SMEC medium which showed sufficiently capable selectivity in the differential cultivation of fecal and non-fecal coliform bacteria group, had following characteristic properties. 1) To detect E. coli with SMEC medium, the incubation temperature ought to be strictly controlled at 44.5±0.1°C. 2) SMEC medium should be used within the available period for Streptomycin in the medium. 3) Efficiency of SMEC medium is stable for more than 4 months at 4°C. 4) Selective ability of SMEC medium depends upon the number of inoculated coliform bacteria group. It is effective, when the viable count of organisms are less than 107 per ml, whereas when the organisms are more than 108 per ml, the selectivity declines. 5) Selective ability of SMEC medium is independent of mixed ratio between the number of E. coli and that of other organisms of coliform bacteria.
The mycoflora of 219 samples of milled rice harvested in 1965 was determined. Samples in domestic channels were collected from each of the nine geographical areas of Japan. To determine the fungal flora, fifty grains of rice from each sample were plated on Peptone-glucose agar containing chloramphenicol. Fungi were found in 143 of the samples (66.3%), whereas 76 (34.7%) did not yield any fungi. Of the 143 positive samples, 119 samples (83.2%) were usually associated with one to five colonies per 50 grains. No particular relation existed between the frequency of occurrence of fungi and the geographical areas. The total number of fungal isolates was 835 involving more than 25 genera. The major genera of fungi among these isolates were Aspergillus (51.0%), Catenularia (11.0%), Penicillium (9.2%), Fusarium (4.9%) and Cladosporium (4.8%). Other genera such as Helminthosporium (4.0%), Trichoconis (2.4%), Phoma (2.4%), and Piricularia (1.1%) were frequently isolated. The comparatively low incidence of fungi suggests that most of the samples had been kept under suitable storage conditions after harvesting and handling. Aspergillus repens (67.1%) was the most prevalent among 426 isolates of Aspergillus, and Penicillium canescens series (27.3%) was the major group of Penicillium spp. but Penicillium citrinum (3.2%) and Penicillium cyclopium (2.3%) were more widely distributed in the samples than Penicillium canescens. On the basis of these results, the milled rice investigated in this study was considered to have been conditioned and handled under satisfactory conditions.
Fungal isolates from rice and flours were tested to determine if aflatoxin producing fungi are present in Japan. One hundred and fifteen strains, including 21 strains of the Aspergillus flavus group, were tested. Fifty one of 115 strains were isolated from rice samples and the others from several kinds of flours including wheat, red bean, and soybean. The isolates were selected from a total of 1300 on the basis of frequency of isolation of species except that all Aspergillus flavus isolates were tested. In primary screening tests, the cultures were grown in shake culture for 96 hours in modified Czapek liquid medium. The presence of aflatoxins were determined by thin-layer chromatography of chloroform extract of the broth. Eleven isolates including 5 belonging to the Aspergillus flavusgroup appeared to produce aflatoxin B1 alone or B1 and B2. Further large scale tests indicated that strains of Aspergillus flavus produced compounds which showed the same thin layer chromatographic behaviours as authentic aflatoxin B1. Production of aflatoxin B1 was confirmed by the trifluoroacetylation procedure of Andrellos and Reids (1964), and by measurement of ultraviolet and infrared spectra. Oral administration to rats of extract of one strain of Aspergillus flavus (WF-3-8) caused histopathological change to the livers accompanied with bile duct hyperplasia similar to that observed in the liver of rats fed toxic peanut meal. This is the first report of aflatoxin producing fungi isolated from foodstuffs in Japan. No aflatoxin producing strains were found among those isolated from rice samples.
It was previously reported by Vogel et al. that EDTA reacted with cobalt ion and hydrogen peroxide with development of reddish purple color, and the reaction was applied to the analysis of EDTA in edibles, e. g. wine or fruit juice. The present experiment was undertaken to see in detail about some conditions necessary for the color reaction, and results obtained were as follows. 1) Amount of cobaltous nitrate solution to be added was found to be equivalent by mole ratio to amount of EDTA. 2) The color intensity was maximum when the concentration of hydrogen peroxide in sample solution was over 1.25M. 3) The color intensity was maximum when sample solution was at pH 3 to 4, whereas at pH 1 to 2 or at pH 5 it was lower than in the former case and at pH 6 the color was decomposed while heating. 4) There was no significant difference of absorbance between the procedure involving heating twice and the one involving heating once, respectively after adding the two reagents. In the latter procedure, the absorbance was stabilized by heating for 10 to 15 minutes at pH 1 to 4, but at pH 5 it reached to the maximum by heating for 5 minutes and declined by following heating. 5) The color produced within the pH range 1 to 4 was stable for 24 hours at room temperature.
