In our previous report, we described the isolation of two unknown subsidiary colors from commercial Sunset Yellow FCF (Y5) and the identification of their chemical structures. The trisodium salt of 6-hydroxy-7-(4-sulfophenyl)-5-(4-sulfophenylazo)-2-naphthalenesulfonic acid (Y5-SA) is one of the subsidiary colors isolated and it has a sulfonatophenyl moiety located at the 7-position of Y5. As it was thought that Y5-SA is generated under the reaction conditions used in Y5 production, we investigated its mechanism of generation. The experimental synthesis of Y5-SA clarified the following points: 1) When a ratio of the the sulfanilic acid diazonium salt and the Schaeffer's salt were changed and then reacted, the quantity of Y5-SA that was generated increased when the quantity of the sulfanilic acid diazonium salt was increased. 2) When the sulfanilic acid diazonium salt was added to Y5, the amount of Y5-SA that was generated depended on time and the quantity of the sulfanilic acid diazonium salt. 3) There was little generation of Y5-SA at pH 8.0 〜 pH 10.0, and generation peaked at pH 12.0, and then decreaced at pH 13.0. Fig.5 shows the reaction pathways of Y5-SA. According to Mazzola et al, Y5 is balanced as shown in Fig.5-A. They also proved that the pKa of phenol is 12.0 by potentiometric titration and from the chemical shift change of ¹³C-NMR. We concluded as follows: ・Y5 at pH 12.0 is balanced as shown in Fig.5-B. Phenoxide ions that are generated are delocalized and stabilized on the naphthalene ring. ・The anion localized at the 7-position of the naphthalene ring is highly reactive. ・This anion reacts with the cation generated by the decomposition of the sulfanilic acid diazonium salt (Fig. 5-C) to produce Y5-SA (Fig. 5-D). When Y5 is manufactured, Schaeffer's salt and sulfanilic acid diazonium salt are coupled in equal amounts. Therefore, to reduce the amount of Y5-SA in Y5, it is necessary to carry out the reaction at pH 10.0, which is the optimum pH for the coupling reaction used in the generation of Y5. Based on these reacts, Y5-SA can be used as a GMP marker during Y5 production.

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The determination of specific serum IgE has been evaluated for the diagnosis of allergy. Although it is possible to detect the proteins capable of binding the serum IgE in vitro by RAST, Western blot and ELISA, the determined protein is not always shown to be a genuine allergen of the allergy in progress. Therefore, it is necessary for the patients to get correct information of allergens. In the present study, we established a method to identify the true allergen within various proteins contained in foods. The concept of this method is based on the principle that an anaphylactic reaction could be induced with any of the proteins in the sensitizing food separated in SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis), if the mouse was sensitized with a certain food. Thus, this method indicative of anaphylactic reaction makes it possible to identify the allergen just cross-linking the IgEs on mast cells in vivo. In this paper, we used hen egg-white lysozyme (HEL) as a model antigen to confirm this method. Normal male ddY mice of 5 weeks of age were sensitized intraperitoneally with HEL emulsified in Freund's incomplete adjuvant (FIA). Nine to 14 days after the sensitization, antigen challenge in abdominal wall was performed in two different forms. After SDS-PAGE, the one was an injection of homogenized segments of acrylamide gel containing HEL, and the other was an injection of HEL eluted from gel segments by electrophoresis or buffer. It was possible to induce the HEL specific anaphylactic reaction by both challenges. These results demonstrated that a small amount of ingredient separated by SDS-PAGE, even in the presence of the gel, could be applied to AW method. Hence, by the present study, the advantage of SDS-PAGE, in addition to the specification of proteins binding to IgE (Western blot), is expanded to the confirmation whether the protein is true allergen of anaphylactic reaction. The extensive application of this method to identify the true allergen in foods that contains varieties of protein is expected.

