Recently, interest in applying natural sweeteners as sugar substitutes has increased in response to consumer desire for health products. One natural sweetener commonly used in Japan is licorice (Glycyrrhiza glabra L.). Licorice sweeteners are commercially available in three forms; licorice extracts, licorice hydrolysates, and enzymatically modified licorice. In addition to GA, they contain a variety of other substances. But the currently used analytical methods only assay for GA. Therefore, it is necessary to develop new, more comprehensive approaches. In the present study, qualitative and quantitative analyses of licorice derived preparations were therefore conducted. In preliminary experiments, it was found that GA and the three forms of licorice sweeteners could be clearly separated into their components by thin layer chromatography (TLC). Consequently we performed reversed-phase high performance liquid chromatography (HPLC) with UV detection at 245 nm with silica gel columns (4.6mm×150mm). And methanol-2% acetic acid (70:30) as the mobile phase. Results are shown in Fig. 5. Each of the three licorice products gave their own characteristics chromatograms. The substances responsible for peaks were identified using liquid chromatography-mass spectrometry (LC/MS) with analysis by negative electro spray ionization (ESI). The peak designated d in Fig. 5. was defined as 23- or 24 hydroxy GA, and f as liquiritic acid. The peak g in fig. 1-b was GA-3-O-glucuronide, and the peaks h throuhg j were assumed to be differently glycocylated GAs. Quantitative analyses of the identified substances were possible by UV-HPLC. The contents of each of the substances were in line with the known concentrations of GA in the three products. The recoveries from food samples spiked with 100μg/g of each licorice product were 76.7〜110%. Furthermore, analysis of GA and licorice related substances in four commercial foods labeled as including added licorice with on method revealed three of them to contain licorice extract and one to have a licorice hydrolysate supplement. No enzymatically modified licorice had been added to any these foods. It is concluded that our present method is very useful for the analysis of licorice related substances including GA.

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味噌及び醤油中のエタノール濃度をバイオセンサーにより測定し、ガスクロマトグラフィーによる測定値と比較した。バイオセンサーの測定値はガスクロマトグラフィーと良い相関を示した。エタノール測定のために酢酸菌を用いてバイオ膜を作製した。バイオ膜の耐久性を調べるために保存試験を行い。5℃では1ヶ月間、室温では3日間保存後のバイオ膜を使用したバイオセンサーで、精度及び再現性良く測定することが可能であった。エタノール添加回収試験では、ほぼ90%の測定値を得ることができた。また、バイオセンサーによる味噌及び醤油10試料のエタノール測定値はガスクロマトグラフィーによる測定値と良く一致した。製造工程でエタノールを用いていない場合は、0.44〜0.77%であった。醤油においては、それぞれ2.44〜3.30%と、0.87〜0.98%の範囲であった。以上の結果から、バイオセンサー法は食品のエタノール測定に適していることがわかった。

