Seisaku Yoshida

新規ミニカラム及び光誘導化HPLCによる生落花生, コーン, そば粉及びとうがらし中のアフラトキシンの迅速分析

A rapid and clean method for the analysis of aflatoxins (AFs) was developed by using a new column and post-column photochemical derivatization HPLC with fluorescence detection. The new cleanup column consisted of magnesia and basic alumina poured on the top of a commercial multi-functional mini-column. It was extremely effective for the cleanup of AFs from raw peanut, corn, buckwheat and red pepper. Fluorescent substances, which interfered with the analysis of AFs from corn, were completely absorbed at the top of the magnesia layer. Recoveries of AFs (B1, B2, G1, G2) added to raw peanuts, corn, buckwheat and red pepper were over 80% at two levels of fortification (higher level: 10, 3, 10, 3 ng/g, respectively, lower level: 1.0, 0.3, 1.0, 0.3 ng/g, respectively). Coefficients of variation were smaller than 12%, except the lower fortified level for red pepper. Limits of detection for AFs in raw peanuts, corn and buckwheat were 0.3 ng/g for B1 and G1, and 0.1 ng/g for B2 and G2. Those in red pepper were 0.5 ng/g for B1, B2, G1 and G2.

Solubility of Manganese in Diet after in vitro Digestion.

Each of four composite samples of one days whole meals were homogenized and digested with pepsin followed by pancreatin. Approximately 40-80% of the total manganese in the diets was extracted into the soluble fraction at pH 1.5 both with and without pepsin. The solubility of the manganese in the diets was not increased by pepsin digestion. The solubility of the manganese components in the diets was reduced to less than 30% by adjustment of the pH from 1.5 to 8. Little increase in the solubility of the manganese components was observed after pancreatic digestion for 1h. The low absorption of manganese from food may therefore depend partly on the low solubility of manganese in the area of the duodenum. Gel chromatography of the soluble fraction after in vitro digestion revealed several kinds of soluble manganese components over a wide molecular weight range. Therefore, for further investigation of the mechanism of manganese absorption in man, it will be necessary to isolate each manganese component in the digest.

Distribution of Zinc and Purification of Copper Complex in the Water-soluble Low-molecular-weight Fraction of Soybeans

The water-soluble fraction of soybean was extracted with 0.01M Tris-hydrochloric acid buffer (pH 7.4). The low-molecular-weight fraction (F-II, MW<10, 000) was separated from the soluble fraction by gel filtration on Sephadex G-50. The distribution of zinc (Zn) and the chemical form of copper (Cu) were studied.The concentrations of Cu and Zn in the water-soluble fraction were 26.6 and 107ppm, respectively. Zn in the water-soluble fraction was observed only as the bound form. Zn in F-II was always eluted together with Cu in F-IIB by gel filtration on both Sephadex G-25 and G-10. The binding affinity of Zn in F-IIB was found to be less than that of Cu. When Cu was added exogenously to the water-soluble fraction, Zn in F-IIB shifted to F-I. However, the addition of Zn did not cause a shift of Cu in F-IIB.The purification of the Cu complex in F-IIB was carried out by the application of anion exchange column chromatography (AG 1×2, DEAE-Sephadex A-25). Cu and Zn in F-IIB were held on the AG 1×2 column and both were eluted with 0.01M acetic acid. The dissociation of Zn from the ligands was observed in the chromatography, but Cu was always eluted together with nitrogen-containing materials which were ninhydrin-positive. The Cu complex in F-IIB gave a blue band on the DEAE-Sephadex A-25 column, but after purification, it did not have any absorption spectrum in the visible region. The Cu complex had the same elution point on Sephadex G-10 before and after the purification.The nitrogen/Cu ratio in the partially purified Cu complex was approximately 4-6 mole/mole. The UV spectrum of the partially purified Cu complex showed strong bands at 250nm and a shoulder at around 225nm. Amino acid analysis gave several major peaks (Asp, Glu, Thr, Ser, Gly, Cys and 1-MeHis). The partially purified Cu complex showed a characteristic ESR spectrum of Cu (II) complexes.

Copper-binding Components in the Water-soluble Low Molecular Weight Fraction of Soybeans

The water-soluble fraction of soybeans was extracted with 0.01M Tris-HCl buffer (pH 7.4). The low molecular weight fraction (F-II, MW. F-IIC>F-IIA).All three Cu complexes in F-II were eluted together with nitrogen-containing materials, which were all ninhydrin-positive, but they were not eluted with sugars or phosphorus compounds.Amino acid analyses of the three Cu complexes in F-II were carried out. The amino acid composition of F-IIA was similar to that of F-IIB, but differed from that of F-IIC. Approximately 35% of the amino acid residues in F-IIC were found to be arginine.Both F-IIA and F-IIB showed ninhydrin-positive spots on a paper chromatogram. The Rf values of the spots from F-IIA and. F-IIB were different, and both were less than those of free amino acids. F-IIC showed several ninhydrin-positive peaks on a Sephadex G-10 chromatogram. Cu in F-IIC was eluted with ninhydrin-positive materials having an Rf value similar to that of arginine.

Ultrafiltration of Copper and Cadmium in Soybeans

As part of a series of studies to clarify the chemical form of heavy metals in food, the interaction between Cu (and Cd) and the water-soluble fraction of soybeans was investigated.1. Of the total Cu and Cd in soybeans, 60% of each was extracted as water-soluble supernatant by ultracentrifugation (×105, 000G).2. The effect of pH on the extraction of the water-soluble fraction was studied. The amounts of Cu and Cd extracted at pH 4 or 5 were minimal. At pH 4 or 5, the amount of total nitrogen and the absorbance at 280nm were also minimum. This suggests that Cu and Cd may bind to a protein (s) which precipitates at its isoelectric point, pH 4 or 5.3. Ultrafiltration of the water-soluble fraction was carried out. Approximately 90% of Cd in the water-soluble fraction was retained in the highest molecular weight fraction (m. w. greater than 40, 000). However, Cu was found in various molecular weight fractions (ca. 21% in m. w. greater than 40, 000, ca. 28% in m. w. 40, 000-10, 000 and ca. 50% in m. w. less than 10, 000).