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.

본 연구는 Aspergillus Oryzae (AO) 및 Saccharomyces cerevisiae (SC)를 첨가하여 제조한 맥주박 위주 발효사료의 발효특성 및 영양학적 특성을 검토하기 위해 실시하였다. 시험구 처리는 시험사료에 AO 를 첨가하여 제조한 발효사료 처리구(FFAO), SC 를 첨가하여 제조한 발효사료 처리구(FFSC) 및 AO 와 SC 를 첨가하여 제조한 발효사료 처리구(FFAS)로 나누었다. 48시간 발효에 따른 조단백질 함량은 처리

본 연구는 Aspergillus oryzae(AO) 혹은 Saccharomyces cerevisiae(SC)를 첨가하여 제조한 맥주박 위주 발효사료의 급여가 한우의 반추위내 미생물에 미치는 영향을 조사하기 위해 실시하였다. 공시동물은 반추위 cannula가 장착된 한우 암소 2두를 이용하였다. 시험은 시판 배합사료 및 corn silage 급여구(대조구), 시판 배합사료 , AO 첨가 발효사료 및 com silage 급여구(TAO)와 시판 배합사료 ,

This experiment was conducted to investigate the effects of mixed culture with mycotoxigenic and non-mycotoxigenic fungi on mycotoxin production. For this work, Aspergillus flavus (aflatoxin producing strain), Aspergillus niger (non-mycotoxigenic strain) and Penicilhum griseofulvum (patulin producing strainvere cultured in 5 ml SLS medium for 15 days under single or mixed culture. Affatoxin was determined by direct competitive ELISA, whereas, patulin was measured by HPLC. The mycelial growth, pH and total acidity were also observed by general methods. The mycelial growth was slightly decreased in the mixed culture, meanwhile total acidity was increased and pH was shown lower than that in single culture. Aspergillus flavus produced 145 ㎍/ml of aflatoxin for 12 days single culture, but in mixed culture, aflatoxin was decreased to 93%, and was shown as 10.16 ㎍/ml level. Patulin production in mixed culture was also decreased to 69.3% and was shown only 23.72 ㎍/ml level as compared with in single culture.

Aspergillus niger KCTC 6144 포자를 alginate bead로 고정화하여 호박배지(포도당 9%, 호박가루 1%, pH 6)를 이용하여 구연산 발효를 수행하였다. 고정화된 A. niger 포자가 bead 내에서 발아하여 균사가 bead를 뚫고 자라났으며 30℃에서 4일간 배양 후 그 크기는 2.0∼2.5㎜에서 6∼8㎜로 커졌다. 30℃에서 5일간 진탕배양(150rpm)으로, 50ml 호박 배지가 든 250ml 삼각 flask를 사용하여. 구연산 생성의 최적 발효 조건을 검토한 결과는 다음과 같다. 탄소원으로 포도당을 12%로 올렸을 때 구연산의 생성이 최고에 달하였고, 질소 및 무기질원으로 1%의 호박가루가 구연산 생성에 가장 알맞는 농도였다. 초기 pH는 6.0, bead의 수가 100개로 하였을 때 구연산 생성이 가장 좋았으며, 포토당 12%, 호박가루 1%로 한 최적 배양 조건에서, 5일만에 구연산이 23.5g/l로 최고로 생성되었다.

This study was performed to investigate the possible effect of Bacillus subtilis which is the predominant species of bacteria in Korean soy sauce, soy paste, and Meju (soybean cake) on the growth and aflatoxin production of Aspergillus parasiticus ATCC 15517. The microorganisms were grown in a modified APT broth and incubated at 30℃ for 12 days. Aflatoxins were determined using high performance liquid chromatography (HPLC). A remarkable inhibition of the growth of Aspergillus parasiticus was observed during the incubation period when in the presence of B. subtilis (mixed culture). Dry mycelial weight in the mixed culture was significantly reduced by 85.3% in comparison to the control at the end of the incubation period (p$lt;0.01). Lower levels of aflatoxins were found in the mixed culture than in the monoculture. At the end of the incubation period aflatoxin production was significantly inhibited by more than 50% (p$lt;0.05). These results indicate that B. subtilis mainly inhibites the growth and aflatoxin production of toxigenic Aspergillus in Meju, soy sauce and soy paste. Although its effect on aflatoxin production was less pronounced, we . could expect more inhibition by another bacteria related with fermentation in Meju.

발효식품이 유해곰팡이에 의한 발암물질(aflatoxin)생성에 미치는 억제효과에 관한 연구의 일환으로 유산균 및 유산균 발효유가 Aspergillus parasiticus ATCC 15517의 성장과 aflatoxin 생성에 미치는 영향을 실험하였다. 발효유를 일정 농도별로 첨가한 YES 배지에서 Asperigillus parasiticus를 배양하여 그 성장과 배양물의 변화를 관찰하고 HPLC에 의하여 aflatoxin을 분석하였다. 그 결과 배양말기에 대조군에 비하여 건조 균체량, 배양물의 PH, 그리고 aflatoxin 생성량 등이 낮게 나타났다(p<0.05). Aflatoxin B₁은 48.6~58.1%가 감소되었으며 G₁은 29.8~34.2%가 감소되었다. 이 발효유의 발효에 사용된 유산간균(Lactobacillus casei)과 A. parositicus를 변형 APT 배지에서 혼합배양한 결과 A. parasitious 단독배양의 경우에 비하여 균체량이 배양 5일째까지는 현저하게 억제되었으나 배양 말기에는 유의한 차이를 보이지 않았다. 또한 배양 말기에 단독배양의 경우보다 pH가 훨씬 감소되고(p<0.05) aflatoxin의 생성량도 감소되었다. 이로부터 발효유는 유해곰팡이인 A. parasiticus의 성장과 aflatoxin 생성을 억제시키는 효과를 가짐을 알 수 있으며, 이는 발효에 관여한 미생물의 경쟁뿐만 아니라 유산균의 대사산물에 의한 영향으로 보여진다.

