Production of Aflatoxins B1 and G1 by Aspergillus flavus in a Semisynthetic Medium

Isolates of Aspergillus flavus produced 0.2 to 63 mg of aflatoxins B(1) and G(1) per 100 ml in a nutrient solution consisting of 20% sucrose and 2% yeast extract. Various factors influencing the fermentation were studied. The maximal amount of toxin was produced by ATCC culture 15548 in 1-liter flasks containing 100 ml of medium incubated as stationary cultures for 6 days at 25 C.     

Production of Aflatoxins B1 and G1 by Aspergillus flavus and Aspergillus parasiticus Isolated from Market Pecans

One hundred and forty-eight isolates of Aspergillus flavus and A. parasiticus were isolated from 5,608 pecans obtained from Chicago and Georgia markets. The percentage of internal contamination by these species was 7.3% in the Chicago market pecans and 1.7% in those from markets in Georgia. Of the 148 isolates, 93% of the A. parasiticus, but only 54% of the A. flavus, were capable of producing aflatoxin. Overall, 57% of the isolates were potentially aflatoxigenic. A. parasiticus isolates generally produced a greater amount of aflatoxins than A. flavus.     

Oxidative and nutrient stability of a standard rodent spaceflight diet during long-term storage

The National Aeronautics and Space Administration's standard spaceflight diet for rodents is the nutrient-upgraded rodent food bar (NuRFB). The shelf life of the NuRFB needs to be determined in order to avoid malnutrition of rodents and confounding of research results resulting from nutritional deficiency. The authors compared the oxidative and nutrient stability of NuRFBs stored at either ambient temperature (26 °C) or at refrigeration temperature (4 °C) for use in long-term rodent feeding experiments. After 0, 3, 6, 9 and 12 months (mo) of storage, lipid oxidation, fatty acid composition and amounts of specific vitamins and amino acids in NuRFBs were analyzed. No oxidative rancidity developed in NuRFBs stored at 4 °C for up to 12 mo, but NuRFBs stored at 26 °C for 6 mo developed oxidative rancidity and had reduced amounts of γ-linolenic acid (18:3n-6). Despite loss of vitamin E, vitamin A and thiamin after storage at 26 °C for 12 mo, vitamin levels in NuRFBs remained at or above the levels recommended for optimal rodent health. The amino acid profile of NuRFBs was unaffected by storage at 4 °C or 26 °C for 12 mo. The results suggest that NuRFBs stored at 4 °C for up to 12 mo and NuRFBs stored at 26 °C for up to 6 mo provide suitable nutrition for rodents in long-term experiments.     

Fate of aflatoxins B1 and G1 residues during biscuit processing

Effect of biscuit processing on the destruction of aflatoxins B₁ and G₁ with and/or without some commonly leavening agents used namely sodium bicarbonate, ammonium bicarbonate and sodium bisulfite and sodium chloride. It was found that mixing step reduced the concentration of aflatoxins B₁ and G₁ by 80.7% and 82.7%, while the effect of baking step being 28.9% and 21.5%. The effect of mixing was found to be more pronounced than that baking step. The highest destruction effect on aflatoxin B1 was observed by adding a mixture composed of sodium and ammonium bicarbonate and sodium bisulfite followed by sodium chloride, sodium bisulfite, ammonium bicarbonate and/or sodium bicarbonate alone, where the reduction values of toxin after mixing were 93.4,91.9,91.7, 88.8 and 86.6% respectively, while the baking effect ranged 17.2 to 34.5% in the presence of different leaving agents added. Concerning aflatoxin G1; the highest destructive effect of toxin was adsorbed by adding a mixture of sodium and ammonium bicarbonate and sodium bisulfite followed by sodium bisulfite, sodium chloride, ammonium bicarbonate and/or sodium bicarbonate alone since the destruction values of such toxin after mixing were 96.2%, 92.8%, 92.6%, 89.0% and 87.7% respectively, while the baking effect ranged 20.9 to 34.5% in all leavening agents added.     

