[³H] Stevioside was administered orally at a dose of 125mg/kg to Wistar rats, and its disposition and metabolism were studied. The level of radioactivity in the blood increased slowly to a maximum of 4.83μg/ml at 8 hours, exhibiting a biological half-life of 24 hours. At 1 hour, the highest concentration was observed in the small intestine, followed by the stomach and cecum in that order. At 4 hours, the concentration in the cecum was markedly higher than those of other tissues. Radioactivity remaining in the body at 48 hours was 30.7% of the dose. At 120 hours, the percentages of radioactivity excreted into the feces and expired air were 68.4% and 23.9%, respectively, while radioactivity excreted into the urine was only 2.3%. Radioactivity excreted into the bile at 72 hours was 40.9% of the original dose. From the results of biliary and fecal excretion, it was concluded that entero-hepatic circulation occurs in the body.
TLC analysis of the intestinal contents, feces and bile showed that stevioside is decomposed by cecal flora to steviol and sugars, indicating that steviol and these sugars are absorbed from the cecum, distributed throughout the whole body, and excreted mainly into feces and expired air.

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The adulterant starches mixed in so-called Yoshino Kuzu, which is one of the common wild vines in Japan, were studied, and a method for evaluating their mixing ratios was investigated.
It was found by means of X-ray diffraction and scanning electron microscope (SEM) analyses that one of the adulterants is a sweet potato starch. The mixing ratio could be evaluated by measuring the particle sizes of starches photographically by SEM, followed by calculating the logarithm of the means of the sizes and statistical comparison.
Examination of six commercial kuzu starches by this method indicated that one of them contained significantly more than 50% of kuzu starch, and three contained less than 50%; the others could not be clearly evaluated.

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A total of 691 specimens of a trumpet shell Charonia sauliae were collected from several locations in Shizuoka Prefecture, and assayed for toxicity of the digestive gland by the official method for tetrodotoxin determination.
1) The frequency of toxic specimens ranged from 30% at Ito to 93% at Shimizu, the average frequency being 66%.
2) The average toxicity of all the specimens assayed was 151MU/g digestive gland. The toxicity score was clearly higher at Shimizu (226MU/g) or Numazu (277MU/g) than at other places.
3) The highest toxicity scores recorded at Shimizu and Numazu were 1, 950 and 2, 580MU/g digestive gland, respectively. At other places, however, fairly toxic specimens (400-500MU/g digestive gland) were found sporadically.
4) The toxicity of trumpet shells in the Shimizu area has tended to decrease in recent years.
5) At Numazu, the toxicity of trumpet shells was higher in spring and summer than in other seasons. Shimizu specimens did not show such a seasonal variation.

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The strictly anaerobic bacterium from a flat sour-spoiled canned “Shiruko” was previously identified as Clostridium sp. In order to classify the species of the isolates, further detailed investigations on the taxonomical characteristics were carried out, in comparison with two reference strains of Clostoridium theymoaceticum DSM 521 and ATCC 31490.
The isolates from flat sour-spoiled canned “Shiruko” showed similar morphological and biochemical characteristics to those of C. thermoaceticum DSM 521 and ATCC 31490. Moreover, flat sour spoilage was observed after “Shiruko” and coffee were inoculated with each of the two reference strains and incubated at 55°C for ten days.
However, several differences were observed between the isolates and the two reference strains as regards the ability to use inositol, ribose and xylose, the pH range for growth, and growth in the medium without glucose.
The above results indicate that the isolates from a flat sour-spoiled canned “Shiruko” should be classified as a xylose nonfermentative biotype of C. thermoaceticum.

