The sodium condition in foods as well as the water condition is an important factor controlling food quality. A ²³Na-NMR imaging probe was devised for this investigation, and distributions and relaxation times of Na ions were measured using phantoms, containing various concentrations of Na ions with different mobilities. The images had a spatial resolution of 0.8×0.8 mm area and 8mm thickness (5.12mm³). The signal intensity obtained was proportional to the concentration of Na ions and it depended strongly on the mobility of the Na ions; Na ions with short relaxation times were difficult to detect because the echo-time for the measurements (36 msec) was comparable to the relaxation time of the Na nucleus. Distribution maps of Na ions in foods, such as a salt-pickled plum and a cucumber pickled with fermented soybean paste, were obtained. They provided different information from that of ¹H-NMR images. Considering that the accumulation of sodium affects the taste and the texture of food materials, ²³Na-NMR imaging may be useful for monitoring food quality in detail such as in the sub-tissue level during storage and processing.

View full abstract

Continuous ethanol fermentation was done to improve soy sauce productivity using immobilized soy sauce yeast cells. The yeast cells were immobilized physically in pores of alumina ceramic beads, and fermentation was done with a filtrate solution from soy sauce mash. The ethanol concentration was controlled by the dilution rate, and the volumetric production rate, being dependent on dilution rate, was found to be much higher than that in batch fermentation. The ethanol production system was mathematically modeled from the basic material balance and the rate equation. The simulated results agreed with the experimental ones. The reactor system with immobilized yeast cells was effective for obtaining ethanol with a high production rate.

View full abstract

The volatile compounds of aged "Nukadoko" were studied using a sample that had been aged for 130 years. The fatty acid, 10-hydroxy-octadecanoic acid, which is considered to be an aging index compound in "Nukadoko", comprised 47% of all the free fatty acids and showed that sufficient aging had been carried out. The volatile components of the "Nukadoko" obtained by freeze drying were quantitatively determined by GC and GC-MS analyses. In the result, 39 esters, 13 acids, 23 alcohols, 13 hydrocarbons, 8 phenols, 9 lactones, 2 aldehydes, 2 ketones and sulfur-nitrogen-containing compounds were found. Quantitatively, 70% of the volatile compounds consisted of acids, which were mainly propionic, butyric and acetic acids in order of magnitude. The next major components were phenethylalcohol, 3-butenyl isothiocyanate, 4-ethyl guaiacol, 2-methoxy-4-cresol, 3-ethylphenol and nona-1, 4-olide. Esters, which formed the large portion of all the volatile constituents, comprised amyl formate, propyl acetate, propyl propionate, ethyl acetate, phenethyl propionate, propyl palmitate and propyl oleate. Of the sulfur-nitrogen-containing compounds, 3-methyl-thiopropanol, 3-butenyl isothiocyanate, phenethyl isothiocyanate and phenylpropiononitrile were found. Limonene, linalool and trans-nerolidol were also detected. Undecan-1, 4-olide, 3-ethyl-phenol and butan-1, 4-olide were evaluated as the character-impact components by sniffing.

View full abstract

The effects of amino acids on IMP production were examined with a mutant strain, KY10895, derived from Corynebacterium ammoniagenes KY13374. L-Proline improved the productivity of IMP more than any other amino acid. The optimum concentration of L-proline for IMP production was 1-2% and the IMP productivity was about 70% more than that in the control medium. The effects of L-proline analogs on IMP production were also examined with the mutant KY10895. DL-3, 4-Dehydroproline inhibited IMP production. Mutants resistant to growth inhibition by DL-3, 4-dehydroproline were derived from strain KY10895. Among mutants thus obtained, strain H-7335 had the highest productivity. The intracellular concentrations of L-proline in strain H-7335 were higher than those of the parental strain, KYI 0895. These findings indicated that an increase in intracellular L-proline was linked with an increase of IMP productivity and strengthening the L-proline synthesis of a strain was an effective method for obtaining a hyper-producer of IMP.

View full abstract

The isoflavone constituents in soybean seeds were investigated, and 9 kinds of isoflavone glycosides were isolated from the hypocotyls of soybean seeds. Three kinds were proved to be malonylated soybean isoflavones named 6"-O-malonyldaidzin, 6"-O-malonylglycitin and 6"-O-malonylgenistin by UV, MS, IR and NMR. The malonylated isoflavone glycosides as major isoflavone constituents in soybean seed were thermally unstable, and were converted into their corresponding isoflavone glycosides. All of the isoflavone components produced intensely undesirable taste effects such as bitter, astringent and dry mouth feeling.

