It is possible to apply DNA sequencing data of A. oryzae RIB40 (Tominaga et al, 2006) to investigation of genomic structure of homologous gene cluster in 210 A. oryzae RIB strains. Using PCR technique, 210 A. oryzae RIB strains were easily classified into groups 1, 2, and 3, and others according to amplified patterns with seven aflatoxin homologous genes. Group 1 (122 strains, 58.1%) strains conserve intact aflatoxin biosynthesis gene homolog cluster. Group 2 (77 strains, 36.7%) and group 3 strains (9 strains, 4.3%) reveal large deletions of the aflatoxin gene homolog cluster, which is more than half. It is possible that the breakpoint within the cluster of group 2 strains would be near the ver-1gene, as described by Kusumoto et al. (2000). Two strains (0.9 %) that could not be classified into group 1, 2, and 3 were called "others". The majority of A. oryzae RIB strains (94.8 %) are categorized as groups 1 and 2. Murakami (1971) has evaluated 20 mycological characters of RIB strains, graded them from 1 to 6 and also proved no aflatoxin production in all strains. To examine the differences between group 1 and group 2 based on phenotype, analysis of variance was performed. Significant differences among 19 characters except for the aflatoxigenic character were recognized with 5 characters (length of stalk, color of old slant culture, roughness of conidia, coloration of hydroquinone, and pink color of conidia in medium with anisic acid). The length of stalk of group 1 was longer than that of group 2 at level of p<0.01 (data not shown). Therefore, this PCR analysis is a useful method for classification at intra-species level. Furthermore, it is safe for the food fermentation and enzyme industry to use A. oryzae especially groups 2 and 3 strains since these strains revealed absolute lack of aflatoxigenic ability at the molecular level. From the results of DNA sequencing analysis between A. oryzae RIB40 belonging to group 1 and RIB62 belonging to group 2, RIB62 shows a large deletion upstream of ver-1 homolog with more than half of the aflatoxin homologous gene cluster being missing. Adjacent to the deletion of the aflatoxin homologous gene cluster, RIB62 has a "unique sequence" of about 8-kb and a telomere. We investigated whether homologues of the unique sequence region of A. oryzae RIB62 were present in other group strains with Southern blot analysis. At first, we performed Southern blot analysis of 210 A. oryzae RIB strains with "no-hit" probe of unique sequence. The results showed that all group 2 strains had an identical size of signal of about 9.4-kb, while in other group strains different size of hybridizing signals from that of group 2 strains or with no signal were detected (data not shown).Subsequently, to confirm the presence of the unique sequence, Southern blot analysis with the four kinds of probe, which were derived from the unique sequence of RIB62 was performed for 16 selected strains from group 1, 2, and 3. Group 1 strains showed various signal patterns; double or single band(s) in most strains and with no signal in RIB40. In addition, the signal pattern of group 1 strains was different according to the probe used. However all group 2 strains showed an identical band of about 9.4 kbin all the cases when the four probes were used. In the group 3 strains, no signal was detected with the four probes. Therefore, 8-kb unique sequence of RIB62 is conserved in A. oryzae group 2 strains and present partially in some group 1 strains. To investigate the chromosomal position of the unique sequence, chromosomal Southern analysis was performed using four kinds of probe, US 1 to 4. Separation of the chromosomes of selected eight of A. oryzae group 1 and group 2 strains by clamped homogeneous electric field (CHEF) revealed different karyotype (data not shown). Among them, RIB62 showed a unique band of about 3 Mb, whereas other strains have no this chromosome. The detected signal(s) in A. oryzae group 1 strains were revealed in two or one chromosome(s) and in no signal in RIB40, while that of group 2 strains showed in single chromosome with four kinds of probes. The signal patterns of group 1 strains were different according to the probe used, while those of group 2 strains were identical. These results are almost identical to those of genomic Southern blot analysis To confirm the structure of the region flanking the partial aflatoxin homologous gene cluster in A. oryzae group 2 strains, we investigated the pattern of PCR amplification in 210 A. oryzae RIB strains with a set of primers designed to amplify between ver-1 and the unique sequence. The oligonucleotide primer for the ver-1 side was common to both RIB 40 and RIB62, while that of the unique sequence side was derived from RIB62. From the results of PCR with this set of primers, a fragment of about 1 kb was amplified from all group 2 strains and none of strains from other group generated PCR products. Therefore, it is possible to distinguish group 2 strains from other group strains with this set of primers. Southern blotting and PCR analysis resulted that all group 2 strains has the identical "unique sequence" and genomic structure of deletion including flaking region. In addition, this characterization of group 2 strains could be applied to distinguish this group strainsfrom the other group strains. The result of chromosomal Southern analysis (data not shown) suggested that the aflatoxin homologous gene cluster and the "unique sequence"existed on the same chromosome in groups 1 strains having these two portions. Taken together, A. oryzae group 2 strains might have differentiated from the ancestral strain due to chromosomal breakage. Although it is extremely difficult to determine the reason for the non-aflatoxigenicity of A. oryzae from the analysis of the genomic structure, this dissertation may provide basic molecular information for the profound approaches. In succession, further research on aflR protein activity or other related signal transduction pathway and the deleted aflatoxin biosynthesis gene homolog cluster of group 2 strains together with group 3 strains may help in clarifying the mechanism of the cluster deletion and differentiation.