The present experiment was aimed at separating EDTA by ion exchange resin method from free amino acids in foods which interfere with the color reaction of EDTA with metal ions based on complex production. By filtration through weak alkaline anion exchange resin column adjusted to pH 2.1±0.1, EDTA was adsorbed on the resin column and interferring amino acids passed through it. The adsorbed EDTA was eluted by 2N HCl. Typical results by this method are given in Fig. 2. Detectable limit of EDTA by the combination of this separation method and the color reaction with cobalt ion and hydrogen peroxide was about 0.4mg.
In order to obtain the basic data to prevent the outbreaks of Shigellosis and Typhoid fever caused by polluted drinking water or foods, the fate of Sh. sonnei and S. typhi in well water and various kinds of food were investigated. Both Sh. sonnei and S. typhi, which can be survived for at least 6 days or longer in well water, were killed within 6 hours by adding 0.4ppm of chlorine. When well water polluted with stool was disinfected with the same amount of chlorine, the both bacteria survived 18-96 hours. The polluted well water in this expemiments was prepared by adding stool in such an amount (0.001%), as to give no perceptible change in tint, turbidity and smell. Further detailed experiments revealed that in successful chlorine disinfection to both bacteria in the above mentioned polluted well water or standing water, such an amount of sodium hypochlorite as to produce 3ppm/ml of free chlorine is necessary. Experiments on food were performed with bread, salad, “Fukujinzuke” (a kind of Japanese pickles), raw cream, commercial milk and cheese, all of which can be taken without any treatment, and beefs untreated and heated at 100°C for 30 minutes. In this experiments, bread and “Fukujinzuke” were preserved at 25°C, and other foods were preserved at 5°C. All of these were inoculated with each 106 cells of Sh. sonnei and S. typhi, respectively, and the fate of these bacteria were pursued. In bread, both bocteria survived as long as 30 days. In salad, they showed slight growth at 4 hours after the inoculation, and remained constant level for the succeeding 20 days, after that the curves weme declined gradually, and in the case of S. typhi, they diminished at the 30th day, but in the case of Sh. sonnei, they could survive little longer. In case of raw cream, both of the bacteria were demonstrated until the end of the 4th week, and in commercial milk, S. typhi survived for 30 days and Sh. sonnei more than 75 days. In raw beef, they survived more than 10 days. In heat treated beef, Sh. sonnei survived for 2 weeks, and S. typhi for 4 weeks. In case of “Fukujinzuke”, the results were quite different from other above mentioned foods, i. e., both bacteria were killed within 48 hours after inoculation. This is considered to be the influence of food preservatives contained in this pickle.
Hydrogen peroxide as a bleaching agent has been used to Japanese noodle and domestic-made fish-paste products (“Hanpen”, “Kamaboko”, “Naruto” and “Chikuwa” etc.) and residual quantity of hydrogen peroxide in such foods has caused an important problem from the viewpoint of toxicity, and at the same time sorbic acid is generally added in the fish-paste products. In view of the facts mentioned above, the authors made some experiments and obtained following results. 1. Most of the fish-paste products showed positive reaction indicating the presence of hydrogen peroxide by using a 5% titanium sulfate solution or a 0.1% V2O5 sulfuric acid solution (4→100) as a reagent. 2. The concentration of hydrogen peroxide was determined by iodometry and the distribution of hydrogen peroxide contained in the commercial products was presented in Table 1, its change in the course of time in Figs. 1, 2, 3 and 4. 3. It was clarified that sorbic acid was decomposed by steam-distillation in the presence of hydrogen peroxide in the case of the determination of sorbic acid in those products, but the decomposition was prevented by adding peroxidase prior to distillation, on the other hand by means of dialysis method sorbic acid was not affected by hydrogen peroxide and could be quantitatively separated from the products. The concentration of sorbic acid in a test solution prepared by the separation method mentioned above was determined by ultraviolet absorption spectrum method. 4. The concentration of sorbic acid was almost never affected by immersing the fish-paste products in a 3% hydrogen peroxide solution as shown in Figs. 3 and 4.