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Food Blue No. 1 (Brilliant Blue FCF, Colour Index No. 42090, FD & C Blue No. 1) is prepared by condensation of 1mol of o-sulfobenzaldehyde (OSBA) with 2mol of N-ethyl-N-benzylaniline sulfonic acid (EBASA). This color contains many kinds of subsidiary colors. In this work, the preparation of two kinds of main lower-sulfonated subsidiary colors of Food Blue No. 1, the identification of two lower-sulfonated subsidiary colors in commercial Food Blue No. 1, and the separation and determination of the subsidiary colors by high performance liquid chromatography were investigated.
One of the subsidiary colors, EA-subsidiary color was prepared by condensation of 1mol of N-ethylaniline (EA), 1mol of OSBA and 1mol of EBASA. Another one, EBA-subsidiary color was obtained by condensation of 1mol of N-ethyl-N-benzylaniline (EBA), 1mol of OSBA and 1mol of EBASA. The infrared (IR) spectra of EA- and EBA-subsidiary colors agreed with those of Bell's EA- and EBA-subsidiary colors. The absorption maxima of EA- and EBA-subsidiary colors in 0.02N ammonium acetate solution were at 629nm and 612nm, respectively (Table 1 and Fig. 3).
Food Blue No. 1, EA- and EBA-subsidiary colors were able to be separated by high performance liquid chromatography (HPLC) on a reversed-phase system under the following conditions. Column, Zorbax ODS; eluant, primary 0.5% ammonium carbonate solution, secondary methanol (40-100% methanol linear gradient at 5%/min), detector, 628nm for Food Blue No. 1, 618nm for EA-subsidiary color, 633nm for EBA-subsidiary color, and 253.7nm. The retention times of Food Blue No. 1, EA- and EBA-subsidiary colors were about 7min, 9min and 11min, respectively (Fig. 7). The peak area of each color determined at the maximal absorbance wavelength was about 4 times that at 253.7nm. Linear calibration curves were obtained in the range of 10-100ppm (50-500ng) for Food Blue No. 1 standard, and EA- and EBA-subsidiary colors (Figs. 8-10). The ratio of the peak areas of EA-subsidiary color at 618nm and 628nm was 1.512 (Table 2). The minimum detectable amount was 2.5ng for Food Blue No. 1 standard, 5ng for EA-subsidiary color and 3ng for EBA-subsidiary color (Fig. 11). The recoveries of EA- and EBA-subsidiary colors added to pure Brilliant Blue FCF (free of subsidiary colors) at levels of 1-10% were 92.7-105.4% and 95.8-103.3%, respectively. (Table 3).
The main lower-sulfonated subsidiary colors isolated from commercial Food Blue No. 1 were identified as EA- and EBA-subsidiary colors from their IR and absorption spectra and chromatographic behavior. Ten commercial samples of Food Blue No. 1 were analyzed by this method. EA-subsidiary color content was determined to be 2.7-10.4%, but EBA-subsidiary color was detected at only a trace level in one of the samples (Table 4 and Fig. 12). This method is considered to be suitable for the analysis of EA- and EBA-subsidiary colors in Food Blue No. 1.

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A method is presented for the systematic determination of mono fatty acid estens of propylene glycol (PE), glycerol (GE), sorbitan (SE) and sucrose (SuE).
The above-mentioned emulsifiers were extracted from foods with tetrahydrofuran. The extract was purified by silica gel column chromatography. Di-, triglyceride and other nonpolar substances were washed out with ether-chloroform mixture (1:99), then PE, GE and isosorbide monoesters (SE) were eluted with methanol-chloroform mixture (5:95) and sorbitan monoesters (SE) and SuE were eluted with methanol. These separated emulsifiers were converted to their trimethyl silyl ether derivatives and determined by gas chromatography.
The recoveries of PE, GE and SE added to ice cream, margarine, soy-milk, pudding-powder, and cake-powder at the level of 0.1% were more than 90.0%. Those of SuE were 38.6-98.0%. The detection limits were 10ppm for PE and GE, and 50ppm for SE and SuE.

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Liver extract products from dogfish are currently on sale as a health food because they contain a large amount of squalene, but the mechanisms of most of the biological functions of squalene still remain unclear. We investigated body weight gain and lipids levels in serum and liver of rats orally administered squalene. Daily oral administration of 0.1 or 0.5ml of squalene for 41 days decreased the body weight gain of rats compared with the control, and the 0.5ml dose decreased HDL-cholesterol and increased the thiobarbiturate-reactive substances value (TBA value) in serum. Increases of triglyceride, phospholipid and TBA value in the liver were observed. The apparent absorption rate of squalene at 48hr after administration of 0.5ml was about 50% as calculated from the amounts of squalene administered and squalene found in feces. Therefore it seems likely that squalene modifies lipids metabolism in rats.

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The component patterns of chlordanes (total of 19 compounds) in seawater, river water, and fish (fresh water fish, 4 species; marine fish, 3 species) and shellfish (marine shellfish, 3 species) were examined by mass spectrometry with selected ion monitoring. The samples were collected from several sampling stations in Tokyo Bay, and in the Tama and Edo Rivers during 1980-1984. The component patterns of chlordanes in river water and seawater samples resembled that of technical chlordane. The patterns in various samples were increasingly different from that of technical chlordane in the following order; river water, seawater, marine shellfish, freshwater fish, marine fish. In general, highly chlorinated components (e. g. traps-nonachlor, cis-chlordane, compound 5) in technical chlordane were more persistent in the aquatic biota. The patterns of chlordanes were similar in all freshwater fish (Pseudorasbora parva) samples collected at four sampling stations in the Tama River, and also in muscle and in viscera of each marine and freshwater fish sample. Different patterns were observed in each species of marine and freshwater fish. The metabolites, oxychlordane and heptachlor epoxide, in these aquatic organisms were present at relatively low levels in relation to chlordanes.

