• 한국균학회소식:학술대회논문집
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• 한국균학회 2006년도 추계학술대회 및 정기총회
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• pp.32-44
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• 2006

To investigate non-aflatoxin-production of A. oryzae at the molecular level, an aflatoxin biosynthesis gene homolog cluster of RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99 % similarity to those of Aspergillus flavus, three genes shared 93 % similarity or less. In addition, although slight expression of aflR, positive transcriptional regulator gene, was detected in some A. oryzae strains having seven aflatoxin biosynthesis homologous genes, other genes related to aflatoxin production were not detected. RIB strains were mainly divided into group 1, having seven aflatoxin biosynthesis homologous genes (aflT, nor-i, aflR, norA, avnA, verB, and vbs), and group 2, having three homologous (avnA, verB, and vbs). Partial aflatoxin homologous gene cluster of RIB62 from group 2 was sequenced and compared with that of RIB40 from group 1. RIB62 showed a large deletion upstream of ver-1 with more than half of the aflatoxin homologous gene cluster missing including aflR, a positive transcriptional regulatory gene. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a unique sequence of about 8kb and a telomere. Southern analysis of A. oryzae RIB strains with four kinds of probe derived from the unique sequence of RIB62 showed that all group 2 strains have identical hybridizing signals. Polymerase chain reaction with specific primer set designed to amplify the junction between ver-1 and the unique sequence of RIB62 resulted in the same size of DNA fragment only from group 2 strains. Based on these results, we developed a useful genetic tool that distinguishes A. oryzae group 2 strains from the other groups' strains and propose that it might have differentiated from the ancestral strains due to chromosomal breakage.

• Mycobiology
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• 제50권6호
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• pp.408-419
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• 2022

Filamentous fungi that could be classified into Aspergillus flavus/oryzae were isolated from traditionally fermented meju commercially available in Korea. The samples were analyzed for aflatoxin B1 and ochratoxin A contamination by HPLC; however, no toxin was detected. In addition, fungal and bacterial metagenomic sequencing were performed to analyze the microbial distribution in the samples. The results revealed that the distribution and abundance of fungi and bacteria differed considerably depending on the production regions and fermentation conditions of the meju samples. Through morphological analysis, ITS region sequencing, and assessment of the aflatoxin-producing ability, a total of 32 A. flavus/oryzae strains were identified. PCR analysis of six regions with a high mutation frequency in the aflatoxin gene cluster (AGC) revealed a total of six types of AGC breaking point patterns. The A. flavus/oryzae strains did not exhibit the high amylase activity detected in the commercial yellow koji strain (starter mold). However, their peptidase and lipase activities were generally higher than that of the koji isolates. We verified the safety of the traditionally fermented meju samples by analyzing the AGC breaking point pattern and the enzyme activities of A. flavus/oryzae strains isolated from the samples. The isolated strains could possibly be used as starter molds for soybean fermentation.

• Journal of Microbiology and Biotechnology
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• 제24권10호
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• pp.1397-1404
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• 2014

A few starters have been developed and used for doenjang fermentation but often without safety evaluation. Filamentous fungi were isolated from industrial doenjang koji, and their potential for mycotoxin production was evaluated. Two fungi were isolated; one was more dominantly present (90%). Both greenish (SNU-G) and whitish (SNU-W) fungi showed 97% and 95% internal transcribed spacer sequence identities to Aspergillus oryzae/flavus, respectively. However, the SmaI digestion pattern of their genomic DNA suggested that both belong to A. oryzae. Moreover, both fungi had morphological characteristics similar to that of A. oryzae. SNU-G and SNU-W did not form sclerotia, which is a typical characteristic of A. oryzae. Therefore, both fungi were identified to be A. oryzae. In aflatoxin gene cluster analysis, both fungi had norB-cypA genes similar to that of A. oryzae. Consistent with this, aflatoxins were not detected in SNU-G and SNU-W using ammonia vapor, TLC, and HPLC analyses. Both fungi seemed to have a whole cyclopiazonic acid (CPA) gene cluster based on PCR of the maoA, dmaT, and pks-nrps genes, which are key genes for CPA biosynthesis. However, CPA was not detected in TLC and HPLC analyses. Therefore, both fungi seem to be safe to use as doenjang koji starters and may be suitable fungal candidates for further development of starters for traditional doenjang fermentation.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 춘계학술대회 및 임시총회
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• pp.25-25
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• 2014

