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문제 정의
- The aim of the present study was to investigate the AFB1-detoxifying activity of two A. oryzae strains isolated from meju. This is the first report showing that A.
제안 방법
- A phylogenetic tree was constructed by a neighbor-joining tree using MEGA5 [37, 38]. DNA data were processed using the Tamura-Nei parameter distance calculation model with gamma-distributed substitution rates in order to analyze the taxonomic positions of the two A. oryzae strains. To determine the support for each clade, bootstrap analysis was performed with 1,000 replications.
- For aflatoxin production testing, YES media and SC media were used, and each fungus was cultured for 3 weeks.
- For qualitative analysis of AFB1 biodegradation, TLC analysis was conducted. AFB1 in culture media, which were cultured as described above, was extracted by liquid-liquid extraction (LLE).
- For quantitative analysis of AFB1 biodegradation, fungal culture media were taken at 0, 1, 3, 5, 7, 10, and 14 days in triplicates. After the culture medium was filtrated through a glass microfiber filter (GF/A; Whatman Inc.
- The beta-tubulin region was selected for molecular identification, and the PCR was performed with two specific primers, Bt2a and Bt2b (5’-GGT AAC CAA ATC GGT GCT TCC-3’ and 5’-ACC CTC AGT GTA GTG ACC CTT GGC-3’), using a PC708 thermal cycler (Astec, Japan)[35].
- 1%). The fungal isolates were tested for AFB1-biodegrading activity by using TLC. Two fungal isolates (MAO 103 and MAO 104) showed AFB1 biodegradation activities; Fig.
- oryzae strains. To determine the support for each clade, bootstrap analysis was performed with 1,000 replications.
대상 데이터
- For the molecular analysis of aflatoxigenicity, genetic analysis of 17 genes in the aflatoxin biosynthetic pathway was conducted according to Tao and Chung [36]. For the aflatoxin production test, yeast extract-sucrose media (YES media: 2% yeast extract, 20% sucrose)[39] and SC media were used. One milliliter of each fungal spore suspension (105/ml) was inoculated into 100 ml of media and incubated for 3 weeks at 25o C with shaking at 130 rpm.
- One hundred thirty-four fungi were isolated from 65 lumps of meju that were collected from the middle and southern regions of South Korea (Table 1). The fungal isolates were divided into five genera by morphological examination: Aspergillus section Flavi, Aspergillus section Nigri, other Aspergillus sp.
- [41]. Salmonella typhimurium strains TA98 (CCARM 0272) and TA100 (CCARM 0273) were used as tester strains. The metabolic activation was performed in a reaction mixture containing 0.
데이터처리
- Data were statistically evaluated using one-way ANOVA. The results were considered significant when p values were <0.
성능/효과
- In conclusion, the AFB1 degradation activity of A. oryzae MAO 103 and MAO 104 was 97.4% and 96.1%, respectively, in 14-day culture media. The loss of AFB1 mutagenicity via the strains was implicated as a potential method for detoxification of AFB1 in food systems.
- The sizes of the PCR product of the beta-tubulin gene of Aspergillus MAO 103 and MAO 104 were 497 and 496 bp, respectively. The Aspergillus MAO 103 and MAO 104 isolates were identified as Aspergillus oryzae by comparison of nucleotide sequences in the PCR products from beta-tubulin with sequences in the same region from strains selected from the NCBI database (NCBI Accession ID: KY020433 for A. oryzae MAO 103, KY020434 for A. oryzae MAO 104). The taxonomic positions of the two AFB1-biodegrading A.
- The amounts of AFB1 detected in both culture broth and mycelia are shown in Table 3. The amounts of AFB1 removed from the total cultures of A. oryzae MAO 103 and MAO 104 were 92.4% and 78.4% of the initial toxin amount, respectively, at 14 days. Considering the total reduction of toxin in the culture broth, the adsorption of AFB1 to fungal mycelia was relatively low in both A.
- oryzae isolates[36]. The two A. oryzae strains possessed only seven aflatoxin biosynthetic genes (aflG, aflI, aflK, aflM, aflO, aflP, and aflQ), and not 10 aflatoxin biosynthetic genes (Table 2). From this result, A.
- oryzae MAO 103 and MAO 104 was investigated by HPLC analysis. The two A. oryzae strains showed time-dependent AFB1 biodegradation activity and were able to degrade more than 90% of the AFB1 in culture media over 14 days (Fig. 4). A.
- The untreated AFB1 control sample, after incubation for 72 h at 30°C, still had strong mutagenic responses in both Salmonella strains, whereas the results from the samples treated with A. oryzae MAO 103 and MAO 104 showed that the mutagenic effects of AFB1 decreased to 5.7% and 6.4% (p < 0.001) in frame-shift mutations of the TA98 strain, respectively.
- This indicates that the AFB1 production in co-cultured soybean-based agar plates with the two AFB1-detoxifying isolates A. oryzae MAO 103 and MAO 104 was significantly reduced to 32.6% and to 45.7% (p < 0.01), respectively, compared with that with A. flavus M 2034.
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