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The natural occurrence of mycotoxins in edible oil and the fate of mycotoxins during oil-refining processes were investigated.
During the 2-year period 1984-1985, 60 samples (corn, peanut, cotton seed, olive, safflower, salad, walnut and sesame seed oil) were analyzed for aflatoxins, trichothecenes and zearalenone. Aflatoxins were detected in 5 of 8 samples of the peanut oil (aflatoxin B₁, 0.52-0.72ppb; B₂, 0.09-0.22ppb; G₁, 0.07-0.08ppb and G₂, 0.01ppb).
Crude oils, obtained from corn germ, were spiked with aflatoxins (0.8-1.0ppm), deoxynivalenol (8ppm), nivalenol (8ppm) and zearalenone (10ppm), then refined through processes simulating the practical oil-refining procedures. No detectable mycotoxins remained in the edible oil. In particular, the neutralization processes including alkaline refining and washing, and decolorizing processes eliminated all measurable traces of mycotoxins from the oil.

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Gel filtration profiles of cadmium in soybeans obtained from a cadmium-polluted field were studied. About 60% of the total cadmium was extracted into the aqueous supernatant. Cadmium was associated primarily with the protein fraction of >20, 000 in molecular weight and in particular with macromolecules of >100, 000 in molecular weight. Gel filtration of the cadmium fraction on a Sephadex G-100 in the presence of sodium dodecyl sulfate resulted in a dissociation of the cadmium-binding component (to a molecular weight range of 30, 000 to 100, 000) accompanied by denaturation of soybean proteins. Separation of the major cadmium fraction by gel chromatography in the presence of 2-mercaptoethanol caused the cadmium to appear in a fraction having a molecular weight of about 10, 000. The binding between cadmium and the component in the presence of 2-mercaptoethanol was stable to treatment with ethylene-diaminetetraacetic acid.

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The effects of sodium dodecyl sulfate, 2-mercaptoethanol and EDTA on the distribution and binding status of copper and zinc in the soybean protein fraction were examined by gel filtration on Sephadex G-100. Copper and zinc in the protein fraction were both associated primarily with the major components of >100, 000 in mol wt. The molecular distribution of zinc in the protein fraction of >100, 000mol wt differed slightly from that of copper. Gel filtration of the protein fraction on Sephadex G-100 in the presence of sodium dodecyl sulfate resulted in conversion of both copper- and zinc-binding components to lower molecular weights (30, 000 to 100, 000) accompanied by denaturation of the soybean proteins. Separation of the major protein fraction of >100, 000mol wt in the presence of 2-mercaptoethanol did not cause any significant denaturation of the large proteins but caused a shift of copper to the fraction of about 10, 000mol wt. Only a small amount of zinc was shifted by 2-mercaptoethanol. Zinc in the protein fraction was removed by EDTA in the absence and presence of sodium dodecyl sulfate and/or 2-mercaptoethanol. Copper in the fraction of about 10, 000mol wt in the presence of 2-mercaptoethanol was not affected by EDTA. The stabilization of copper binding by 2-mercaptoethanol was also observed in the presence of sodium dodecyl sulfate.

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Theobromine in foods was determined by gas chromatography (GC). The following procedure was used to clean up samples. The homogenized sample was boiled in alkaline aqueous media, then fat was extracted with n-hexane. The aqueous layer was acidified with diluted hydrochloric acid and sodium chloride was added. Theobromine was extracted from this treated aqueous solution with dichloromethane and the extract was evaporated to dryness. The residue was redissolved in dichloromethane containing internal standard. GC analysis was performed on a column packed with 1% cyclohexane dimethanol succinate on Gaschrom Q, with FID.
In this method, the average recoveries were 99-101%, the coefficient of variation was less than 3% and the limit of determination for theobromine in foods was about 0.005%.

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Benzoic acid (BA) of natural origin in fermented milk is known to be derived from hippuric acid (HA) in raw milk. Since it is forbidden to fortify fermented milk with BA, BA in fermented and HA in raw milk should be precisely determined in order to assess the source of BA.
Our method of simultaneous analysis for both organic acids consists of extracting with acetone, making the extract alkaline with sodium bicarbonate solution, concentrating the mixture, and injecting a sample into a high performance liquid chromatography (HPLC) apparatus. HPLC was performed on a Zorbax ODS column, with 2% acetic acid-acetonitrile (78:22) as the mobile phase and o-methylhippuric acid as an internal standard. The detection limits of BA and HA were each 1ng, and the average recoveries of BA and HA added to milk at levels of 5-40ppm were not less than 98%.

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[in Japanese]

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