Koji molds are comprised of yellow, black and white. Black and white koji molds were recently re-visited by this author and it is concluded that they consists of Aspergillus luchuesnsis, A. niger and A. tubingensis, and the most important species for alcoholic beverage production is A. luchuensis. In the case of yellow koji mold, it is comprised of Aspergillus oryzae, A. sojae and A. tamari. In the case of A. sojae, the species is scarcely isolated from nature and rarely used for industry in Korea. Aspergillus tamari is often isolated from traditional Korean Meju, a fermented soybean product, and the classification of the species is clear. However, in the case of A. oryzae, differentiation between A. oryzae and A. flavus is still in controversy. In this study, we collected 415 strains of Aspergillus flavus/oryzae complex from air, rice straw, soybean, corn, peanut, arable soil and Meju in Korea and we examined the aflatoxin producing capacity of the strains. The norB-cypA, omtA and aflR genes in the aflatoxin biosynthesis gene cluster were analyzed. We found that 367 strains (88.4%) belonged to non-aflatoxigenic group (Type I of norB-cypA, IB-L-B-, IC-AO, or IA-L-B- of omtA, and AO type of aflR), and only 48 strains (11.6%) belonged to aflatoxin-producible group (Type II of norB-cypA, IC-L-B+/B- or IC-L-B+ of omtA, and AF type of aflR). In the case of A. flavus/oryzae strains from Meju, almost strains (178/192, 92.7%) belonged to non-aflatoxigenic group and only 14 strains (7.3 %) belonged to aflatoxin-producible group. It is proposed in this study that non-aflatoxigenic strain from Meju is classified as A. oryzae, considering that Meju is food material.

• Mycobiology
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• 제51권3호
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• pp.139-147
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• 2023

Aspergillus sojae has long been considered a domesticated strain of Aspergillus parasiticus. This study delineated relationships among the two species and an Aspergillus PWE36 isolate. Of 25 examined clustered aflatoxin genes of PWE36, 20 gene sequences were identical to those of A. sojae, but all had variations to those of A. parasiticus. Additionally, PWE36 developmental genes of conidiation and sclerotial formation, overall, shared higher degrees of nucleotide sequence identity with A. sojae genes than with A. parasiticus genes. Examination of defective cyclopiazonic acid gene clusters revealed that the PWE36 deletion pattern was identical only to those of A. sojae. Using A. sojae SMF134 genome sequence as a reference, visualization of locally collinear blocks indicated that PWE36 shared higher genome sequence homologies with A. sojae than with A. parasiticus. Phylogenetic inference based on genome-wide single nucleotide polymorphisms (SNPs) and total SNP counts showed that A. sojae strains formed a monophyletic clade and were clonal. Two (Argentinian and Ugandan) A. parasiticus isolates but not including an Ethiopian isolate formed a monophyletic clade, which showed that A. parasiticus population is genetically diverse and distant to A. sojae. PWE36 and A. sojae shared a most recent common ancestor (MRCA). The estimated divergence time for PWE36 and A. sojae was about 0.4 mya. Unlike Aspergillus oryzae, another koji mold that includes genetically diverse populations, the findings that current A. sojae strains formed a monophyletic group and shared the MRCA with PWE36 allow A. sojae to be continuously treated as a species for food safety reasons.

• Mycobiology
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• 제44권3호
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• pp.155-161
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• 2016