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シソ科植物26およびムラサキ科植物4種、合計30種のハーブにおけるコーヒー酸誘導体類の生産について調査した。分析した化合物はrosmarinic acid (RA), lithospermic acid (LA)およびlithospermic acid B (LAB)の3種のポリフェノール類であり、これらの化合物は抗酸化物質としての利用のみならず、腎機能改善や抗ウイルス等の薬用生理活性物質としても期待されている。分析には、高速液体クロマトグラフィーを用いた。シソ科植物については、調査したすべての植物種においてRAの生産が認められた。含量については、幅があり、0.009%(Mentha spicata)から4.574% (Melissa officinalis)まで認められた。RAに比べて含量は少ないが、LAとLABの生産もいくつかの種で認められた。LAおよびLABの最高値は、Salvia miltiorrhizaで認められた(LA, 1.535%およびLAB, 5.106%)。ムラサキ科植物では、シソ科植物と比べると低含量ではあるが、多くの植物でRAの生産が認められた。Symphytum officinaleでは、分析した3種の化合物は検出されなかった。Lithospermum erythrorhizonの根では高含量のLA (4.735%)とLAB (2.882%)が認められた。調査した、両科の植物において、これらフェノール類の器官特異的な蓄積は認められなかった。今回の結果は、過去のいくつかの分析例と必ずしも一致するものではないことから、分析植物のサンプリングの磁気、栽培条件の違いなどにより、これらフェノール類含量はかなり変動するものと思われる。シソ科植物11種およびムラサキ科植物1種、合計12種のハーブについて、茎葉培養系を作製し、その培養体におけるRA, LAおよびLABの生産についても調査した。調査した植物種のうち、Coleus forskohlii, Ocimim basiticum 'Sweet' およびHeliotropium pervianumの3種以外の植物種については、圃場栽培した親植物体の方が、in vitro培養体よりも高含量のRAを含んでいた。O. basilicum 'Sweet'とH. pervianumではin vitro培養体の方が親植物体よりもRA含量が高く、またC. forskohliiにおいては、in vitro培養体および親植物体ともほぼ同等のRA生産を示した。このことは、シソ科およびムラサキ科植物を、特殊な環境条件下(in vitro)で培養した場合、そのRA代謝に与える影響に関して、品種間に差のあることを示していた。O. basilicum 'Sweet' in vitro培養体は、RA含量の最高値(5.012%, 葉部、乾燥重量あたり)を、またH. pervianumは、LAB含量の最高値(1.125%, 根部)を示した。これら2種の培養植物体は、RAおよびLABの高生産系、また、生合成研究材料として有用かもしれない。5種のシソ科ハーブについて、カルス培養系を作製し、その培養系における上記コーヒー酸誘導体類の生産についても検討した。調査した5種の植物のうち、Melissa officinalis以外の4種のカルスにおいて、親植物体よりも高いRA生産が認められた。Hyssopus officinalisとOcimim basilicum 'Sweet'の2種のカルスでは、茎葉培養体よりもさらに高いRA生産が認められた。M. officinalisは、茎葉培養体およびカルスの両方において、親植物体に比較して、低いRAの生産を示した。この植物種では、組織培養条件下は、RA生産には適していないと思われる。以前の報告で、O. basilicum 'Sweet' の根培養系ではより高いRA生産が認められていることから、H.officinalisにおいても、同様の培養系においてRAの高生産が期待される。

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Allergy is one of the serious problems in the present world, so it is very valuable that we could regulate the allergy with foods we eat every day life. To establish simple and rapid methods of searching anti allergic substances in foods, it is important to have proper test systems. Previously, we established an experimental system, in which we could examine the inhibitory effect of food components on the antigen specific and IgE dependent anaphylactic hypotension induced in mice on the 9th day of active immunization. Considering the principle of the above assay system based on the anaphylaxis, it might be possible that similar phenomena could be observed more rapidly by a passive immunization of IgE antibody. In our present study, normal mouse was sensitized with mouse monoclonal anti DNP IgE antibody and 24 hours later, challenged with DNP-HSA (dinitrophenyl human serum albumin). Test samples prepared from foods were possible to be orally administered before challenge. Then blood pressure was measured in the same way as previous method as shown in Fig. 1. In this system, both diphenydramine and dexamethasone as the standard anti allergic substances, administered intravenously. 1 hour before being challenged, showed the inhibition of the anaphylactic hypotension about 70% at the dose of 10mg/kg respectively. Anti allergic activities of foods were examined by oral administration of extracts of food samples 1 hour before challenge. As shown in Fig. 2, beefsteak tree, green tea, oolong tea and maidenhair tree showed 69.2%, 69.7%, 88.8% and 70.5% inhibition of the anaphylactic hypotension. None of them had hypotensive activities on the normal mice, therefore, their inhibition of anaphylactic hypotension are thought to be related with any anti allergic effects.