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Carrageenan is one of a few high-molecular weight substances, which have a lot of sulfur atoms in their molecules. It may be possible to measure easily average molecular weight and content of carrageenan in vivo by GPC/ICP-AES method, which can detect sulfur selectively in high-molecular weight substances. In this paper, we described the average molecular weights and the contents of fecal carrageenan in rats orally given λ-type refined carrageenan measured by the method. Seven-week-old, Sprague-Dawley rats (one male and one female) were given the mixed diet powder containing 5% of λ-type refined carrageenan for one day. After that, they were given the basic diet powder without carrageenan for 2 days. The feces of rats were collected at the points of 1, 2 and 3 days after the beginning of the mixed diet administration, and stored at -20℃. The consumption of carrageenan sample was 1.2 g/day in male, and 0.91 g/day in female. The number average molecular weight (Mn) of carrageenan sample in feces collected at 1 day after administration was 355 kDa, and the weight average molecular weight (Mw) was 782 kDa in average of two rats. The Mn of fecal carrageenan sample collected at 2 days after admimistration was 303 kDa and the Mw was 718 kDa. It was considered that almost all of λ-type refined carrgeenan fed rats was not degraded via the gut, because the Mn of carrageenan sample in the mixed diet was 338 kDa and the Mw was 832 kDa, measured by GPC/ICP-AES method. The amount of carrageenan sample in feces collected at 1 day after administration was 659 mg in male and 466 mg in female, at 2 days after administration, 377 mg in male and 329 mg in female, and at 3 days after, 10 mg and 6.3 mg, respectively. Therefore, it was considered that almost all of carrageenan sample taken by rats was excreted in feces within 1 day, regarding the schedule of administrations and fecal collections. The recovery of fecal carrageenan sample against the taken was about 90%. It was considered that the recovery of carrageenan sample from feces might depend on the extraction ratio of carrageenan from feces, and the extraction ratio of carrageenan sample would be a further subject.

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The present report concerns results of a 1-year oral toxicity study of Chinese bayberry extract, one active component of which is myricitrin, a flavonoid. F344 rats (20rats/group both sexes) were given diet containing Chinese bayberry extract at doses of 0, 0.5, 1.5 or 5.0%. Decrease in monocytes on differential counts of WBCs and increase of reticulocytes were observed in the 1.5 and 5.0% male groups. Significant decrease in relative lung weights was apparent in the 0.5, 1.5 and 5.0% males. However, these changes were considered incidental effects. No treatment related effects were noted regarding clinical observation, body weights, food and water consumption, ophthalmologic findings, clinical chemistry and pathology. In conclusion, 1 year dietary treatment with Chinese bayberry extract at levels up to 5.0% demonstrated no toxicologically significant effects in either male or female rats. It was thus inferred that the no-observed adverse effect level (NOAEL) was up to 5.0% in both sexes.

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We already investigated the total daily intakes of nitrate and nitrite from the foodstuffs purchased in Japan by the market basket method in 1995 and 1996. This research was also carried out using the same method, that is, three hundred and forty-four kinds of the processed foods and one hundred and thirty-two kinds of the raw foods were collected from markets according to the average intake, then separated and grouped into twelve categories in 1998 and 1999, respectively. Each group was homogenized after addition of water, and analyzed. Nitrate and nitrite in prepared samples were determinded by HPLC with electric conductivity detector and by diazo process, respectively. The total daily intakes of nitrate and nitrite were 190 mg/day and 0.9 mg/day, respectively. Both total daily intakes were slightly lower than those in 1995 and 1996. Nitrate and nitrite were intaked more from the raw foods and the processed foods, respectively. Table 1 Total daily intakes of nitrate and nitrite from processed foods and raw foods [table]

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Stevia extract (Ste) and α-glucosyltransferase-treated stevia (Gts) are included in the List of Existing Food Additives in Japan. A rapid and simple method for simultaneous determination of the stevia sweeteners by HPLC coupled with UV and MS was developed in the present study. Both Ste and Gts contain steviol glycosides as major sweetening components. It was possible to distinguish the HPLC peaks of not only stevioside and rebaudioside A available as purified standard compounds, but also 10 other coexisting glycosides with the aid of LC/MS, owing to the characteristic structural profiles of the glycosides. Analysis with UV-HPLC using ODS column as the stationary phase and MeCN-water (30:70) as mobile phase and detecting at 210nm was commonly profitable for separation of many components of stevia. Further separation of overlapped peaks was achieved by hydroxyapatite (HA) column for Ste (MeCN-water 78:22) and for Gts (gradient from 78:22 to 70:30). Calibration curves drawn by total areas of the peaks of glycosides of Ste and Gts were almost rectilinear. The limits of detection for Ste were 2.0 μg/g (ODS) and 5.0 μg/g (HA), while those for Gts were 2.5 μg/g (ODS) and 25 μg/g (HA), respectively. The recoveries of the constituents of Ste added to several foods were 83.4 〜 95.0 % by ODS, and 80.7 〜 100.9 % by HA, and those of Gts were 84.7 〜 98.5 % by ODS, and 87.8 〜 95.0 % by HA, respectively. Along with the TLC study, the present UV-HPLC method was applied to determine the kind and amount of the sweetener in commercial foods. Pickles contained 23 μg/g of Ste, on the other hand, soft drink and rice cracker contained 269 μg/g, and 24 μg/g of Gts, respectively. Finally, constituents were measured by the present UV-HPLC method in experiments incubating the stevia sweeteners with extract of normal rat's feces. Major glucosides of both Ste and Gts were found to be converted to steviolbioside, possibly through hydrolyses by intestinal microbes. Thus, the present method was shown to be useful to examine the biotransformation of stevia sweeteners.

