The determination of diethylcarbonate (DC) and its formation from diethylpyrocarbonate (DEPC) were studied.
1. Distillation-method: 50ml of a sample solution containing 1.5-25mg of DC was directly distilled, and the distillate was subjected to gas chromatography (GC) with a 1 meter column of C₂₂ supporting 10% of polyethylene glycol 1000 at 100°C (HFID detector). When isobutanol was contained in the sample, the peak of DC was piled up that of isobutanol. Therefore, the method of alkali-treatment of DC was studied. This method was suitable for the determination of DC at the concentration more than 25ppm.
2. Pentane extraction-method: Shake 50ml of a sample solution containing 0.1-2.0mg of DC with 100ml of pentane two times respectively. After careful evaporation to 2-20ml of the extract, the extract was subjected to GC. In parallel with the test, the blank sample not containing DC should be examined with the same method, because a small parts of isobutanol in sample was extracted with pentane (for example, the amount of DC corresponding to isobutanol extracted from 100ml of “Sake” or wine was 0.2-0.3mg). The recovery was 85-90%.
3. About 5% of DEPC was found as DC on gas chromatogram. Therefore, for the determination of DC in asample containing larger amounts of DEPC, n-butylamine should be added to pentane extract in order to decompose DEPC.
4. The higher the content of alcohol, the more was DC produced from DEPC.
5. An amount of DC in “Koji” extract was decreased during the fermentation by yeast.

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イソキサンテン系食用色素ローズベンガル, フロキシン, エリスロシンおよびエオシンとウシ血しょうアルブミン, 卵アルブミン, カゼインおよびファゼオリンの間のメタクロマジーを調べた. この結果, タンパク質ではウシ血しょうアルブミン, 色素ではローズベンガルが強いメタクロマジーを示した. また, タンパク質では卵アルブミン, 色素ではエオシンが弱いようである. しかし, メタクロマジーの発現はかなり多様で, それぞれの系およびpHその他の条件で調べる必要がある.

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タートラジンおよびエロー2G, アゾ系色素レッド2Gおよびレッド6Bとウシ血漿アルブミンの間のpH変化による結合数を調べ, 結合に関与する基を推定した. また, 前記色素にポンソーR, 3RおよびSXを加え, 最大結合数および結合の自由エネルギーを求めたが, タートラジンは, 他の色素に比べ結合数も少なくまた結合力も弱いことがわかった.

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In this paper, the Arnon Iiquid culture method (A); the ameliorated Arnon culture method by adding 0.1% Mg (OAc) ₂ and 1% glucose (B) to Chlorella vulgaris; the Henneberg liqiud culture method to Saccharomyces cerevistae var. Sake the Brewing Institute No. 6 were respectively applied to toxicity test of twenty two food pigments at the concentration of 0.1 and 0.01 percentages of these dyes, and during 4 days as the culture period for the method (A) and 7 days for the method (B) to Chlorella at about 25°C under 10W white fluorescent lamp and during 4 days as the culture period at ca. 35°C to Saccharomyces, the following four points were inspected: (1) the observation of the outlook by naked eye, (2) the micrometric determination of cell number by Thomas' method and the ratio Sample/Std., (3) the micrometric determination of cell diameter and the ratio Sample/Std., and (4) the microscopic observation of inner structure and coloration grade of these organisms.
Results of the test were as follows:
1. The coloration of the cell was more or less a bad phenomenon to the life cycles and appeared in thirteen cases: Red No. 1, 3, 101, 103, 104, 105, 106, Orange No. 1, 2, Yellow No. 2, Green No. 1, Blue No. 1 and Violet No. 1.
2. Red No. 4, Yellow No. 1 and Blue No. 2 had also influence to inhibit the life cycles without coloration.
3. From the results of the examination, four pigments-Red No. 2 (Amaranth), 102 (New Coccine), Yellow No. 4 (Tartrazine) and No. 5 (Sunset Yellow FCF) -had almost no influence on the life cycles and three of them except New Coccine are ones having the highest safety grade among all pigments reported hitherto by the Expert Committee of Food Additives of FAO/WHO.
4. The coloration of the cell by pigment was not always similar in both cases of microorganisms. Which term of the life cycle was affected by the pigments was different in each case but generally the addition of pigment had more or less bad influence on all over the life cycle when it colored. For example, on the test of Saccharomyces, the bubbling of carbon dioxide and then the ethanol fermentation decreased in the cases of Red No. 104, 105 and Orange No. 1 and especially an infragrant smell was emitted in the case of Red No. 105.
5. The adequate concentration of pigment in the test medium which contained 10⁶-10⁸ cells was about 0.1 percentage in all cases because the toxicity test was effectively examined. The pH of medium changed somehow to acidic area but at least remained at 4.5. At this pH figure, there appeared almost no influence on the life cycle and as one of the special case, Blue No. 2 discolored during the culture but this discoloration did not depend on the acidic pH.
In view of the above description, it will be summarized that any pigment may not be available or desirable as food additive when some bad influences appeared on the life cycle of the microorganisms in this method. None the less, as this method is aimed at inspection for only four items at some constant and not various concentrations of pigment, so the result does not always show direct concern with deciding on the availability of food additives.

