A thiamine-binding protein was purified from the extract of rice bran acetone powder by conventional procedures of acid precipitation, a series of column chromatography on DEAE-Sephadex A-50 and DEAE-cellulose, and gel filtration of Sephadex G-200. The purified thiamine-binding protein was nearly homogeneous as judged by disc gel electrophoresis and the molecular weight was estimated to be 94, 000 by gel filtration on Sephadex G-200 and 50, 000 by sodium dodecylsulfate (SDS) gel electrophoresis, suggesting that the protein is composed of two identical subunits. The apparent Kd and Bmax of the binding for [14C]thiamine was 0.44±0.05 μM and 17.2±0.7 nmol/mg of protein, respectively. The optimal pH for the binding is between 8.0 and 9.0. From the competition experiment using several thiamine derivatives, high binding specificity of the protein for thiamine was presumed.
A simplified and accurate method for the determination of vitamin D in foods, feeds and pharmaceuticals was established by highperformance liquid chromatography (HPLC) using successively reversedphase and straight-phase columns. About 1-2g of a sample was accurately weighed and directly saponified. The extracted unsaponifiable matter was subjected to preparative HPLC using a reversed-phase column and a vitamin D fraction was collected. The fraction was subsequently subjected to analytical HPLC using a straight-phase column and vitamin D was assayed by estimating the peak height. The proposed method was applied to various kinds of samples, e.g., fishery products, fish meals, mixed feeds for fish farming and chicken farming, egg yolk, milk products, cattle liver, Shiitake, fortified foods and multivitamin preparations. The results showed that the proposed method was useful for the determination of vitamin D in such samples, because the peak of vitamin D in the profile of the second HPLC was always clearly separated from other concomitants and good recovery was obtained. Therefore, we think that the method is useful as a routine one for the determination of vitamin D in foods, feeds and pharmaceuticals.
The free amino acid profile of the fruiting bodies of Pleurotus ostreatus was studied in detail. The first evidence for the occurrence of Nδ-acetyl-L-ornithine in the mushroom was presented, and 33 ninhydrin-positive compounds were determined, besides Nδ-acetyl-L-ornithine. Alanine, glutamic acid, valine, glutamine, glycine and leucine were predominant protein amino acids occurring in the free form. As regards the non-protein amino acids, ornithine, y-aminobutyric acid, saccharopine, α-aminoadipic acid, ethanolamine, cystathionine, and N-(γ-glutamyl)ethanolamine were mainly detected. The sum of the nitrogen of the three abundant amino acids, alanine, glutamic acid and ornithine, accounted for about 40% of the total nitrogen in the amino acid fraction.
The antioxidative activity of a 70% methanol extract of sweet potatoes was estimated in a linoleic acid=aqueous system. The extract had a markedly strong antioxidative activity. Major phenolic components contained in the 7% methanol extract were identified as chlorogenic acid and isochlorogenic acid-l, -2 and -3 by using high-performance liquid chromatography. The other minor free phenolics were identified, or tentatively identified, as caffeic acid and 4-o-caffeoylquinic acid. Chlorogenic acid and/or isochlorogenic acids, however, had only slight antioxidative activity. From the results of the addition of chlorogenic acid, isochlorogenic acids and the other coexisting components contained in the sweet potato extract, the effective antioxidant activity of the sweet potato extract was proposed to be mainly based on the synergistic effect of phenolic compounds with amino acids.