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Food allergy caused by food allergen, is a hypersensitive reaction of a vital immune system, and is presently a social health problem. In general, subjects with high food specific IgE tend to suffer from food allergy mediated by IgE. On the other hand, it has been reported that some subjects with positive IgE-RAST values against egg white also show positive for chicken meat. It is important to elucidate the cross reactivities between the several food stuffs for the prevention of their cross allergic reactions. Previously, we proposed an in vivo assay system on the basis of the mouse anaphylactic reaction using the abdominal wall (AW method). Further, this system has been developed for identification system of the true allergen in foods by challenge with samples separated by the procedure of SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis). In this study, the cross-reactivity between egg white and chicken meat was evidenced by both in vitro (Western bloting technique) and in vivo (AW method) tests. Moreover, it was ascertained that a heating of chicken meat diminished both its allergenicity and the crossreactivity to egg white.

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One of bacteria on the surface of a soybean was isolated and identified as Bacillus subtilis. The bacterium was named Bacillus subtilis MF920. We studied effect of natural materials to its growth. Growth inhibition by glycine has been known. However, when bacteria were heated with some amino acids, L-serine sterilized more bacteria than glycine. Amino acid analysis clarified that heating did not cause the decomposition of L-serine. This indicated that L-serine itself caused the growth inhibition.

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This study investigated the cause of the illegally high levels of Bisphenol A (BPA), as established by the Food Sanitation Law, found in polycarbonate (PC) tableware for children in 1997. An analysis of the metals in the material of these articles revealed titanium and zinc at levels of 4000 to 7000 ppm. A correlation between the levels of Zn and BPA was also found. Most of the substandard articles had a label stating that they had been treated with an anti-bacterial agent-N, which contains ZnO. Therefore, we investigated the effect of the addition of metal oxides such as TiO₂, ZnO, Fe₂O₃, Al₂O₃, SiO₂ and MgO and anti-bacterial agent-N on the formation of BPA in the PC material. Pellets and test pieces were prepared by adding 0, 0.5, 1.0 and 2.0 % of these oxides and anti-bacterial agent-N under the same conditions to the PC tableware. When the test samples were prepared without the anti-oxidant, the increase in the level of BPA in the material corresponded to the amount of metal oxide that was added except for the SiO₂. ZnO was shown to have the strongest effect on the BPA formation, and when it was added to the PC resin in the ratio of 0.5%, the BPA content in the material exceeded the Food Sanitation Law level (500 ppm). Treatment with more than 1% anti-bacterial agent-N showed the same result. Test samples that were prepared by adding equal amounts of ZnO and anti-oxidant, P-EPQ, suppressed the formation of BPA, while the anti-oxidant, Irgafos-168, showed no effect. Based on these experiments, we concluded that the high level of BPA detected in the PC tableware was caused by the addition of anti-bacterial agent-N, the main component of which is ZnO, and the subsequent choice of the anti-oxidant.

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Contents of strophanthidin and digitoxigenin glycosides in a growing stage of Corchorus olitorius (Moroheiya) were investigated in order to ensure a safety of C. olitorius and its products. Total contents of strophanthidin glycosides were scarcely changed, but those of digitoxigenin glycosides were gradually decreasing in the period from sproutings to seed leaves (1-11 days after seeding). About 80 % of strophanthidin glycosides were detected in seed leaves during the period from seed leaves to four leaves (8-25 days after seeding). Both glycosides were not detected in leaves, stems, roots and buds in the period of harvesting (93 days after seeding) and budding (106 days after seeding). Both strophanthidin and digitoxigenin glycosides in fruits were not detected for 20 days after blooming, but were detected 35 days after blooming. It is difficult to distinguish safe fruits from not safe ones on visual observation in this period. Therefore, it is neccessary to pay atttention not to contain fruits in edible leaves. Strophanthidin glycosides were first detected 35 days after blooming and then increase to reach plateau 60 days after blooming. Digitoxigenin glycosides were detected 35 days after blooming and then no apparent increase was observed over experimental period.

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