidity 26
II-2-2. Genomic DNA extraction and pyrosequencing 27
II-2-3. Isolation and identification of isolates strains from doenjang 30
II-2-4. Analysis of biogenic amine level of doenjang and Statistical analysis 31
II-2-5. Evaluation of protease, amylase, and lipase activities 33
II-2-6. Fibrinolytic activity 34
II-2-7. Antibiotic susceptibility test 35
II-2-8. Evaluation of the potential production of enterotoxin of isolated strains 36
II-2-9. Cytotoxicity assays 38
II-2-10. Quantitative evaluation of anti-pathogenic activities 38
II-2-11. Quantitative evaluation of biogenic amines production of isolated strains 39
II-3. Results and Discussion 40
II-3-1. General features of doenjang samples 40
II-3-2. Bacterial communities of doenjang samples 42
II-3-3. Biogenic amine contents of doenjang samples 48
II-3-4. Structure of cultural bacterial communities isolated from traditional doenjang 53
II-3-5. Evaluation of protease, amylase, and lipase activities 59
II-3-6. Evaluation of essential and toxic elements of doenjang 62
II-3-7. Quantitative assay of the enzyme activities of B. subtilis strains 65
II-3-8. Detection of enterotoxin genes and cytotoxicity activity 68
II-3-9. Antibiotic susceptibility 70
II-3-10. Quantitative evaluation of the anti-pathogenic activities 73
II-3-11. Biogenic amine production by 12 B. subtilis strains 75
II-4. Conclusions 78
References 80

Chapter III. Comprehensive phylogeny and metabolic functions of Bacillus subtilis related strains using pan-genome analysis 86
III-1. Introduction 87
III-2. Materials and Methods 89
III-2-1. Bacterial strains and DNA isolation 89
III-2-2. Whole genome sequencing, de novo assembly and annotation 89
III-2-3. Collection of genomic data and quality assessment 95
III-2-4. Relatedness analysis based on average nucleotide identity (ANI) and in silico DNA-DNA hybridization values 95
III-2-5. Phylogenetic analysis based on 16S rRNA and whole genomes 96
III-2-6. COG protein function analysis and KEGG pathway analysis 97
III-2-7. Data deposition 98
III-3. Results and Discussion 99
III-3-1. Genome sequencing and general features of B. subtilis 99
III-3-2. Phylogenetic analysis based on 16S rRNA 101
III-3-3. Relatedness based on ANI and in silico DDH 103
III-3-4. Phylogenetic analysis based on whole genomes 109
III-3-5. The pan- and core-genomes analysis 113
III-3-6. Ratio (%) of genes assigned in each COG category 122
III-3-7. Metabolic and regulatory pathways in B. subtilis 125
References 129

Chapter IV. Effects of Aspergillus oryzae SNU-HR on microbial communities and metabolites during meju fermentation 133
IV-1. Introduction 134
IV-2. Materials and Methods 138
IV-2-1. Preparation of starter culture 138
IV-2-2. Preparation of meju samples and sampling 138
IV-2-3. pH, water content, and bacterial counting 139
IV-2-4. Quantitative PCR to estimate the number of bacteria and fungi 140
IV-2-5. Evaluation of protease, amylase activities 141
IV-2-6. Genomic DNA extraction and pyrosequencing 142
IV-2-7. Sequencing processing and data analysis 145
IV-2-8. Statistical analysis 146
IV-2-9. Metabolite analysis 146
IV-3. Results 148
IV-3-1. Changes in pH and water content 148
IV-3-2. Bacterial and fungal abundance during meju fermentatio 151
IV-3-3. Changes in amylase and protease activities meju fermentation 152
IV-3-4. Changes in bacterial and fungal diversity during the meju fermentation 155
IV-3-5. Changes in bacterial and fungal communities during the meju fermentation 161
IV-3-6. Multivarite statistical analysis 169
IV-3-7. Metabolite changes during the meju fermentation 174
IV-4. Discussion 179
References 185
국문초록 191
dc.format.extent3623651 bytes-
dc.publisher서울대학교 대학원-
dc.subjectBacillus subtilis-
dc.subjectAspergillus oryzae-
dc.subjectbacterial community-
dc.titleAnalyses of microbial communities and metabolites in Korean fermented soybean foods, meju and doenjang, and Bacillus subtilis pan-genome-
dc.title.alternative한국의 전통 콩 발효식품인 메주와 된장의 미생물 군집 및 대사체 분석과 Bacillus subtilis 판지놈 분석 연구-
dc.contributor.AlternativeAuthorHye Rim Kim-
dc.citation.pagesvi, 197-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
Appears in Collections:
College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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