Sugar accumulation in chemically debudded potato tubers during cold storage

Potato tuber buds may be excised by immersion of the tubers in a mixture of EtOH-Me₂CO (1:1) for 4 hr. This enabled the study of the effect of tuber aging (at 17°) on the starch-to-sugar conversion during storage at 4°, in the absence of complications due to sprouting. Sugar accumulation during a two-week period of storage at 4° decreased with increasing time of prior storage at 17°.     

Characterization of the Critical Amino Acids of an Aspergillus parasiticus Cytochrome P-450 Monooxygenase Encoded by ordA That Is Involved in the Biosynthesis of Aflatoxins B1, G1, B2, and G2

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin to aflatoxins B₁, G₁, B₂, and G₂ requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA gene, was identified in the aflatoxin-biosynthetic pathway gene cluster by chromosomal walking in Aspergillus parasiticus. The ordA gene was a homolog of the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B₁. Complementation of A. parasiticus SRRC 2043, an OMST-accumulating strain, with the ordA gene restored the ability to produce aflatoxins B₁, G₁, B₂, and G₂. The ordA gene placed under the control of the GAL1 promoter converted exogenously supplied OMST to aflatoxin B₁ in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasiticus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three point mutations which resulted in three amino acid changes (His-400→Leu-400, Ala-143→Ser-143, and Ile-528→Tyr-528). Site-directed mutagenesis studies showed that the change of His-400 to Leu-400 resulted in a loss of the monooxygenase activity and that Ala-143 played a significant role in the catalytic conversion. In contrast, Ile-528 was not associated with the enzymatic activity. The involvement of the ordA gene in the synthesis of aflatoxins G₁, and G₂ in A. parasiticus suggests that enzymes required for the formation of aflatoxins G₁ and G₂ are not present in A. flavus. The results showed that in addition to the conserved heme-binding and redox reaction domains encoded by ordA, other seemingly domain-unrelated amino acid residues are critical for cytochrome P-450 catalytic activity. The ordA gene has been assigned to a new cytochrome P-450 gene family named CYP64 by The Cytochrome P450 Nomenclature Committee.     

Changes in potential denitrification and respiration during the cold storage of soils

The denitrification potential in moderately fertilized soil sampled four times during 1995 decreased significantly after cold storage, at 4 +/- 2 degrees C for 1 week. Prolonged storage (up to 24 weeks) resulted in a further decrease of denitrification potential which dropped to 38-54% of the original values. Similarly, denitrification potential decreased substantially during the first week of storage in differently fertilized soils. After 24 weeks of storage, denitrification potential dropped to 29-55% of that in fresh soils. The effects of storage at 4 +/- 2 degrees C on denitrification potential and respiration (determined as carbon dioxide evolution) were in general the same in moderately fertilized soils from four different sites: in all soils, depression of both the denitrification potential and potential respiration was found after 8 weeks. However, the extent to which the parameters were decreased differed from case to case. Not only the duration and storage conditions but also unidentified soil parameters are important for the persistence of biological activity in stored soils.     

Liver preservation by cold storage with hyperosmolar solutions for twenty-four hours.

Canine livers were successfully preserved in an ischemic state for 24 hr under hypothermic storage with hyperosmolar colloid or crystalloid solutions. Livers preserved with a colloid hyperosmolar solution (MSGF) showed slightly better survival results than the ones preserved with a crystalloid hyperosmolar solution. It is possible that hyperosmolarity associated with hyperkalemia is an important factor for liver preservation for transplantation.     

Salting the charged surface: pH and salt dependence of protein G B1 stability

This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is or=18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.     

