Chicken embryo testThe destruction and detoxification of aflatoxins B,, G,, B, and G2 (50 &mL in 4% dimethyl sulfoxide) with ozone were confirmed by thinlayer chromatography and chicken embryo assay. Aflatoxins B1 and G, were sensitive to ozone and easily degraded with 1.1 mgiL of ozone within 5 min at room temperature. The result of Ames test showed the inactivation of the mutagenic activities of the toxins by ozone. Also, the deleterious acute effect of ozone-treated aflatoxin B, was not detected in the rat. On the other hand, aflatoxins B2 and G2 were rather resistant to ozone, requiring SO-60 min to degrade them completely with 34.3 mg/L of ozone.

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To characterize the factors involved in the production of volatile aldehydes during mashing, a model mashing experiment was done. After we inactivated the endogenous lipoxygenase (LOX) activity in the mash by mashing at 709 C for 30 min, further incubation with recombinant barley LOX-1 stimulated the accumulation of 2( E )-nonenal; however, this eŠect was signiˆcantly reduced by boiling the mash sample. The result suggests that both LOX-1 and a heat-stable enzymatic factor are involved in the production of 2( E )-nonenal during mashing. Malt contained fatty acid hydroperoxide lyase-like activity (HPL-like activity) that transformed 9-hydroperoxy-10( E ),12(Z )-octadecadienoic and 13-hydroperoxy-9(Z ),11( E )-octadecadienoic acid into 2( E )-nonenal and hexanal, respectively. Proteinase K sensitivity tests showed that they are distinct factors. 9-HPL-like activity survived through the mashing at 709 C for 30 min but was inactivated by boiling, suggesting it will be the heat-stable enzymatic factor found in the model mashing experiment.

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To increase the thermostability of beta-amylase, seven kinds of single-mutant plasmids were constructed with an expression vector of barley beta-amylase by mutagenesis. The remaining activity versus temperature curves were used to determine the temperatures (T50) at which 50% of the initial activity was lost during a 30-min heating period. These mutations increased the T50 values by amounts ranging from 0.8 to 3.2 degrees C. To express the sevenfold-mutant beta-amylase in Escherichia coli, plasmid pB927 was constructed. E. coli harboring plasmid pB927 produced sevenfold-mutant beta- amylase. The T50 value of purified sevenfold-mutant beta-amylase (69.0 degrees C) was higher than that of not only the original recombinant beta-amylase (57.4 degrees C) by 11.6 degrees C but also soybean beta-amylase (63.2 degrees C) by 5.8 degrees C. The intragenic amino acid replacements were found to have simple additive effects on the thermostability of beta-amylase. The sevenfold-mutant beta-amylase was found to be stable at pHs up to 12.5, while the original recombinant beta-amylase was unstable at pHs above 9.5. The data obtained from kinetics studies suggested that the sevenfold-mutant beta-amylase acquired enhanced thermostability, but its function as a beta-amylase remained unchanged.