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Previously, we proposed a standard concept for quantification of the intensities of mouse anaphylactic responses using fall in blood pressure (mouse anaphylactic hypotension test; MAHT) after treatment with food allergens. In the present study, optimal rout for elicitation of an anaphylactic response in the MAHT were examined with comparison of intravenous (i.v.) and intraperitoneal (i.p.) administrations. Since only water-soluble fractions were appropriate for i.v. administration, food samples were extracted with three kinds of buffers at different pHs. The water-insoluble fractions which could not be extracted in this way, therefore could not be assessed for allergenicity. In contrast, whole food samples could be administered i.p. in saline. To determine whether i.p. administration was appropriate to detect allergenicities, several standard antigens such as hen egg-white lysozyme, ovalbumin and ovomucoid were tested. All looked similar response to i.v. administration (Fig. 3). Further examination of foodstuffs known to be potent allergens in man, including egg-white (raw, boiled), skimmed milk, mackerek, soybean, wheat flour, rice and buchwheat gave clear positive results, the response to i.p. administration in challenge often being greater than that observed with i.v. administration. This was especially the case with wheat flour, rice and buckwheat samples (Fig. 6). In addition i.p. administration allowed easy quantification of allergic intensity of water-insoluble substances which could not be tested by the i.v. route. In summary, by applying i.p. route of administration, allergenicity of whole food samples, including water-insoluble fractions in saline, could be detected. Accordingly, this very simple and rapid method can be recommended for administering samples for challenge tests.

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This study was performed to estimate the maximum no-effect dose level and the 50% effective dose (ED₅₀) lactitol in causing diarrhea. Fifty-six healthy male and female subjects at the ages of twenties to fifties were enrolled in study. The maximum no-effect dose level of lactitol was 0.075g/kg in males and 0.15g/kg in females. The ED₅₀ was 0.36g/kg in males and 0.43g/kg in females. (Fig. 2) Major abdominal symptoms caused by ingestion of lactitol were borborygmus, abdominal distension, and flatus, and lower abdominal pain with large doses. (Fig. 6).

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Phosphate and its salt are added to many food as food additives such as pH adjustants. Phosphates also affect the taste of food in many ways. To beer products, phosphates are added to help fermentation, and they may contribute to taste of beer. So we examined the effect of phophoric anion on the taste of beer. Beer samples were purchased from the market and the reagents used in this experiment were of analytical grade. Phosphoric anion was determined by Shimadzu PIA-100 ion chromatograph equipped with Shim-pack IC-A3 (S) column (2.0mm i.d. ×150mm), electric conductivity detector. The mobile phase was 8mM p-hydroxybenzoate and 3.2mM Bis-Tris mixture solution, and elution rate was 0.2ml/min. Sample was degassed and filterered (0.45μm) before injection. Injection volume was 10μl. The taste study was conducted according to common methods and 12 volunteers per each beer sample were randomly selected. The kind of taste or feelings of beer samples tested were bitter and gulpy feeling. The results were as follows: 1) The chromatographic separation of several stansard anions following conditions described above was good enough to determine each anions. The recovery rate of 12.5ppm phosphoric anion added to beer sample was 90.4-109.6% (99.2% mean). 2) The concentration of phosphoric anion ranged from 200 to 400ppm and differed from brand to brand even if they were brewed at the same company. But there were little difference among the lot of same brand. 3) The concentration of phosphoric anion and the extent of bitter taste were positively correlated with correlation coefficient of 0.7473 as shown in Fig. 1. There was no correlations between the content of phosphoric anion and other factors examined in this study.