View full abstract

Guanidinoacetate kinase (ATP: guanidinoacetate N-phosphotransferase, EC 2.7.3.1) was extracted and purified to homogeneity from a polychaete, Perinereis sp., by ammonium sulfate fractionation and chromatographies with Butyl-Toyopearl 650C, Sephadex G-200, and DEAE-Sephacel gels. Perinereis guanidinoacetate kinase was separated into two active fractions (I and II) by DEAE-Sephacel column chromatography, which had similar specific activities, and so some properties of guanidinoacetate kinase II (a major fraction) were investigated. The molecular weight of the enzyme was 90, 000 on gel filtration and it was composed of two non-identical subunits (47, 000 and 45, 000). The enzyme showed optimum activity around pH 8.1 and was stable from pH 5.5 to 9.5 in the forward reaction. It was strictly specific for guanidinoacetate, and ATP was the most effective phosphoryl group donor for the forward reaction. The enzyme was activated by metal ions such as Mg²⁺ or Mn²⁺ while Ca²⁺ was 29% as active as Mg²⁺. The Km values were 4.1 mM and 0.80 mM for guanidinoacetate and ATP, respectively. The enzyme activity was strongly inhibited by sulfhydryl-group-blocking agents, Hg²⁺, N-bromosuccinimide, and SDS. ADP and agmatine were competitive inhibitors.

View full abstract

Cytosolic serine hydroxymethyltransferase [L-serine: tetrahydrofolate 5, 10-hydroxymethyltransferase, EC 2.1.2.1] from Euglena gracilis z was purified by precipitation with ammonium sulfate followed by column chromatography on DEAE-cellulose, and by affinity chromatography on an L-adsorbent. Through this procedure the cytosolic enzyme (s-SHMT) was purified 420-fold with a yield of more than 26.8%. The preparation gave a single band with an M_r of 45, 000 on SDS-PAGE. The native enzyme contained 2 mol of pyridoxal phosphate/mol of enzyme, and had an M_r of 90, 000 on gel filtration, indicating a dimeric structure. Several other properties of the enzyme were also studied.

View full abstract

In the course of a search for an alkaline stable protease for industrial use, an alkaline protease (protease BYA) was isolated from an alkalophilic Bacillus sp. Y, and its properties were characterized. Its optimum pH was pH 10.0-12.5, when casein was used as a substrate. In addition to the stability of protease BYA at pH 6.5-13.0, it was also very stable towards various surface-active agents, such as sodium dodecyl sulfate and sodium linear alkylbenzene sulfonate. Protease BYA was most active at 70°C. The isoelectric point (pI) of protease BYA was about 10.1. Protease BYA was characterized as a serine protease because of its sensitivity to phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The protease seems to be related to proteases of the subtilisin family, such as subtilisin BPN', subtilisin Carlsberg, and No. 221 protease.

View full abstract

A 5.5-kb HindIII fragment of Synechocystis PCC6803 containing a liverwort (ORF316) homolog encoding a putative zinc finger protein was cloned. Nucleotide sequence analysis showed that the homology of the amino acid sequence deduced from the ORF326 of Synechocystis PCC6803 with the counterparts of a liverwort and tobacco was 50% and 46%, respectively. Synechocystis ORF326 also showed 38% homology with the dedB gene in Escherichia coli. The gene organization of the region in these species of organisms was quite different. This suggests that the Synechocystis ORF326 and liverwort ORF316 genes may be related to a common regulatory gene, but not photosynthetic gene characteristic to chloroplasts.

View full abstract

The protective effect of orally administering immune milk, which was obtained from cows immunized with a mixture of various bacteria, on radiation-induced lethality was studied in mice exposed to a lethal dose of irradiation. The mice were given immune milk or control milk for 7 days, exposed to X-irradiation, and then given either milk daily to the end of the observation period. The irradiated mice given immune milk showed a higher survival rate than the irradiated mice given control milk. The number of Enterobacteriaceae in the feces was almost the same in both groups of mice, whereas the number of Lactobacilli was higher in the mice given immune milk. The concentration of IgA in the supernatant of a cell culture derived from Peyer's patches, mesenteric lymph node and spleen was higher in that prepared from the mice given immune milk than in that prepared from the mice given control milk. The activity of Kupffer cells for bacterial killing in vivo was higher in the mice given immune milk than that in the mice given control milk. Bacteria in the liver after irradiating appeared in a lower number in the mice given immune milk than in the mice given control milk. These results suggest that immune milk prevents mice from endogenous infection by augmenting the defense activity against the invasion of intestinal bacteria.

View full abstract

IMP and GMP were degraded during storage in the presence of methyl Nucleate (ML), this degradation being caused by the peroxidation of ML. The mechanisms for degradation in the first period (0-7 days) and in the second period (8-15 days) of storage were different. The primary products (hydroperoxides) of peroxidized ML contributed to the degradation in the first period, and the secondary products (carbonyl compounds) contributed in the second period. Especially, IMP reacted with the secondary products to form its base (hypoxanthine) by cleavage of the N-C bond.