The most economically important species used in a wide range of fermentation industries throughout Asia belong to Aspergillus section Flavi, which are morphologically and phylogenetically indistinguishable, with a few being toxigenic and therefore a major concern. They are frequently isolated from Korean fermentation starters, such as nuruk and meju. The growing popularity of traditional Korean alcoholic beverages has led to a demand for their quality enhancement, therefore requiring selection of efficient non-toxigenic strains to assist effective fermentation. This study was performed to classify the most efficient strains of Aspergillus section Flavi isolated from various types of traditional wheat nuruk, based on a polyphasic approach involving molecular and biochemical evaluation. A total of 69 strains were isolated based on colony morphology and identified as Aspergillus oryzae/flavus based on internal transcribed spacer and calmodulin gene sequencing. Interestingly, none were toxigenic based on PCR amplification of intergenic regions of the aflatoxin cluster genes norB-cypA and the absence of aflatoxin in the culture supernatants by thin-layer chromatography analysis. Saccharification capability of the isolates, assessed through ${\alpha}-amylase$ and glucoamylase activities, revealed that two isolates, TNA24 and TNA15, showed the highest levels of activity. Although the degrees of variation in ${\alpha}-amylase$ and glucoamylase activities among the isolates were higher, there were only slight differences in acid protease activity among the isolates with two, TNA28 and TNA36, showing the highest activities. Furthermore, statistical analyses showed that ${\alpha}-amylase$ activity was positively correlated with glucoamylase activity (p < 0.001), and therefore screening for either was sufficient to predict the saccharifying capacity of the Aspergillus strain.

• Mycobiology
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• 제45권4호
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• pp.240-254
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• 2017

Cereal grains are the most important food source for humans. As the global population continues to grow exponentially, the need for the enhanced yield and minimal loss of agricultural crops, mainly cereal grains, is increasing. In general, harvested grains are stored for specific time periods to guarantee their continuous supply throughout the year. During storage, economic losses due to reduction in quality and quantity of grains can become very significant. Grain loss is usually the result of its deterioration due to fungal contamination that can occur from preharvest to postharvest stages. The deleterious fungi can be classified based on predominance at different stages of crop growth and harvest that are affected by environmental factors such as water activity ($a_w$) and eco-physiological requirements. These fungi include species such as those belonging to the genera Aspergillus and Penicillium that can produce mycotoxins harmful to animals and humans. The grain type and condition, environment, and biological factors can also influence the occurrence and predominance of mycotoxigenic fungi in stored grains. The main environmental factors influencing grain fungi and mycotoxins are temperature and $a_w$. This review discusses the effects of temperature and $a_w$ on fungal growth and mycotoxin production in stored grains. The focus is on the occurrence and optimum and minimum growth requirements for grain fungi and mycotoxin production. The environmental influence on aflatoxin production and hypothesized mechanisms of its molecular suppression in response to environmental changes are also discussed. In addition, the use of controlled or modified atmosphere as an environmentally safe alternative to harmful agricultural chemicals is discussed and recommended future research issues are highlighted.

• 한국균학회지
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• 제42권4호
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• pp.282-288
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• 2014

본 연구에서는 순창지역에서 만들어지는 장류에서 곰팡이를 분리하고 동정하여 보다 안전하고 기능성이 높은 발효제품을 위한 균주를 확보하고자 하였다. 순창지역에서 생산되는 장류 제품으로부터 곰팡이를 분리하여 ${\beta}$-tubulin 유전자 분석 통해 10개의 균주가 Aspergillus oryzae/flavus complex임을 알 수 있었다. 보다 정확한 동정을 위하여 아플라톡신 클러스터 유전자 중에 하나인 omtA의 염기서열을 증폭하여 A. oryzae와 A. flavus 표준 균주의 omtA 서열과 함께 계통 분류한 결과, A. oryzae의 표준 균주와의 유연관계가 높음을 알 수 있었다. 또한 norB-cypA 사이의 염기서열을 증폭한 결과 500 bp이 증폭 산물이 확인되었는데 이는 표준 균주인 A. oryzae의 norB-cypA 사이의 염기서열 증폭 산물과 동일한 크기임을 확인할 수 있었다. A. oryzae로 확인된 10균주를 활용하여 코지를 제조하고 ${\alpha}$-amylase 활성과 protease 활성을 측정하였다. Protease 활성은 6, 13, 17, 27, 37, 그리고 38 균주로 제조된 코지는 대조구(시판되고 있는 종균으로 제작한 코지)보다 2배 정도 높은 protease 활성을 보였으며, ${\alpha}$-amylase 활성은 257~320 U/mL로 측정되었다. 식품안전성을 위한 아플라톡신 분비 확인 결과, 63번 균주로 제조된 코지를 제외한 모든 코지에서 아플라톡신을 만들지 않는 것으로 확인되어, 순창에서 분리된 A. oryzae는 추후 메주 접종균으로 개발할 수 있음을 보여주었다.