Food additives have contributed to safe and stable supply of food. Although acceptable daily intake (ADI) has been used for the safety assessment of each food additive, their synergistic and/or combined effects on our health are unclear. Actually, several food additives are usually used together in a food, therefore, they may have some adverse effect on cellular levels and/or whole animals even if their individual used levels are below ADI, or commercially used levels. In this paper, we carried out to investigate their cellular effects as well as whole body effects in case of combined use on ADI and daily intake level. Two commonly used preservatives (potassium sorbate and sodium benzoate) and four artificial colors (Yellow No. 4 and 5, Red No. 102 and 106), which have been certified by the Japanese Standard of Food Additives, were chosen and used. Each preservative was dissolved in Ca²⁺, Mg²⁺ free Tyrode buffer pH 7.4 at final concentrations corresponded to 10mg/mL (potassium sorbate) and 1.77mg/mL (sodium benzoate), and each color was used at concentrations of 0.01%. Washed rabbit platelets were prepared by the method described. An aliquot (2x10⁷ platelets/mL) was incubated with calcium ionophore A-23187 (10^-6M) or thrombin (1U), or without either agonist in the presence or absence of preservative and coloring mixture. In addition, commercially used amount of each preservative and color were added together to drinking water and administered to a rabbit for 5 days, then washed platelets were prepared and subjected to incubation. Incubation lasted for 5 min at 37℃ with vigorous shaking and was terminated by placing the reaction mixture on ice followed by centrifugation. The magnitude of platelet activation was determined by the amount of thromboxane B₂ (TXB₂) synthesis and the extent of platelet aggregation. In spite of an inhibitory effect of these adiitives when each of them was used, in combinations of preservative and 2 or 3 artificial colors used in this experiment, no inhibitory effect was observed on both agonists induced TXB₂ synthesis (Table 1, 2) or platelet aggregation in vitro. On the other hand, in an ex vivo experiment, an inhibitory effect was observed at A-23187 or thrombin induced TXB₂ synthesis in combination of potassium sorbate and 2 (Yellow No. 4 and 5) or 3 (Yellow No. 4, 5 and Red 102, Yellow No. 4, 5 and Red 106) colors (Fig. 1, 2). Platelet aggregation was also inhibited or delayed in combinations of these additives. In contrast, no significant change was observed in blood biochemical assay data (Table 5). These results indicate that it is important to consider both an interactive effect of food additives used together and an effect on cellular level when we make safety assessment of food additives.

View full abstract

An analytical method for γ-oryzanol from foodstuffs was studied. γ-Oryzanol from rice bran is a mixture of several ferulate esters, cycroartenyl ferulate and 24-methylenecycloartanyl ferulate are major components. A reverse-phase HPLC method with electrochemical (ECD) and ultraviolet (UV) detection was used for separating the individual components of γ-oryzanol. As the pretreatment, the use of six kinds of solid-extraction cartrides for the extraction of γ-oryzanol was examined. C18 and Florisil types of solid-extraction cartridges were satisfactory for γ-oryzanol extraction from foodstuffs. The recovery of γ-oryzanol (major components) was more than 85 %. ECD was about 6-fold more sensitive than UV.

View full abstract

A test method was developed for determining di(2-ethylhexyl) phthalate (DEHP) and di-isononyl phthalate (DINP) in polyvinyl chloride (PVC) products including articles for food contact use and toys. On the material test, they were extracted with acetone and n-hexane mixture (3:7) by soaking overnight at 37℃, then diluted by acetone and analyzed by GC/MS or GC-FID. By comparison of the extraction method and the dissolution method, the determined levels of DEHP and DINP in the PVC products were almost the same. The recoveries of the spiked DEHP and DINP were between 94.9% and 100.8%. DINP has two CAS number, No.28553-12-0 and No.68515-48-0. The former one is used more than the latter. For the migration test, samples were soaked in n-heptane at 25℃ for 60 min, and then directly analyzed by GC/MS or GC-FID. For a sample determined to be exceeding the standard value by GC-FID, it should be confirmed by GC/MS.

View full abstract

Three subsidiary colors (Sub-A, Sub-B, and Sub-E) were isolated from commercial Food Green No. 3 (G-3) by using preparative HPLC and their structures were determined by MS and NMR. The structures of Sub-A, Sub-B, and Sub-E were the disodium salt of 2-[[4-[N-ethyl-N-(3-sulfophenylmethyl)amino]phenyl][4-[N-(3-sulfophenylmethyl)amino]phenyl]methylio]-4-hydroxybenzenesulfonic acid [abbreviated as HSBA-(m-BASA) (m-EBASA)], the disodium salt of 2-[bis[4-[N-ethyl-N-(4-sulfophenylmethyl)amino]phenyl]methylio]-4-hydroxybenzenesulfonic acid (abbreviated as p,p-G-3), and the disodium salt of 2-[[4-[N-ethyl-N-(2-sulfophenylmethyl)amino]phenyl][4-[N-ethyl-N-(3-sulfophenylmethyl)amino]phenyl]methylio]-4-hydroxybenzenesulfonic acid (abbreviated as o,m-G-3), respectively. HPLC analyses revealed that the contents of seven subsidiary colors (Sub-A, B, C, E, F, G, and H) including the four subsidiary colors (Sub-C, F, G, and H) reported previously in 4 commercial G-3 products were 0.45〜1.20%, 0.19〜2.60%, 8.49〜27.0%, 0.05〜1.52%, 1.15〜2.37%, 0.05〜0.22%, and ND〜0.24%, respectively.

