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Selection of Mycotoxin Non-producing Aspergillus Strains and the Characteristics of GABA Enriched Doenjang (Soybean Paste)

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dc.contributor.advisor지근억-
dc.contributor.author김남연-
dc.date.accessioned2017-07-13T16:51:27Z-
dc.date.available2017-07-13T16:51:27Z-
dc.date.issued2015-02-
dc.identifier.other000000024866-
dc.identifier.urihttps://hdl.handle.net/10371/120328-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 식품영양학과, 2015. 2. 지근억.-
dc.description.abstractTo date, edible fungi such as Aspergillus flavus var. oryzae (A. oryzae) and black Aspergillus (Aspergillus niger aggregates) has been considered as safe. However, it has been recently reported that some strains have a mycotoxin biosynthetic capability. Based on this reason, a re-evaluation of the mycotoxin biosynthetic capability is required to determine the safety of edible fungi.

In the present study, comprehensive evaluation on the potential mycotoxin producing strains of Aspergillus strains which are frequently used in traditional Korean fermented foods was preformed. In return, mycotoxin non-producing Aspergillus strains were selected, which will contribute to the development of the fermented foods with enhanced safety levels.

Then, we have newly developed γ-aminobutyric acid (GABA) enriched fermented soybean paste by serial fermentation using mycotoxin non-producing A. oryzae FMB S46471 and GABA producing L. brevis GABA 100. Furthermore the soybean paste developed in present study included low salt contents as L. brevis GABA 100 could inhibit the growth of spoilage bacteria.

Furthermore the production of biogenic amines (BAs) as well as GABA in the fermented soybean pastes with enhanced functional benefits of GABA were characterized since concerns that the production of GABA may be accompanied by the increase in the production of BAs were raised.

In conclusion the present result showed that the newly developed fermented soybean paste using A. oryzae and L. brevis produced considerable levels of GABA whereas the level of BAs was relatively lower than the conventionally fermented soybean pastes. Taken together, this study will contribute to the development of the fermented foods with enhanced safety and provide useful information on the development of GABA enriched fermented foods.		-
dc.description.tableofcontentsContents
Abstract……………………………………………………………………...........i
Contents…………………………………………………….....................…...….iv
List of Tables………………………………………………………….......…......ix
List of Figures………………………………………………………..........……. x
List of Abbreviations…………………………………………………..........….xii

Chapter 1 Literature review..............................................................................…1
1.1 The production of mycotoxins……………..............…….……….............2
1.2 GABA production by lactic acid bacteria.…........................................…10
1.3 The production of biogenic amines...........................................................13

Chapter 2 An evaluation of aflatoxin and cyclopiazonic acid production in
Aspergillus oryzae ..............................................…….……..…………............…17
2.1 Introduction………………………………………………....……...........18
2.2 Materials and methods…………………………………….….......…......20
2.2.1 Fungal strains and cultures………………………………….............20
2.2.2 Fungal collection for DNA isolation ……………………….....…....23
2.2.3 PCR amplification …………………………….…………......…......23
2.2.4 Extraction of mycotoxins ……………………….……......…..........26
2.2.5 Detection of mycotoxins using HPLC methods…………….....…...27
2.2.6 ELISA ..........................................................……….....……........…28
2.3 Results……………………………………………………....……….......28
2.3.1 PCR analysis, HPLC analysis, and ELISA for the detection of
aflatoxin from Aspergillus strains ………..............................................…28
2.3.2 PCR and HPLC analyses for the detection of CPA from Aspergillus
strains..........................................................................................................32
2.4 Discussion ………………………..………………………….......….......38

Chapter 3 Reliable and simple detection of ochratoxin and fumonisin production in black Aspergillus ……………………………………..........……42
3.1 Introduction……………………………………………………..........….43
3.2 Materials and methods………………………………………….......…...44
3.2.1 Strains and growth conditions ……………………….........………..44
3.2.2 DNA isolation and PCR amplification of mycotoxin biosynthetic
genes...........................................................................................................47
3.2.3 HPLC analysis for ochratoxin A ……...............................……..…..48
3.2.4 HPLC analysis for fumonisins ……………..........…......………..…49
3.3 Results……………………………………………...…………............…50
3.3.1 PCR analysis for the detection of ochratoxin and fumonisin from A.
niger aggregates …………...................................................................…..51
3.3.2 HPLC analysis for the detection of ochratoxin and fumonisin from
A. niger aggregates......................................................................................53
3.4 Discussion………………………………………..…………….........…...57