This study analyzed the changes in physicochemical components of Astragalus membranaceus (AM) fermented with Phellinus linteus. Moisture content, pH, total acidity, total reducing sugar content, extraction yield, free sugar content, free amino acid and isoflavonoid (calycosin, formononetin) were investigated. The moisture content was increased during fermentation with Phellinus linteus. The pH level increased while the total acidity significantly decreased during fermentation. The reducing sugar content were in the range of 0.32~0.61%. The extraction yield using water was higher than that using 80% ethanol. The major free sugars were identified as glucose, fructose, sucrose and the content of free sugars decreased through fermentation. However, the glucose and sucrose contents of the water extracts were increased. In addition, the free amino acid increased significantly during fermentation. Finally, calycosin and formononetins contents in water extracts of after 30 days of AM fermentaion with Phellinus linteus were (3.91 mg/100 g) and (1.38 mg/100 g), respectively. These results suggest that fermentation with Phellinus linteus could be used to increase the bioactivity of AM. The mycelium-fermented AM could be a valuable source of functional material and edible resource for industry.

This study analyzed the physicochemical characteristics of Astragalus membranaceus (AM) fermented with seven different mushroom mycelia. Physicochemical characteristics, such as contents of moisture, pH, total reducing sugars, free sugar, and isoflavonoid, were investigated. The moisture content was increased in most of the samples. The pH values of AM fermented with Phellinus linteus and Flammulina velutipes were increased, while the pH of other samples were similar to that of non-fermented AM. The reducing sugar content was in the range of 211.69~391.74 mg/100 g. The extraction yield using water was higher than that when extracted with 80% ethanol. The free sugar content was increased through fermentation with mushroom mycelia. However, the glucose contents of the 80% ethanol and water extracts were decreased. Finally, the calycosin and formononetin contents in 80% ethanol and water extracts of AM fermented with Phellinus linteus were 2,549.24 mg/g, and 827.66 mg/g for calycosin, and 1,366.69 mg/g and 221.28 mg/g for formononetin, respectively. These results suggest that fermentation with mushroom mycelia could be used to increase the bioactivity of AM. The mycelium-fermented AM might be a valuable source of functional material and edible resource for industry.

버섯 재배는 자동화된 공장처럼 관리되고 있다. 배지를 만들어 서 수확하는 1배치의 생명 주기가 2개월 안에 종료되며, 이러한 배 지를 만드는 작업이 보통 1일 2회 내지 3회 정도 농가별로 진행하고 있어서 3회 기준으로 볼 때 1년에 1,095(3배치 * 365일)배치를 반 복하는 것이다. 즉 1,095 배치에 대하여 각 배치당 생산량의 차이에 대한 원인을 분석하고, 많은 생산량을 가진 배치의 생장 환경을 기 준으로 하여 다른 배치의 생장 환경을 조정한다면 생산량의 확대를 가져올 것이다. 이러한 개념에 기반을 두어 진행한 것이 본 연구이 며 시스템 구축 사업을 통하여 개념을 실현하였다. 버섯 생장 환경 수집 대상은 온도, 습도, CO2 뿐만 아니라, 배지의 습도, 오염 정도 등이 있으며 수집 방법으로는 일부는 설치된 센서를 통하여 자동으 로, 일부는 작업일지를 통하여 수동(예를 들어, 오염 정도, 폐기량 등)으로 이루어진다. 수집된 데이터는 누적되어 보관되고 있으며, 결과적으로 볼 때 즉 최대 생산량을 가진 배치의 과거 수집된 생장 환경 정보를 분석하는 것이다. 분석 데이터의 보편성을 위하여 약 60회(임의로 설정) 이상의 배치를 분석하여 최종 판단하는 것이고 적합하다고 판단하였으며, 이를 기반으로 자동으로 자동 분석 정보 가 도출되며, 이를 기반으로 수동으로 담당 전문가(버섯 연구소 담 당자)가 분석한 결과에 최종 의견을 넣은 보고서를 작성하여 해당 농가에 제공하는 것(버섯생장분석시스템)으로 구성하였다. 이와 같은 시스템의 적용은 본 연구에서는 느타리와 새송이에 적용하였 지만 모든 버섯 생산에 적용할 수 있을 것이다.