By using carbon-13 (¹³C) nuclear magnetic resonance (NMR) spectrometer, we have developed a easy quantitative method for determining the molar ratio of partial acylglycerines <1, 2-diacylglycerine (DG), 1, 3-DG, 1-monoacylglycerine (MG), and 2-MG> to triacylglycerine (TG) and/or glycerin. For refinement of some measurement conditions, we set the five kinds of palmitoyl acylglycerines (TG, 1, 2-DG, 1, 3-DG, 1-MG, and 2-MG) and glycerine as model compounds. After examining the solubility of these model compounds, we decided that pyridine-d₅ was suitable as an NMR measuring solvent. We also set the delay time of the pulse repeating at 15s, which is five times longer than the longest spin-lattice relaxation times (T₁) of proton (¹H) NMR signals of the glycerine moieties protons and ¹³C NMR signals of the glycerine moieties carbons. Good agreement was observed between the result of the ¹³C NMR and that of the reverse-phase high performance liquid chromatography (HPLC) by the model compounds. When the characteristic peaks are quantitatively measured in ¹³C NMR of the commercial diacylglycerins product, each molar ratio of the derivatives, such as TG, 1, 2-DG and 1, 3-DG, can easily be determined.

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In the present study, we investigated the tolerance of Lactobacillus gasseri NY0509 and L. casei NY1301 against simulated digestive juice in vitro, and the inhibition of growth of harmful intestinal bacteria, Escherichia coli and Clostridium perfringens by these bacteria. In the tolerance test of the gastric juice (containing 0.04% pepsin), the viable cell counts of both L. gasseri NY0509 and L. casei NY1301 maintained these initial levels in pH 3.0 for up to 4hrs. In the bile tolerance test, L. casei NY1301 could grow in 0.3% oxgall, and L. gasseri NY0509 also showed bile tolerance. When L. gasseri NY0509 and/or L. casei NY1301 were mixed-cultured with E. coli JCM 1649^T or C. perfringens JCM 1290^T, the viable cell counts of E. coli JCM 1649^T and C. perfringens JCM 1290^T were remarkably decreased, respectively. These results suggest that L. gasseri NY0509 and L. casei NY1301 reach the intestine alive after ingestion, and inhibit harmful intestinal bacteria.

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For the rapid purification of catechins from tea leaves, soybean protein was applied. Amongst catechins [(-)-epigallocatechin (EGC), (+)-catechin (C), (-)-epicatechin (EC), (-)-epigallocatechin 3-O-gallate (EGCG), (-)-epicatechin 3-O-gallate (ECG)] in a tea leaf extract, the catechin gallates (EGCG and ECG) were specifically extracted by complex formation with soybean protein. The procedure is as follows: 1) Soybean protein was prepared from autoclaved de-fatted soybean. 2) The protein extract was mixed with a water extract of tea leaf and the pH of the mixture was adjusted to 4.5. 3) Catechin-protein complex, precipitated in the mixture, was filtrated and dried. 4) Catechins were extracted with acetone-water (1:1) from the catechin-protein complex. The complex of the catechin and soybean-protein would be a new food and/or pharmaceutical product, which is attractive by its bio-activities originated from both catechins and soy protein.

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Two α-amylase inhibitors (KAI and FAI) were purified to apparent homogeneity from the respective seeds of Phaseolus vulgaris L. cv Kintoki and P. vulgaris L. cv Fukushirokintoki, and their properties and subunit structure were examined. Isoelectric points of KAI and FAI were 4.6 and 4.5, respectively, and each inhibitor was found to be a glycoprotein with a molecular weight of about 45000. Amino acid compositions of the inhibitors were similar to each other and characterized by no cysteine content. SDS-PAGE of each inhibitor showed at least four kinds of polypeptide bands corresponding to molecular weights between 14000 and 20000. On the other hand, size-exclusion HPLC indicated that each inhibitor was separated into two protein peaks, I and II under a denaturing condition. Amino acid compositions of I and II of KAI were very similar to those of I and II of FAI, respectively. N-terminal 20 amino acid sequences of I and II of KAI were the same as those of the correspondings of FAI. Comparison of I and II of each inhibitor exhibited that they were different from each other in their amino acid compositions and sequences. These findings suggest that each of KAI and FAI is composed of two protein subunits, which showed heterogeneity in molecular size due to different degrees of glycosylation.

