The object of this investigation was to determine and to identify the substance obtained as a transformation product of DHA in the boiled or stored acid solutions. For this purpose paper- and thin-layer-chromatographies, ultraviolet spectrometry, polarography, and gas-chromatography techniques were employed. And the results obtained were as follows: 1) DMP was identified as a transformation product of DHA in soft drinks, fermented milk, and other acid juice and solutions. 2) A pathway is suggested for the transformation of DHA via diacetylacetone to DMP. But the fact that small amount of 2, 6-dimethyl-4-pyrone-3-carboxylic acid was isolated from the same solutions might be the evidence for the existence of an other pathway. 3) The oral LD50 for DMP in mouse was found to be 1.67g/kg (95% confidence limits 1.41-1.88g/kg).
Propionic acid, which is admitted as a preservative of bread, was separated by steam distillation and determined by liquid column chromatography. The conditions of liquid column chromatography were as follows: Apparatus JLC. A Type (Nihon Denshi) Fixed phase Dowex 1-X8 Eluent 0.1N HCl Flow rate of eluent 0.6ml/min Column: for separation 0.8×50cm (40°C) for detection 0.8×8cm (35°C) Detector Thermal detector Sensitivity ±1/100°C or ±3/1000°C (Signal×3) Chart speed 60mm/hr The recovery ratio was about 90% in the case of addition experiment to bread, but when propionic acid was previously added before baking, the recovery ratio decreased to about 70%. It was clarified that the low recovery ratio was not due to the consumption of propionic acid by yeast, but the volatilisation of propionic acid while baking. The minimum limit concentration of detectable propionic acid was 25ppm.
The 198 strains of Clostridium perfringens isolated from fishes were tested whether they serologically agree with Hobbs' Types. The results obtained from the experiments are as follows. 1) The 32 strains (16%) of isolated Cl. perfringens 198 strains were in agreement with Hobbs' Types in agglutination test, that is, Type 2-2 strains, Type 3-one strain, Type 4-8 strains, Type 5-7 strains, Type 6-one strain, Type 8-one strain, Type 9-7 strains, Type 12-one strain and Type 13-4 strains were detected (Table 1). 2) The 11 strains belonging to Hobbs' Types, although they came in small numbers, were detected in heated (80°C, 20min) specimens. Moreover, heat-resistant (100°C, 60min) Hobbs' Type strains, namely the 5 strains from body-surface specimens of fishes and the one strain from alimentary canal specimens of those were selectivery isolated by applying the heat-treatment (Tables 1 and 2). 3) In unheated specimens, Hobbs' Type strains were detected, in a low percentage through the period from June to September (Table 3). However, every one of the 21 strains (18 strains from body-surface and 3 strains from alimentary canal) identified as Hobbs' Type strains did not possess heat-resistance in cooked meat cultures (Tables 1 and 2).
Fourteen strains of five species of fungi were inoculated on margarine to which no preservatives were added, and then stored at 25°, 20° and 5°C for 30 days. Periodical measurement was carried out on the growth of fungus, production of off-flavor and determination of acid and peroxide values. The same experiments were also carried out with margarine to which common salt was added in to the rate of 1, 2 and 3%, and maintained at 25°C for 30 days. Fungi growth was observed in all the samples kept at 25°C, and the growth was especially marked with Penicillium, Aspergillus and Monascus species. Addition of 1% of common salt prevented growth of four strains of fungi, while addition of 2% of salt allowed slight growth of four strains of fungi. There was substantially no fungi growth in any of the samples added with 3% of salt.
Five strains of fungi were inoculated in margarine to which 0.5, 1 or 2g/kg of either dehydroacetic acid or its sodium salt, sorbic acid, its sodium or potassium salt was added. These samples were kept at 25°C for 30 days and periodical measurements were for made fungal growth, production of off-flavor and acid value peroxide value. Dehydroacetic acid was more effective against Penicillium roqueforti but there was no significant difference between dehydroacetic acid and sorbic acid against the other four strains. Sodium and potassium salts showed somewhat weaker antifungal effectthan the free acids.
Five strains of fungus were inoculated on margarine samples to which 0.5, 1 or 2g/kg of benzoic acid, its sodium or potassium salt was added. The samples were kept at 25°C for 30 days, and periodical examinations were made on fungal growth, production of off-flavor and determination of acid and peroxide values. There was no difference in the antifungal effect of benzoic acid on the five fungi strains from that of sorbic acid. Effect of sodium and potassium salts was slightly weaker than those of dehydroacetic dcid and sorbic acid.
In market fermented milk approximate ten ppm level of benzoic acid is detected commonly. The previous paper has indicated the presence of benzoic acid in skimmilk as the material of fermented milk, and also benzoic acid as a fermented productaby Lactobacillus in skimmilk medium. This paper showed that the precursor of benzoic acid, metabolite by lactic acid bacteria, is hippuric acid. It was found that when Lb. casei was grown in a skimmilk medium, benzoic acid was formed in the culture broth while hippuric acid disappered. The conversion of hippuric acid into benzoic acid was demonstrated with a good equivalent relation between the amount of hippuric and benzoic acids before and after incubation. Such activity of hippuric acid degradation was confirmed to be present widely in Lactobacillus, Streptococcus and Leuconostoc.
