A method for the extraction and identification of natural dyes in Kamaboko (fish paste products) prepared as imitation crab was developed.
A sample (5g) cut into small pieces was incubated with sodium dodecyl sulfate (0.3g) and 50ml of 0.2% protease solution (Pronase P) at 37°C for 2 hours, then the mixture was centrifuged. The supernatant fluid was extracted with ether. DEAE-Sephadex was added to the lower aqueous layer, and this mixture was stirred for 10min. The DEAE-Sephadex was filtered off and washed with water and 0.1% hydrochloric acid, then the dyes were eluted with 5% sodium chloride solution-ethanol (1:3). The eluate was evaporated to dryness on a steam bath and the dyes were extracted with ethyl acetate from the residue. The extract was filtered, the solvent was evaporated off and the residue was dissolved in ethyl alcohol.
The dyes from the ether and aqueous layers were identified by thin-layer chromatography. The recoveries of monascus pigments, cochineal, laccaic acid and norbixin added to Kamaboko prepared as imitation crab were over 80%.
The method was applied for the detection of natural dyes in commercial imitation crab products prepared from Kamaboko, and monascus pigments, laccaic acid and norbixin were found.

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An improved method was developed for the estimation of viable bacterial counts for raw meat stored at low temperature, and was used to analyze the influence of various factors affecting the viable bacterial count. The viable bacterial counts of 40 samples each of commercial cut beef, pork, and chicken meat stored refrigerated or frozen in the raw state were estimated comparatively by using three methods of sample inoculation, i. e., the surface plate method and the pour plate method poured at 50 and 60°C with standard plate count agar, at six incubation temperatures (7, 25, 30, 32, 35, and 37°C).
1) Among the three methods of inoculation, the viable bacterial count was the highest in the surface plate method, regardless of temperature of culture. As regards the temperature of culture, the count was the highest in the culture at 25°C, regardless of the method of inoculation used. Therefore, the count was the highest when the surface plate method was performed at 25°C.
2) The viable bacterial count was significantly higher when estimated by the surface plate method at 25°C than when estimated by the method prescribed in the Official Guideline of Sanitary Tests of Food with culture at 35°C.
3) There was a significant correlation between the viable bacterial count estimated by the surface plate method at 25°C and that at any other temperature.
4) The mode of storage (refrigeration or freezing) was the most influential factor upon the viable bacterial count. The next most important factor was the kind of meat (beef, pork, or chicken), followed by the temperature of culture, and the method of inoculation in decreasing order of influence upon the count.

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An experiment was performed to study the characteristics of bacteria of raw meat from the viewpoint of the range of temperature required for bacterial growth; 1, 056 strains of bacteria isolated from refrigerated and frozen raw meat by incubation at 7, 25, and 37°C were used. They were observed for growth at 7-37°C and identified.
1) Of the strains isolated by incubation at 7°C, two (0.4%) belonged to the category of so-called obligate psychrophilic bacteria, which grew only within a range of temperature from 7 to 20°C.
2) Among the strains isolated by incubation at 7 and 25°C, 61.8% and 32.5%, respectively, could not grow at 35°C. These strains belonged to the groups of bacteria growing at 7-25°C, 7-30°C, or 7-32°C.
3) All the strains isolated at 7 and 37°C could grow at 25°C, except for the two mentioned in 1) above. Therefore, incubation at 25°C was presumed to result in detection of almost all the bacteria that could grow on incubation at 7 and 37°C.
4) Principal components of the groups of bacteria classified on the basis of the range of temperature for growth were as follows: Lactobacillus in the group of bacteria growing at 7-20°C, coryneforms in those growing at 7-25°C and at 7-30°C, coryneforms and Pseudomonas in those growing at 7-32°C and at 7-35°C, coryneforms, Lactobacillus, and Pseudomonas in that growing at 7-37°C, and Micrococcus in that growing at 25-37°C.

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An experiment was conducted to elucidate the food-hygienic significance of bacteria of raw meat; 1, 056 bacterial strains isolated from refrigerated and frozen raw meat were examined for ability to decompose fat and protein. Some representative strains were also observed for growth characteristics at low temperature.
1) The ratio of lipolytic and proteolytic bacteria to those isolated varied to some extent with the temperature at which isolation was done, the kind of meat (i. e.; beef, pork, or chicken meat), and the mode of storage (i. e., refrigerated or frozen meat).
2) When the isolated bacteria were analyzed by genus, more than 70% of bacteria belonging to Alcaligenes, Acinetobacter, and Pseudomonas showed lipolytic activity, and nearly 80% of bacteria belonging to Pseudomonas showed proteolytic activity.
3) The isolated bacteria were analyzed for range of growth temperature to be divided into 7 groups. Each group was examined for the rates of lipolytic and proteolytic bacteria. The highest rate of lipolytic bacteria was obtained from the group growing in a range of 7-32°C. The highest rate of proteolytic bacteria was obtained from the group growing in a range of 7-25°C.
4) The rates of growth at 1 and 5°C were the highest in some of the coryneforms and Pseudomonas and rather low in Lactobacillus and Micrococcus. The growth rate was not parallel with the upper limit of the growth temperature in the bacterial strains examined.