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Nine strains of Lactobacilli which were isolated from rice and processed rice products were tested for fermentation, survival of an artificial digestive juice and growth in medium of human diet model. They were compared with Lactobacillus acidophilus and L. gasseri which were used for fermented milk preparation or isolated from feces and intestines. Tested Lactobacilli were six strains of L. casei subsp. casei and three strains of L. plantarum. In most of plant material, plant origin lactic acid bacteria showed increase in the cell number and its fermented products were lowered under pH 5. In a stomach model test, the viable cell count maintained its initial level at pH 3 except L. acidophilus L-54. However the viable cell count decreased in many strains at pH 2.5. In a intestine model test, L. casei subsp casei, L. plantarum and L. acidophilus 1132 showed tolerance or growth under the condition of 0.2-0.4% bile powder, but the number of viable cell was smaller than the condition without bile. To compare growth of Lactobacilli on human diet, Japanese and American diet model medium containing 0.4% bile powder were prepared. L. casei subsp. casei and L. plantarum grew in both medium and the number of cell increased by passage in the medium. These results showed plant origin lactic acid bacteria has ability of probiotics.

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In relation to fruit development, we examined the differences in the degrees of insolubility of soluble tannins by acetaldehyde vapor treatment in the fruits of three types of astringent persimmon (Diospyros kaki Thunb.); pollination-variant and astringent (PVA) type, pollination-constant and astringent (PCA) type, and pollination-variant and nonastringent (PVNA) type with a small number of seeds. Insolubilibed tannins in the flesh cubes of these three types of persimmon fruit after acetaldehyde vapor treatment at three stages of fruit development similarly became soluble by extraction with HCl-MeOH, irrespective of the fruit type and maturity. These results confirm that the ease of removing astringency in all of the three types of astringent persimmon may depend on the amount of acetaldehyde accumulated in the flesh.

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The stimulative effect of Lactobacillus gasseri NY0509 and L. casei NY1301 on the in vitro growth of Bifidobacterium species, human intestinal bacteria, was investigated. L. gasseri NY0509 and/or L. casei NY1301 were mixed-cultured with four strains of Bifidobacterium species (B. longum JCM 1217^T, B. adolescentis JCM 1275^T, B. breve JCM 1192^T, and B. bifidum JCM 1254) in 10% skim milk. L. gasseri NY0509 and L. casei NY1301 markedly increased the numbers of B. longum JCM1217^T, B. adolescentis JCM 1275^T, and B. breve JCM 1192^T at early stationary phase in comparison with monoculture of Bifidobacterium, respectively. On the contrary, the growth rate of B. bifidum JCM 1254 was only stimulated in the mixed-culture with L. casei NY1301. These results suggest that L. gasseri NY0509 and L. casei NY1301 stimulate the growth of Bifido-bacterium species, beneficial bacteria in human intestine.

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Nine strains of plant origin lactic acid bacteria were isolated from rice and processed rice products to examine their antimutagenic effect. Tested Lactic acid bacteria were six strains of Lactobacillus casei subsp. casei and three strains of L. plantarum. Used mutagens were 3-amino-1, 4-dimethyl-5H-pyrido (4, 3-b) indole (Trp-P1), 3-amino-1-methyl-5H-pyrido (4, 3-b) indole (Trp-P2), 2-aminoanthracene (2-AA) and N-methyl-N' nitro-N-nitrosoguanidine (MNNG). Antimutagenic activity on living and killed cells of lactic acid bacteria were measured using Escherichia coli WP2uvrA. All strains showed antimutagenic activities against Trp-P1, Trp-P2 and 2-AA. The activity against Trp-P1 was higherthan the other mutagens. Living and killed cells of lactic acid bacteria showed similar activity against Trp-P1 and Trp-P2. Four strains of killed cells of L. casei subsp. casei had higher activity than the living cells against 2-AA. Only living cells of L. casei subsp. casei had antimutagenic activities against MNNG. This result was similar case to our previous report using 4-nitro-quinoline-1-oxide.

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