View full abstract

Silver zeolites were developed from an invention titled "Methods of producing hard water-softening agents with germicidal action" patented in 1938. The purpose of the invention was to decrease the hardness of drinking water, and to obtain aseptic water. Using natural or synthetic zeolites as carriers, anti-bacterial zeolites are prepared by the ion exchange reaction of metal ions having an anti-bacterial effect such as silver, zinc, and copper ions. Compared with organic anti-bacterial agents which are alcohol, quaternary ammonium salt, thiabendazole, and ortho-phenylphenol, the inorganic anti-bacterial agents have excellent properties such as wide anti-bacterial spectrums, a high safety, long effective duration, and high heat-resistance. Zeolite and silver have been registered as food additives, and among the anti-bacterial zeolites, the zeolite A-silver, zinc, and ammonium compound zeolite was registered as an indirect food additive by the United States FDA in June 2000. Silver is a common metal, which has been used for tableware, spoons, costume accessories, and coins. With regard to the anti-bacterial effect of metals, Nepli discovered in 1893 that 0.1 ppm silver ions killed spirogyra, and it has been reported that silver, mercury, zinc, copper, tin, lead, bismuth, cadmium, and chromium are also anti-bacterial metals. Although the characteristics of the anti-bacterial zeolites significantly depend on the combination of the types of zeolites and those of the anti-bacterial metals, the specific combination has been determined based on experience. In this study, the combination types were investigated by preparing anti-bacterial zeolites using the zeolite A, which is a typical zeolite, and using silver, zinc, and copper, which are comparatively safe metals, and then comparing their anti-bacterial effects. It was found that the anti-bacterial effects of silver ion were quite high, showing that silver is an essential metal for anti-bacterial zeolites. Furthermore, the anti-bacterial effects of the zeolite A increased with the concentration of silver. To investigate the influence of the pore size of the zeolites on the anti-bacterial effects, silver zeolites were prepared using zeolite Y as carriers, and natural zeolite produced in Miyagi prefecture, which is directly used for water purification equipment, and then comparing the anti-bacterial effect of each zeolite with that of zeolite A. No differences in the anti-bacterial effects were observed regarding these types of zeolites.

View full abstract

The newly notified analytical method for aflatoxins was established by improving the previous reports. An inter-laboratory validation studies were conducted to validate the analytical method. Aflatoxins B₁, G₁, G₂ and B₂ spiked into peanuts, pistachio nuts, corn, red pepper and nutmeg at the level of 10 ng/g were analyzed in replicates in 5 laboratories. Mean recoveries of aflatoxin B₁ were 77.9〜92.7% and those of aflatoxins G₁, G₂ and B₂ were 81.5〜92.5%, 84.7〜82.2% and 83.7〜89.1%, respectively. Repeatability relative standard deviations of aflatoxins B₁, G₁, G₂ and B₂ in five crops were in the ranges of 2.9〜7.3%, 2.1〜6.5%, 1.3〜5.2% and 1.9〜4.6%, respectively. Reproducibility relative standard deviations of aflatoxins B₁, G₁, G₂ and B₂ in five crops were in the ranges of 6.5〜10.1%, 2.5〜14.2%, 6.5〜10.1% and 5.3〜10.6%, respectively. These results suggested that the proposed method is reliable and reproducible for the inspection of aflatoxin levels in peanuts, pistachio nuts, corn, red pepper and nutmeg.

View full abstract

Lactosucrose, fructo-oligosaccharide, isomalto-oligosaccharide and lactulose have been increasingly used as foods for specified health uses in Japan. On the other hand, Agaricus Blazei Murill has been shown commercially to have many beneficial effects on health. However, there is little information concerning safety of these food ingredients in liver disease. In this study, the effects of these health food ingredients on hepatic injury induced by carbon tetrachloride and galactosamine hepatitis in rats were examined. Male Wistar rats were injected with carbontetrachloride (50% in olive oil, 1ml/kg body weight) twice a week for 1 month and then were fed the experimental diets containing 5% oligosaccharides or 1% Agaricus for 10 d. In another experiment, rats were injected with D-galactosamine solution (600mg/kg body weight), and 24 h later the rats were orally administered the experimental materials using stomach tube. The serum transaminase activities were measured after 48h from the injection of D-galactosamine. All experimental materials did not change the serum transaminase activities in those experiments. These results suggest that these dietary supplements did not change the liver injured under these experimental conditions, but also had no obvious toxicity.

View full abstract