Aflatoxin production of Aspergillus parasiticus I F 0 30179 and Aflavus var columnaris S46 on black pepper, white pepper and red pepper was investigated and the results compared with those obtained using polished rice. The levels of aflatoxin B, production on cooked and uncooked black pepper and white pepper were negligible. However, a little aflatoxin production was observed on red pepper even when uncooked. The inhibition of aflatoxin production was also observed in extracts of black pepper, white pepper and red pepper. Irradiation of the spices did not affect aflatoxin production.Key words: aflatoxin B, , spice, irradiation, Aspergillusjavus, A parasiticus.Serious damage can be caused to stored spices or cereal grains by mould growth which occurs under unfavourable conditions during long storage or shipment at high humidity. Certain moulds growing on spices or cereal grains produce mycotoxins, which may be acutely toxic, carcinogenic or teratogenic to consumers. Aflatoxin B, is the most carcinogenic of many kinds of mycotoxins, and is produced by Aspergillusflavus and A parasiticus. In Japan, the natural occurrence of aflatoxin has been reported in imported agricultural products such as edible nuts, corn and some spices. Previously (Muhamad et a1 1986), the authors have reported the isolation of many strains of A flaous from spices, and more than 10% of these isolates demonstrated the ability to produce aflatoxin B, under laboratory conditions. Therefore, this study was carried out to evaluate the possibility of aflatoxin production on black pepper, white pepper and red pepper and the results were compared with those obtained with polished rice. The influence of irradiation of spices on their inhibitory effects was also ascertained.* To whom correspondence should be addressed. Spores of Aspergillus parasiticus I F 0 30179 and a wild strain of AJlauus var colurnnaris S46 isolated from black pepper were harvested from Difco potato dextrose agar plates after incubation at 30°C for 7 days. Spore suspensions were prepared by adding sterilized distilled water to give a concentration of c a 1 x lo6 spores mi-'. For determination of aflatoxin, a SL (synthetic low salts) broth containing the following was prepared as a liquid medium: sucrose, 85 g; L-asparagine, 10 g; (NH,),Mo702, . 4H20, 2 mg; Na,B,O, , 2 mg; FeSO, . 7H20, 2 mg litre-'; pH 4.5. This SL broth was dispensed into 200 ml cottonstoppered Erlenmeyer flasks, each flask receiving 20 ml of medium, and was sterilised at 110°C for 10 min.Unground polished rice, black pepper, white pepper and red pepper (20 g each) in cotton-stoppered Erlenmeyer flasks (200 mi) with 10 ml of distilled water were autoclaved at 121°C for 15 min or irradiated at 5 kGy, using the 160 kCi (6.0 PBq) 6oCo irradiator in this institute. For the study of the effects of spice extracts on aflatoxin production, extracts of black pepper, white pepper and red pepper (unirradiated and irradiated at 10 kGy) were obtained by steeping 3 g of powdered spice in 30 ml of ethanol for 24 h at ...
Effects of gamma-irradiation, given in the range of 5 to 30 Gy on Caski cells (Epitheloid carcinoma from the cervix) were investigated by the MTT (3-(4,5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) method. Results were compared with data assessed simultaneously from cell number counts. The sizes of cells irradiated with 10 to 30 Gy were larger than those of unirradiated ones, and each irradiated cell reduced a larger amount of MTT than did each unirradiated cell. Irradiation in the above range, therefore causes Caski cells to lose their ability to divide, but the effect on the mitochondria was very slight. Application of the MTT method to the irradiated cells should be done with care. Because, in the irradiated cells depending on the irradiation dose, the MTT activity does not correlate to the cell number.
Orotate phosphoribosyltransferase (OPRTase, EC2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). To obtain heat-stable OPRTase and to elucidate the mechanism of heat stability, this enzyme from Thermus thermophilus was expressed in Escherichia coli and purified. The pyrE gene of T. thermophilus which encodes OPRTase, contains an open reading frame of 549 base pairs with 69% G+C content. Since this gene expressed itself inefficiently in E. coli, the 5' and 3' ends of the coding regions were replaced with synonymous codons which contain more A+T and corresponds to major codons for E. coli. Introduction of the modified gene fragments into a plasmid having a tac promoter resulted in production of a polypeptide of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) in E. coli. This protein represented as much as 16% of the bacterial total protein and showed the OPRTase activity. Three purification steps, consisting of heat treatment at 65 degrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypeptide. The optimum activity of the purified OPRTase was observed at 150 mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 microM PRPP. The activation energy of this enzyme reaction was 20.3 kJ/mol. The Km of this enzyme for orotate as a substrate was 75 microM and the maximum specific activity was 300 units/mg protein under the optimum conditions. The purified OPRTase was stable for 20 min at 85 degrees C.
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