Oxidative status of human milk and its variations during cold storage

Breastfeeding and human milk are widely accepted as optimal for human infants' nutrition. Nowadays lifestyle often makes it difficult to maintain or even initiate human lactation. This situation is mostly related to the workload of women away from home. New approaches are needed to enable maternal lactation under these circumstances. Human breastmilk storage for differed use is one possibility. The aim of this study was to assess changes in glutathione peroxidase (GPx) activity and in the concentration of the lipid peroxidation marker, malondialdehyde (MDA), when human milk was kept refrigerated or frozen. Thirty-two human milk samples were assayed for GPx activity and MDA concentration. Samples were divided in three aliquot portions, the first to be immediately analysed, the second to be refrigerated at 4 degrees C and analysed 24 h thereafter, and the third to be frozen at -20 degrees C and assayed after 10 days. GPx activity was significantly decreased in refrigerated and in frozen milk, when compared to their control samples. MDA was increased only in refrigerated milk but not in frozen samples. Thus, freezing seems better than refrigeration in order to prevent lipid peroxidation in stored human milk samples.     

Improved functional and nutritional properties of tomato fruit during cold storage

The use of synthetic antioxidants has been associated with serious concerns for human and environmental health. During ripening stages, tomato fruit is exposed to different abiotic stresses which not only influence its nutritional, mechanical, and functional properties at harvest, but also affect the quality and shelf life of the fruit during storage. This study investigated the pattern of changes in dietary antioxidants during various ripening stages of tomato fruit (cv. Red Rose) and their impact on storage behavior of the fruit during cold storage. Tomato fruits were harvested at mature green, breaker, turning, pink, light-red and red stages of maturity. Then, they were analysed for flesh firmness, soluble solids content, titratable acidity, total sugars, pH, dry matter content, lipophilic (lycopene, β-carotene, and total carotenoids), and hydrophilic (ascorbic acid, phenolic and flavonoids) antioxidants. Additional fruits were harvested at each maturity stage and divided into three equal lots, then were subjected to low-temperature (10 ± 1 °C) storage with 80 ± 5% RH, for 7, 14, and 21 days. Flesh firmness, and the levels of dietary antioxidants were analysed following the subsequent storage periods. The results revealed that the peak of hydrophilic antioxidants such as ascorbic acid, phenolic compounds, and flavonoids was between the ‘pink’ and the ‘light-red’ stages of fruit maturity. Whereas tomatoes harvested at the ‘red’ stage of maturity had the highest levels of lycopene and β-carotene. Both the stage of fruit maturity at harvest and duration of cold storage influenced flesh firmness, organoleptic and functional properties of ‘Red Rose’ tomato fruit. In conclusion, the results of the current investigation have practical implications in formulating foods with improved functional properties at processing industries.     

Biosynthetic relationship among aflatoxins B1, B2, G1, and G2

Aspergillus parasiticus NIAH-26, a UV-irradiated mutant of A. parasiticus SYS-4 (NRRL 2999), produces neither aflatoxins nor precursors. When sterigmatocystin (ST) or O-methylsterigmatocystin was fed to this mutant in YES medium, aflatoxins B1 (AFB1) and G1 (AFG1) were produced. When dihydrosterigmatocystin (DHST) or dihydro-O-methylsterigmatocystin was fed to this mold, aflatoxins B2 (AFB2) and G2 (AFG2) were produced. The reactions from ST to AFB1 and DHST to AFB2 were also observed in the cell-free system and were catalyzed stepwise by the methyltransferase and oxidoreductase enzymes. In the feeding experiments of strain NIAH-26, the convertibility from ST to AFB1-AFG1 was found to be remarkably suppressed by the coexistence of DHST in the medium, and the convertibility from DHST to AFB2-AFG2 was also suppressed by the presence of ST. When some other mutants which endogenously produce a small amount of aflatoxins (mainly AFB1 and AFG1) were cultured with DHST, the amounts of AFB1 and AFG1 produced were significantly decreased, whereas AFB2 and AFG2 were newly produced. In similar feeding experiments in which 27 kinds of mutants including these mutants were used, most of the mutants which were able to convert exogenous ST to AFB1-AFG1 were also found to convert exogenous DHST to AFB2-AFG2. These results suggest that the same enzymes may be involved in the both biosynthetic pathways from ST to AFB1-AFG1 and DHST to AFB2-AFG2. The reactions described herein were not observed when the molds had been cultured in the YEP medium.