In continuation of the grevious work (Shokuhin Eiseigaku Zasshi, 8, 427, 434 (1967)), the decomposition products of tetrachlorofluorescein and phloxine were studied. If the decomposition of these dyes is similar to that of fluorescein, resorcinol and 2- (2′, 4′-dihydroxybenzoyl) -tetrachlorobenzoic acid (2′, 4′-diOHB-tetra Cl-BA) may be yielded from tetrachlorofluorescein, and 2, 4-dibromoresorcinol and 2- (3′, 5′-dibromo-2′, 4′-dihydro-xybenzoyl) -tetrachlorobenzoic acid (2′, 4′-diOH-3′, 5′-diBrB-tetra Cl-BA) from phloxine. However, when tetrachlorofluorescein is hydrolyzed, it yields resorcinol, but 2′, 4′-diOHB-tetra Cl-BA could not be obtained. In the case of phloxine, it yielded 2, 4-dibromoresorcinol and 2-brornoresorcinol mainly, but 2′, 4′-diOH-3′, 5′-diBrB-tetra Cl-BA was not detected. Therefore, 2′, 4′-diOHB-tetra Cl-BA and 2′, 4′-diOH-3′, 5′-diBrB-tetra Cl-BA were synthe-sized and the behavior of these compounds in alkaline medium were studied. On heating in alkaline medium, these compounds transformed into fluorescent substances, alkaline solution of 2′, 4′-diOHB-tetra Cl-BA giving two spots of a greenish fluorescent product (Rf 0.20) and a bluish fluorescent product (Rf 0.06) on a thin layer chromatogram, using a solvent system of chloroform+glacial acetic acid (4: 1), while that of 2′, 4′-diOH-3′, 5′-diBrB-tetra Cl-BA gave a greenish fluorescent product (Rf 0.47) and a bluish fluorescent product (Rf 0.17). These greenish fluorescent products were isolated from these reaction solution and their structures were determined by infrared and nuclear magnetic resonance spectra, together with their analytical data. A greenish fluorescent product obtained from 2′, 4′-diOHB-tetra Cl-BA was 2, 3, 4-trichloro-6-hydroxyxanthone-1-carboxylic acid while that obtained from 2′, 4′-diOH-3′, 5′-diBrB-tetra Cl-BA was found to be 2, 3, 4-trichloro-5, 7-dibromo-6-hydroxyxanthone-1-carboxylic acid. From these results it became clear that 2′, 4′-diOHB-tetra Cl-BA was not obtained when tetrachlorofluorescein was hydrolyzed with 50% sodium hydroxide solution, and 2′, 4′-diOH-3′, 5′-diBrB-tetra Cl-BA, when phloxine was hydrolyzed.
Cells of Clostridium botulinum type A strain 190 grown in thioglycolate medium (GYPT medium) autolysed after having reached a maximum growth. This strain was dissociated into large and small colony-forming types in semisolid media. The cells obtained from the large colony type autolysed more rapidly than those from small one. Washed cells harvested at logarithmic growth phase lysed in phosphate buffer at 37°C within 2-3 hours. Autolysis rose above pH 6.0 and was optimal near pH 7.0. The potential for autolysis reached a maximum toward the end of the logarithmic growth phase and thereafter the cells became resistant to autolysis. The autolytic activity was destreyed by heating the cells at 60°C for 10 minutes and was slightly affected by cysteine (10-2M), N-ethylmaleimide (10-2M) and mercaptoethanol (10-1M). During autolysis nitrogen, protein, nucleic acids, reducing sugars, amino sugars and botulinum toxin were released from the cells as the reduction of the turbidity in cell suspension occurred. Electron microscopic observations on the process of autolysis revealed that the partial lysis of walls occurred first at the end of the organism and the cytoplasmic contents were lost through such lesions. The lysis of the wall centripetally spread and finally the morphological entity of the wall was completely lost. From these findings it is suggested that the autolysis may be proceded by auto-digestion of the cell wall at the end of the organism.