Quantitative and bacteriological analysis were made on plant air and flow-back milk, in order to reveal the sources of contamination at the head tank, where the first stage of psychrotrophic bacterial contamination had been recognized in the bottling process of market milk. Air inhalation and milk flow-back through air pipes were found to take place at the head tank in vacuum filling system, and it was found that 280-760L of air was inhaled in a minute from the outside of bottling machine and 0.68-1.15L/min of flow-backed milk was mixed into head tank contents, during c. a. 200 bottlings of 180ml market milk. In case psychrotrophic bacteria were found in number of 7800/L in the flow-backed milk, 4-130 (average 41) in the 10L plant air and the milk volume passing through the head tank was 37L/min, these kinds of bacteria also could be demonstrated in number of 140-250/L in the head tank contents. These results suggest that almost a half of all psychrotrophic contaminants in the head tank might have been derived from the flow-backed milk, so the flow-back milk is regarded as one of the important source of contamination. On the air-borne bacteria, if all the psychrotroph present in incoming air of bottling machine were caught by milk, the number of contaminants would not be supposed to reach the contamination level of head tank contents. From the results of bacteriological determination in several plants, it was confirmed that smallness in number of psychrotroph in flow-back milk and plant air resulted in less contamination level of head tank contents. And it was presumed that the psychrotroph contamination level above 6/L in head tank contents gave the psychrotroph positive in all the bottled products.
Antimicrobial effect of water-soluble derivatives of p-hydroxybenzoic acid esters on the growth of Hiochi bacteria was investigated, among which mainly a p-heptoxycarbonyl phenyl phosphate (PP-7) was used. (1) From the experiments on various kinds of factor for the antimicrobial activities, the PP-7 was found to inhibit the growth of Hiochi bacteria at the concentration near 35ppm in Sake, and to have 5-10 times strong antimicrobial activity compared with that of salicylic acid (SA). (2) The antimicrobial action of PP-7 against Hiochi bacteria in Sake was bacteriostatic, similarly to that of SA. (3) By the results of sensory test, PP-7 did not show any bad influence on the qualities of Sake at the concentration near 35ppm. It is concluded, thus, that the PP-7 seems to be a new and good preservative for Sake.
In the case of determination of preservatives in soft drinks, some differences were often observed between the dosage of preservatives and their recoveries from soft drinks. In such cases, these differences are generally considered to have resulted from errors of determination or misdosing of preservatives. In order to clarify this problem, the soft drinks containing preservatives were stored at 25°C and 37°C for 7 months, and changes in recoveries of the preservatives were measured during the storage periods. The results after 7 months, the recoveries of butyl p-hydroxybenzoate and benzoic acid were 80% and 61%, respectively regardless of their storing temperatures. But the recoveries of dehydroacetic acid were strongly depending on the storing time and temperature; thus after 7 months' storage at 25 and at 37°C, 30 and 23% of dehydroacetic acid were recovered, respectively and furthermore, after 8 months at 37°C only 10% was recovered.
As previously reported, the decrease in recoveries of dehydroacetic acid was observed in citric acid solution allowed to stand at 25 and 37°C for several months. This time, the changes in ultraviolet absorption spectra were investigated on continuous heating of dehydroacetic acid in citric acid solution. As the result, upon continued heating, absorption maxima of dehydroacetic acid at 224 and 307mμ were found to decrease in their intensities while the newly appeared absorption maximum at 249mμ increased in its intensity. And these phenomena were found to be irreversible. From these facts, it was concluded that the decrease in recoveries of dehydroacetic acid in citric acid solution could not be attributed to the successive decomposition or degradation of dehydroacetic acid but that it was caused by the transformation of dehydroacetic acid to the substance having the absorption maximum at 249mμ. After 34 hours' heating, whole dehydroacetic acid were transformed into this substance in the citric acid solution, but mold-static activity of this solution for Aspergillus niger still remained in strength from 1/100 to 1/10 of the original solution.
A review work was done to find out simple and rapid precipitation test, for the routine differentiation inspection of household meat, which would reduce the laboriousness as well as the cost. Comparative studies were made on the tube method, Ouchterlony technique, micro-transmigration and micro-gel-diffusion. Sensitivity and accuracy of the micro-transmigration and microgel-diffusion were carried out on 54 cases of unknown meat samples in which some adulterations were suspected. It was found out that the antigenic substance of raw meat extract detectable by the precipitation test was a quite similar substance with that found in corresponding serum albumin. The antigenicity of raw meat extract lowered very rapidly when it was heated at 80°C in wet condition.