쌀에 대하여 aflatoxin 생성을 위한 기질로서의 능력을 알아보기 위하여 현미(청청벼) 시료에 Aspergillus parasiticus를 접종하고 조건을 달리하여 저장하면서 aflatoxin B₁의 생성을 관찰하였다. 시료중의 aflatoxin B₁의 분석은 ELISA를 이용하여 수행하였다. 현미 시료에서 aflatoxin B_1 생성에 가장 좋은 온도는 28℃였으며, 시료의 수분함량을 15.8%로 증가시킨 경우 aflatoxin B₁의 생성이 유의하게 증가하였고 (p<0.05), 고압증기멸균시킨 시료는 aflatoxin B_a 생성에 보다 효과적인 기질이 되었다. 실온에서 3개월 동안 저장한 현미에서는 15일 동안 저장한 경우에 비하여 aflatoxin B₁ 생성이 유의한 증가를 보였다(p<0.05). 따라서 저장온도 및 수분함량이 쌀에서도 aflatoxin B₁ 생성에 영향을 미치는 바를 나타내었으며, 또 시료의 상태 및 저장기간도 쌀에서 aflatoxin 생성 위험요인으로 작용할 수 있음이 제시되었다. 이러한 결과로부터 쌀이 aflatoxin 생성에 좋은 기질이 될 수 있는 것으로 평가된다.

Effect of allylisothiocyanate on the enzyme activities including malate dehydrogenase, isocitrate dehydrogenase, NADPH and acetyl CoA which were related to atlatoxin production of Aspergillus parasricus R-716 were invetigated. The activities of malate dehydrogenase (EC.1.1.1.37), isocitrate dehydrogenase (E.C.1.1.1.42) and NADPH oxidase (E.C.1.6.99.1) indicated relatively high in the 50 ppm allylisothiocyanate-added-culture. In contrast, the activity of acetyl CoA in the 50 ppm allylisothiocyanate-added-culture showed rather lower level through the cultivation.

Aspergillus fumigatus IFO 5840이 생산하는 cytosine deaminase에 미치는 cytosine analogue의 영향을 조사하여 다음의 결과를 얻었다. 1.Cytosine deaminase의 강한 저해를 나타내는 cytosine analogue는 2-thiouracil, 2-thiocytosine, 2-mercaptopyrimidine, 및 6-azacytosine이었다. 2.본 효소에 대한 2-thiocytosine 및 6-azacytosine의 50% 효소활성 저해농도(HIC)는 각각 0.80mM 과 1.15mM 이었다. 3. 2-thiocytosine은 일정한 수준에서 본 효소의 반응을 정지시키는 반면, 6-azacytosine은 반응시간에 비례하여 일정한 비율로 저해하였다. 4. 2-thiocytosine과 6-azacytosine은 cytosine이나 5-fluorocytosine의 기질 종류에 관계없이 본 효소의 저해제로 작용하였으며 2-thiocytosine이 6-azacytosine보다 약 2배 정도의 높은 저해를 나타내었다. 5. cytosine을 기질로 하였을 경우, 2-thiocytosine과 6-azacytosine에 의해 경쟁적 저해를 나타내며 이들에 대한 K_i값은 각각 4.5 × 10^-4M 과 1.756 × 10^-3M 이었으며 cytosine, 2-thiocytosine 및 6-azacytosine에 대한 Hill 게수(Hn)는 각각 1.80, 1.81, 2.45로 나타났다.

One kind of fungus was isolated and identified from comtaminated red-ginseng in order to give fundermental data for improving hygienic quality of ginseng product. The isolated strain was identified as Aspergillus sp. Hyphae of the strain had septum structure. The strain showed vesicle and aterigmata structure which were typical characteristics of Aspergillus species. The growth of the strain was slightly inhibited by sodium benzoate and potassium sorbate at a concentration of 0.05%. The strain showed no growth at 4.0% potassium sorbate. The isolated strain Aspergillus sp. showed no significant degradation in the presence of red-ginseng saponins.

The effect of allylisothiocyanate, the major compound of radish on the growth of Aspergillus parasiticus R-716 and aflatoxin production, was investigated. An increase in the level of allylisothiocyanate results in a decrease both growth and aflatoxin per myclial weight, and the addition of 125 ppm allylisothiocynate completely inhibited the growth of this strain. The addition of allylisothiocyanate to the culture of R-716 strain delayed the production of atlatoxin. The inhibition of aflatoxin was more B-group than G-group and M-group during cultural period. The growth of strain and aflatoxin production were greatly affected by the addition of allylisothiocyanate.