Most of the cells of Clostridium botulinum type A strain 190 harvested at logarithmic growth phase in GYPT medium were converted autolytically to spheroplasts in 0.5M sucrose-phosphate buffer within 2-3 hours at 37°C. Electron microscopic observations on the formation of spheroplasts demonstrated that the cytoplasmic contents were extruded through partly dissolved walls at the end of the organism and formed spherical bodies. Crude wall fraction, isolated from logarithmic phase cultures by sonication and fraction-ation, rapidly autolysed in phosphate buffer. The wall fraction isolated from the large colony type of the strain lysed more rapidly than those from the small one as reported on the whole cells. Reducing sugars and amino sugars being main constituents of the wall were released from the wall fraction as wall-autolysis occurred. Electron micorscopic study showed that the rigid structure of the wall was completely lost and only fragile membranous or amorphous components remained as residues. Heated wall preparations digested with trypsin and nagarse were dissolved by a soluble wall-autolysate, but not by a soluble cytoplasmic fraction. It seems likely that autolytic enzyme system may exist at or near the cell wall.
The present studies were undertaken to determine the population of toxigenic fungi in domestic milled rice harvested in 1965. Of a total of 835 isolates, the toxigenicity of 82 representative strains has been investigated by subcutaneous administration with cultural filtrates of the fungi and methanol extracts obtained from mold-rice substrates. The experimental animals, male mice weighing 15-18g, were subcutaneously injected with 0.5ml/mouse of the cultural filtrates and with 25mg, 250mg and 1, 250mg/kg of the methanol extracts, respectively, in a single dose. Results of the tests indicated that fungal metabolites from one strain of Penicillium expansum and Penicillium islandicum were highly toxic, and fungal metabolites from one strain of P. cyclopium, P. cylclopium var. echinulatum, P. martensii, P. decumbens, P. expansum and Aspergillus versicolor, and 2 strains of P. roqueforti were mildly toxic. Toxic effects were not found in the other fungi, including the groups belonging to Ascomycetes and Fungi Imperfecti. Thus, highly toxic fungi were found in 2 of representative strains, a frequency of 2.4%. The incidence of mild toxic fungi was 9.7%. This is the first report of the prevalence of toxic fungi in domestic rice. Known mycotoxins such as patulin, luteroskyrin, and islanditoxin and two different unknown were detected. Except for a few cases of Fusarium toxicoses, human mycotoxin poisoning has not ever been happened in Japan. These facts could be considered as evidence for a qualitative interpretation of mycotoxicosis in the Japanese people who consume rice as their main food.
During a population survey of toxic fungi in foodstuffs, two strains of highly toxic fungi and nine strains of mildly toxic fungi were detected. Toxic metabolites of these 11 strains of fungi were administrated subcutaneously to male mice weighing 15-18g, respectively, and seven days after the single administration, experimental animals were killed and histopathological observations of various organs were carried out. The nature and the degree of the induced histopathological changes of various organs of mice were throughly investigated and described. Hepatic injuries, e. g., irregularity of the nuclear size of hepatic cells, occasional mitotic figures, and inflammatory changes were commonly recognized as an induced pathologic change by all fungal toxic metabolites used in this study, however, the hepatic changes seemed to be nonspecific reactions. The most conspicuous hepatic injuries were noticed in mice administered toxic metabolites of the Penicillium islandicum. Nephrotoxity of the Penicillium islandicum, and neurotoxity of the Penicillium decumbens and the Penicillium cyclopium were indicated. As it has been demonstrated in the previous toxicity study, it was noteworthy that even with toxic metabolites obtained from the same fungus, different methods of obtaining the toxic metabolites caused a different pathological effect. Individual differences in susceptibility among experimental animals to the fungal toxic metabolites were also observed.
Propylene oxide has been known to be effective against insects, fungi and bacteria. The authors determined gas chromatographically propylene oxide residue in various foods treated with propylene oxide vapor. Fumigation and gas chromatographic conditions are as follows: fumigation temperature 40, 50°C time 60, 120, 180, 240min pressure 1atm vacuum 60mm/Hg gas chromatograph instrument Shimadzu GC-2C column length 1.5m, internal diameter 3mm packing propylene glycol 6000 (25%) support Chromosorb W (30-60 mesh) carrier gas He flow rate 60ml/min detector TCD temperature column 65°C injection port 100°C chart speed 10mm/min The results of the determination were shown in. Table 1. The residue of propylene oxide was found in some fumigated foods. The residue was probably caused by being dissolved in the fat contained in food or being adsorbed on the surface of food.