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Intakes of organochlorine compounds and organophosphorus pesticides from restaurant and box lunches in Shiga Prefecture were estimated in 1993 and 1994 respectively. Estimated intakes of these compounds from restaurant (n=40) and box lunches (n=21) were shown in Table 1 and Table 2. If the concentration of compound in sample was less than the detection limit, the intake value was calculated as zero. High values of these compounds were almost found in the menu of the fishes, but the high value of T-DDT were found in the menu of Hamburg. It is thought that the contamination of fished has continued and Hamburg has been contaminated. The average intakes of organiochlorine compounds from restaurant lunched were higher than those from box lunches, further value of dieldrin and T-chlordane for restaurant lunches were higher than daily intakes estimated by market basket method. Organophosphorous pesticides were below the detection limits. The maximum intakes of organochlorine compounds from restaurant and box lunches and the acceptable daily intake (ADI) were compared. The maximum intake of dieldrin for restaurant lunches corresponded to 30.38% of ADI value, and the intakes of other compounds were considerably lower than ADI values. Intakes of these contaminants from restaurant and box lunches in Shiga Prefecture were at low levels, but the continuous study is necessary to monitor these contaminants in the meals.

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Daily intakes of organochlorine compounds and organophosphorus pesticides in Shiga Prefecture were estimated by the market basket and duplicate portion methods. The results of the market basket method for the period from 1984 to 1996 indicate that daily intake of organo chlorine compounds is on the decrease. In 1996, estimated daily intakes of these compounds were 0.038μg for heptachlorepoxide (HCE), 0.897μg for total DDT (T-DDT), 0.069μg for total HCH (T-HCH), 0.038μg for dieldrin, 0.035μg for HCB, 0.153μg for total chlordane (T-chlordane) and 0.541μg for PCB. These compounds were entirely derived from fish, reflecting contamination of the aqueous environment. The average daily intakes (n=77) of organochlorine compounds, determined by the duplicate portion method in 1991, 1992, were 0.142μg for HCE, 0.660μg for T-DDT, 0.612μg for T-HCH, 0.076μg for dieldrin, 0.074μg for HCB, 0.240μg for T-chlordane and 0.869μg for PCB. Regional differences in daily intakes were found on examination of 6 sites. High daily intakes were noted of HCE for Ohtu city, HCE, T-DDT, T-HCH and PCB for Ohmihatiman city, T-chlordane for Tsutiuama town, T-DDT and T-HCH for Kounan town, HCB for Biwa town and Torahime town and PCB for Kutsuki village. The highest daily intake of T-DDT was 11.4μg, but the value was only 4.57% of the acceptable daily intake (ADI), and maximum daily intakes of the other compounds were well under the ADI values, too. MEP was detected only in one sample by the duplicate portion method and other organophosphorous pesticides were below the detection limits. In conclusion, daily intakes of these contaminants are at low levels in Shiga prefecture, but contained measurement of levels in meals is still necessary.

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Fifty four hydocolloids from natural sources (Table 1) were measured for contaminant microorganisms by the classical agar plate assay and the ATP bioluminescence assay. The agar plate assay revealed that most of these are contaminated with bacteria within the range of 10 to 1,900 counts (as the general bacterial count) per g of the sample (Table 2). Especially, all or some of guar gum, traganthgum, carrageenan, rocastbean gum, gellan gum and xanthan gum gave more than 500 counts per g. All the three pectins tested gave no such count. Some samples were contaminated with funji. Especially, karaya gum 1-2 gave 420 counts of funji per g. Yeast, Escherichia coli and Staphylococcus aureus were not detected at all. All the samples gave a significant luminescence on the ATP bioluminescence assay (Table 3), which shows that these contained ATP. The radiation efficiency with authentic ATP added to the reaction mixture as the internal standard varied remarkably depending on the kind of the samples used (Table 3). A statistical caluculation on 25 samples giving more than 50% of the efficiency indicated a significant correlation between the bacterial count on the agar plate assay and the ATP content obtained from the ATP bioluminescence assay (Table 4, Figs. 1 and 2).

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天然着色料ベニバナ黄色素の主色素成分であるsafflomin Aの構造は、従来報告されていた1 (Fig. 1)ではなく、hydroxy-safflon yellow Aと同じ4 (Fig. 2)であることを、主にNMRとHPLC-PDAの分析結果より明らかにした。また、HPLCによる定量結果より、市販製品中の主色素成分は、4とsafflomin B (3: 別名safflor yellow B)であり、両色素ともほぼ等モルずつ含まれていることが判明した。

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