Chapter 4 Characterization of the production of biogenic amines and γ-aminobutyric acid in the soybean pastes fermented by aflatoxin non-producing Aspergillus oryzae and γ-aminobutyric acid producing Lactobacillus brevis................................................................................................61
4.1 Introduction…………………...………………………….....….………..62
4.2 Materials and methods…………………………………..….....….…..….65
4.2.1 Fungal strains and cultures ……………………………....…...….…65
4.2.3 Viable cell counts of LAB and A. oryzae...........................................65
4.2.4 Small-scale cultivation conditions for an evaluation of GABA production by GABA producing LAB.…………................................…....66
4.2.5 The production of GABA rich soybean pastes in scaled-up fermentation and the preparation of soybean pastes for comparative analysis on the production of GABA and BAs …....................................................67
4.2.6 Extraction of aflatoxins from fermented soybeans............................68
4.2.7 Detection of aflatoxins using HPLC analysis ……………..........….69
4.2.8 Protease assay.………………………….................................……...69
4.2.9 Qualitative analysis of GABA and glutamic acid by TLC analysis...70
4.2.10 Quantitative analysis of free amino acids by HPLC analysis..........71
4.2.11 The analysis of BAs ……………………...............………..….…...72
4.3 Results…………………………………………………....……….....…..74
4.3.1 Characterization of soybean fermented by an aflatoxin non-producing
A. oryzae................................................................................................….74
4.3.2 Viable cell counts of LAB and fungus from the fermented
soybean .............................................................................................….…76
4.3.3 Evaluation of GABA production by GABA-producing LAB under
small-scale cultivation conditions ….....................................................….78
4.3.4 Optimization for the GABA production in scaled-up fermentation...81
4.3.5 Determination of GABA in the 12 kinds of fermented soybean …...86
4.3.6 Determination of BAs in the fermented soybean ……...............…...89
4.4 Discussion…………………………………………………………..…....93

Chapter 5 Conclusion………...…………….……………………………...........98
References.................………...…………….…………………………....….......102
Appendices ………………………………………………………..………....…123
Korean abstract…………………………………………………………...........128















List of Tables
Table 2.2.1 A. oryzae and A. flavus strains used in this study.................................21
Table 2.2.3 Primers used in this study ………………..............................….....….25
Table 2.3.2 Summary of PCR patterns and HPLC analysis for the detection of aflatoxin and CPA from A. oryzae and A. flavus strains..........................................35
Table 3.2.1 Aspergillus aggregates used in this study………….......…….….........45
Table 3.3.2 Summary of PCR patterns and HPLC analysis for the detection of ochratoxin and fumonisin from A. niger aggregates……………........…….……..55
Table 4.3.2 Viable cell counts of LAB and A. oryzae in the experimental fermented soybean pastes.…..........................................................................................….….77
Table 4.3.5 The determination of BAs and GABA in the experimental fermented soybean pastes...…………..................................................................................…87
Table 4.3.6 Characteristics in the experimental fermented soybean pastes ...........92
Table A.1 Library of aflatoxin non-producing Aspergillus oryzae strains............124
Table A.2 Library of ochratoxin/fumonisin non-producing Aspergillus niger aggregates..............................................................................................................126




List of Figures
Figure 1.1.1 Molecular structure of aflatoxins B1, B2, G1, and G2...........................3
Figure 1.1.2 Molecular structure of ochratoxin A ...................................................5
Figure 1.1.3 Molecular structures of fumonisin B1 and fumonisin B2.....................7
Figure 1.1.4 Structure of α-CPA..............................................................................9
Figure 1.2 Structure of γ-aminobutyric acid...........................................................12
Figure 1.3.1 Structure of histamine, tyramine, putrescine, and cadaverine............14
Figure 1.3.2 Biogenic amine biosynthesis pathways in bacteria............................16
Figure 2.3.1 Multiplex PCR analysis for the detection of aflatoxins from Aspergillus strains...................................................................................................30
Figure 2.3.2 PCR analysis for the detection of CPA from Aspergillus strains. ..…33
Figure 3.3.1 PCR analysis for the detection of ochratoxin and fumonisin from A. niger aggregates …………………….............................................................…....52
Figure 4.3.1 Protease activity in soybean fermentation...………..................…….75
Figure 4.3.3.1 Evaluation of GABA production by L. brevis GABA100……...…79
Figure 4.3.3.2 High-performance liquid chromatogram for GABA production depending on the inoculation of lactic acid bacteria.........................................…..80
Figure 4.3.4.1 Characterization of GABA production in scaled-up fermentation..82
Figure 4.3.4.2 Dynamic changes in the levels of GABA and glutamic acid...........83
Figure 4.3.4.3 GABA production in scaled-up fermentation .............................…85
Figure 4.3.5 GABA production in the experimental fermented soybean pastes.....88
Figure 4.3.6 BA production in the experimental fermented soybean pastes...........90		-
dc.formatapplication/pdf-
dc.format.extent80977682 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectAspergillus-
dc.subjectgamma-Aminobutyric Acid-
dc.subjectBiogenic Amine-
dc.subjectLactobacillus brevis-
dc.subjectMycotoxins-
dc.subjectSoybean Paste-
dc.subject.ddc641-
dc.titleSelection of Mycotoxin Non-producing Aspergillus Strains and the Characteristics of GABA Enriched Doenjang (Soybean Paste)-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pagesxii, 129-
dc.contributor.affiliation생활과학대학 식품영양학과-
dc.date.awarded2015-02-
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