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According to the previous method, twelve sorts of official food preservatives and antimicrobials, chloramine B and T, n-butyl and n-propyl benzoate, p-oxybenzoate, propionic acid, butylsalicylate, and other carboxylic esters were examined. Results of the test were as follows:
1. By the disturbance of life cycles of Chlorella and Saccharomyces, the bacteriostatic or bactericidal action was inspected easily as a comparative reference to bacteria.
2. This method might be available as a comparative safety test of food preservatives or antimicrobials when used by the multiple dilution culture.
3. In the case of acidic preservatives such as benzoic acid, salicylic acid or sorbic acid, the correlation of pH between additives and medium or food had great influence to the preservative action.
4. Esters of acidic preservatives indicated generally a greater bacteriostatic action than that of the free acids.
In this paper, the effect of preservatives or antimicrobials was tested only by addition of sugar as one of five principal nutrients.

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Thin layer chromatography has been applied to the analysis of reduction products of 12 monoazo dyes (Table 2).
The dyes were reduced with stannous chloride and then spotted on cellulose plate. Mixtures of methyl acetate, glacial acetic acid and water (8: 2: 5 or 9: 2: 4), chloroform, methyl alcohol and water (1: 4: 1), and benzene, methyl alcohol and water (2: 6: 1) were employed as developing solvents for the reduction products. The location of the reduction products was carried out by irradiation of ultraviolet light and by spraying such chromogenic agents as 1-naphthol-4-sulfonic acid, 1- (4-sulfophenyl) -3-methyl-5-pyrazole, phenylhydrazine-p-sulfonic acid and pyridine-2-aldehyde. 1-Naphthol-4-sulfonic acid gave good result for diazo component, and phenylhydrazine-p-sulfonic acid or pyridine-2-aldehyde for amines obtained from azo components respectively.
Infrared spectra of 1-amino-2-naphthol-3, 6-disulfonic acid, 1-amino-2-naphthol-6, 8-disulfonic acid, 1-amino-2-naphthol-6-sulfonic acid, and 2-amino-1-naphthol-4-sulfonic acid produced from the monoazo dyes are shown in Figs. 1-4. The Rf values for the reduction products of monoazo dyes are summarized in Table 2.

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The structure of xanthene dyes can be clarified by hydrolyzing the dyes with caustic alkalies and by analyzing products resulted from the hydrolysis. The present paper describes the separation and detection of the hydrolysed products by means of thin layer chromatography.
When fluoresceine is hydrolyzed with 50% sodium hydroxide solution, it yields resorcinol and 2- (2′, 4′-dihydroxybenzoyl) -benzoic acid. The structure of the latter compound has been made clear by the infrared and nuclear magnetic resonance spectra. These two products could be separated on a thin layer of sillica gel, using a solvent system of dioxane+dichloromethane+pyridine (5: 18: 0.8). The location of the spots was achieved by spraying the chromatoplate with a solution of diazotized sulfanilic acid and then by exposing to NH₃ vapour. Resorcinol was revealed as a brownish-yellow colored spot and 2- (2′, 4′-dihydroxybenzoyl) -benzoic acid as a brown colored spot.
In the case of eosine, its hydrolysed products, i. e. 2, 4-dibromoresorcinol and 2- (3′, 5′-dibromo-2′, 4′-dihydroxybenzoyl) -benzoic acid have been confirmed by the infrared and nuclear magnetic resonance spectra. They could be separated on a thin layer of silica gel each of them, using ethyl alcohol+benzene+glacial acetic acid (25: 90: 4) and the location of the two spots was carried out in the same way. Chromatograms are shown in the Figs. 1 and 6.

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When dibromofluoresceine is hydrolyzed with 50% sodium hydroxide solution, it yields 2-bromoresorcinol and 2- (3′-bromo-2′, 4′-dihydroxybenzoyl) -benzoic acid. The structures of these two products have been clarified by the measurement of their melting points respectively, and their infrared and nuclear magnetic resonance spectra. These two products could be separated each other on a thin layer of silica gel, using a solvent system of isopropyl ether+glacial acetic acid (10: 0.1). The location of the spots was achieved, using diazotized sulfanilic acid as a chromogenic agent.
Various solvent combinations were examined for the separation of resorcinol, 2- (2′, 4′-dihydroxybenzoyl) -benzoic acid, and their bromine derivatives. The solvent system of chloroform+glacial acetic acid (4: 1) gave the best result. One of the developing solvents examined was dichloromethane, which was found to give the best separation for resorcinol and its bromine derivatives. There was a linear relationship between the Rf values and the number of bromine in these compounds. The compounds studied and Rf values obtained by use of four solvent systems are given in Table 3.

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Several kinds of determination methods of diethylpyrocarbonate (DEPC) have been published, but most of them are indirect methods, for example, the neutralization titration method.
In this paper, the new direct determination method is described. The principle of the method bases on the fact that DEPC is converted to phenylurethane on reacting with aniline, which is determined by gas chromatography. Chromosorb W containing DC 550 (10%) is suitable as the column and 2-ethoxynaphthalene or 1-methylnaphthalene as the internal standard substance.
This method was applied to “Sake”, wine, beer and fruit juice, in which 100ppm of DEPC were added.
The gas chromatogram was not interfered by the components contained in foods and the recovery was over 95%.
The rate of decomposition of DEPC in the foods was about 90% after three hours at 20°C and almost 100% after 24 hours.

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Lane-Eynon volumetric method, the Japanese official method for the determination of iactose in milk, was compared with Chloramine-T method, designated as international standard by IDF, and Munson-Walker gravimetric method.
Chloramine-T and Munson-Walker method gave 0.15% and 0.23% higher lactose values respectively than Lane-Eynon method in the case of market milk. The differences in resuits by the three methods were significant at 1% level of probability. The standard deviations of the three methods were 0.057 for Lane-Eynon, 0.058 for Chloramine-T, and 0.043 for Munson-Walker method.

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