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In order to clarify the effect of dietary fats on the acute toxicity and metabolism of aflatoxin B₁ in animals, we have attempted to use fertile eggs because of technical ease in the experimental procedure.
Four-day-old fertile eggs (White Leghorn) were injected into the yolk sac with 0.4ml of soybean oil, beef tallow or saline and after 2 weeks the pretreated embryos were sacrificed to prepare the hepatic soluble microsomal fraction (S-9). When aflatoxin B₁ was treated with these S-9's in vitro, the amounts metabolized in 10 minutes were in the order of soybean oil-injected group (77%), saline-injected group (62%) and beef tallow-injected group (42%). The amounts of aflatoxin B₁ metabolized by the S-9's were well correlated with the abilities of the S-9's to detoxify aflatoxin B₁, determined from the survival rates of chick embryos injected into the air cells with S-9-treated aflatoxin B₁. Estimation of the activities of the mixed function oxygenases in the S-9 and of cytoplasmic aflatoxin B₁ reductase showed that the levels of p-nitroanisole demethylase, aryl hydrocarbon hydroxylase and cytoplasmic aflatoxin B₁ reductase were increased in the soybean oil-injected group and decreased in the beef tallow-injected group. This suggests that soybean oil injected into the yolk sac of chick embryo might induce these hepatic enzymes and stimulate the metabolism of aflatoxin B₁, resulting in detoxification in terms of acute toxicity. These results are very similar to those previously obtained with rats.
The chick embryo was concluded to be a very useful tool for this type of study.

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Pyrimethamine (PM) in chicken muscles and eggs was quantitatively determined by high performance liquid chromatography.
The acetonitrile extract of samples was cleaned up by the use of a SEP-PAK C₁₈ cartridge with acetonitrile-water-acetic acid (30:70:2) as an eluent. To exclude interfering substances in the chicken muscles, the extract was passed through an alumina column and then treated with SEP-PAK. PM was eluted on a column packed with Nucleosil 5 C₁₈ by using acetonitrile-water-acetic acid (30:70:1) as a mobile phase, with detection by absorbance measurement at 232nm. A linear calibration curve was obtained in the range from 2.5 to 100ng of PM. The limit of detection was 2.5ng of PM and 0.025ppm in samples. The recoveries of PM added to chicken muscles and eggs were 91.6% and 97.3%, respectively.

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A method for the determination of carbonate in sodium hydroxide solution was developed by using a total organic carbon (TOC) analyzer based on combustion-infrared analysis.
The pH of sodium hydroxide solution was adjusted to between 8.0 and 9.2 with 2M ammonium buffer solution (pH 9.0) and hydrochloric acid to avoid loss or uptake of gaseous carbon dioxide. A portion (20μl) of the solution was injected into the TOC analyzer using an automatic sample injector. In the analyzer, the carbonate was converted to carbon dioxide by heating at 900°C in the combustion tube, and the absorbance at 4.3μm was measured and recorded on a chart. A linear relationship was observed between peak height of carbon dioxide and concentration of sodium carbonate in the range of 0-240μg/ml. The relation was applied to the determination of carbonate in sodium hydroxide solution.
The determination limit of the present method was 0.02% as Na₂CO₃ in sodium hydroxide, about one-tenth of that of the titration method described in JSFA-IV.

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A method was developed for the determination of amygdalin in apricot kernels by high performance liquid chromatography (HPLC). Amygdalin was extracted from apricot kernels with methanol, and 10μl of the filtrate was injected into a high performance liquid chromatograph. HPLC was performed on a stainless steel column packed with Zorbax ODS, with acetonitrile-water (14:86v/v%) as the mobile phase. The retention time of amygdalin was about 7min. The average recovery of amygdalin was 97.4% and the lower limit of determination was 50ng.
This method was also applied for the determination of amygdalin in commercial almond jelly and other apricot products. Almond jelly prepared in this study from apricot kernels contained 5.07ppm amygdalin. The amygdalin content had decreased to 30.4% in first process of preparing the almond jelly and to 0.3% finally.
No amygdalin was detected in other foods tested.

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A simultaneous clean-up method for analysis of residual organochlorine and organophosphorus pesticides in vegetables and fruits was established as follows. The pesticides were extracted from vegetable or fruit homogenates with acetonitrile. The extract was concentrated on a rotary evaporator under reduced pressure after addition of sodium p-toluenesulfonate solution, then the residue in the concentration flask was washed with ethanol and applied to an Amberlite XAD-8 column. The column was washed with 200ml of water and 100ml of 5% sodium chloride solution-methanol mixture, and then eluted with 200ml of methanol.
By this method, the organochlorine and organophosphorus pesticides tested were simultnaeously eluted, and substances interfering with the gas liquid chromatographic determination were efficiently removed. The recoveries of the pesticides added to vegetable or fruit homogenates were in the range of 76.1-93.0% for organochlorine pesticides and 79.8-98.9% for organophosphorus pesticides. This clean-up method can be applied as a simple quantitative method for residue analysis of organochlorine and organophosphorus pesticides in vegetables (except aromatics) and fruits.

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[in Japanese]

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