View full abstract

The main storage seed protein of Amaranthus hypochondriacus, the oligomeric salt-soluble globulin, was isolated and purified to homogeneity by Sephacryl S-300 gel filtration and Separon Hema-Bio 1000 DEAE anion-exchange chromatography. The globulin had two heterogeneous forms, a major and minor species with apparent molecular masses of 398, 000 and 337, 000 daltons, respectively. Each of the two species were analyzed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (PAGE) and by native-PAGE (without prior reduction of disulfide bonds). Only slight differences were observed in their subunit patterns although appreciable differences in mobility and dissociation behaviour were noted on native-PAGE. The major protein fraction was oligomeric, consisting of a tetrameric and hexameric moiety held together by weak secondary forces. The minor fraction, a hexamer of slightly higher apparent molecular mass was a polymeric species derived in part from a sulfhydryl-disulfide interchange reaction.

View full abstract

The protein particles in soymilk were fractionated in size by differential centrifugation. Particles of more than 100 nm in diameter (LSP) constituted 40% of the total protein in raw soymilk, 70% of the protein components being US globulin. LSP was not formed in the presence of 2-mercaptoethanol and sodium ascorbate. LSP was decreased by heating, and particles of 100-40 nm in diameter (MSP) were increased. The formation of MSP was not due to any degradation of LSP but to the combination of supernatant proteins of less than 40 nm in diameter with each other. MSP formed by heating contained the β subunit of 7S and the basic subunit of 1 IS as main components. The particles of more than 40 nm in diameter (LSP+MSP) constituted 50% of the total protein in both raw soymilk and soymilk.

View full abstract

The enzyme of the first step in ubiquinone biosynthesis, 4-hydroxybenzoate-polyprenyltransferase was solubilized with 0.3% Triton X-100 from a membrane fraction of Pseudomonas putida IAM 1219. The enzyme had an optimum pH at 7.0-7.2 in 50 mM imidazole buffer and the apparent Km was 1.4 × 10^-4 M for nonaprenylpyrophosphate and 0.54 × 10^-5 M for 4-hydroxybenzoate. The enzyme required magnesium ion, and was completely inhibited by Cd²⁺, Cu²⁺, Ag⁺, Hg²⁺ at 0.1 mM concentation, and was also strongly inhibited by the thiol reagents, 4-chloromercuribenzoate and N-ethylmaleimide, indicating that the transferase is a thiol enzyme. The enzyme was strongly activated by adding phospholipids extracted from the bacterial cells, of which phosphatidylethanolamine was most effective.

View full abstract

4-Hydroxybenzoate-polyprenyltransferase, an enzyme in ubiquinone biosynthesis, from Pseudomonas putida was partially purified by ion-exchange and gel filtration column chromatography. The enzyme required phospholipid as an essential factor for activity. Hexaprenyl pyrophosphate(-PP) and pentaprenyl-PP as well as nonaprenyl-PP were used as polyprenyl donors, but tetraprenyl- and farnesyl-PPs were scarcely transferred to 4-hydroxybenzoic acid. No inhibition was observed by the end-product, ubiquinone-9 of P. putida. Long chain acyl-CoA, free fatty acids, or isopentenyl-PP strongly inhibited the enzyme activity. A possible regulatory role of the enzyme in bacterial ubiquinone biosynthesis is discussed.

View full abstract

Effects of trialkyltin chlorides on isolated rat hepatocytes were examined. Incubation of isolated hepatocytes with trialkyltin chloride at concentrations of 1-100μg/ml for a 2-hr period resulted in a rapid decrease of cell viability. While both triethyl- and tributyltin chloride intensely inhibited oxygen uptake in isolated hepatocytes, tributyltin chloride proved to be much more toxic than triethyltin chloride. Triethyltin chloride strongly inhibited mitochondria! respiration at the low concentrations. Cells treated with tributyltin chloride leaked large amounts of magnesium, protein, and 260-nm-absorbing materials. Cells treated with triethyltin chloride also showed same leakages, but to a lesser degree. Electron microscopic observation showed that the surfaces and intracellular structures of isolated hepatocytes were badly damaged by tributyltin chloride.

View full abstract

The bioavailability of iron is described from a ferric complex of 3-epi-hydroxymugineic acid (epi-HMA), a ferric-specific ligand which works in plants. Epi-HMA is a mugineic acid analogue which dramatically enhances iron absorption in gramineous plants. The retention of ⁵⁹Fe in the whole body on day 1 after dosing ⁵⁹Fe(III)-epi-HMA in the stomach of anemic rats was 78.3%, while that of rats given ⁵⁹FeSO₄ was 95.9%. Thereafter, the retention of both groups decreased slowly at approximately the same rate, and the rates on day 9 were 63.3 and 70.6%, respectively, a difference which was not significant. The distribution of ⁵⁹Fe among the organs and tissues of rats administered with ⁵⁹Fe(III)-epi-HMA was similar to that of ⁵⁹FeSO₄. A study on intraduodenal dosing of ⁵⁹Fe(III)-epi-HMA suggested that most of the ferric complex of epi-HMA had dissociated into Fe(III) and epi-HMA in the stomach before transfer to the duodenum, and that subsequent absorption of Fe(II) occurred in the intestine. These results indicate that iron from a ferric complex of epi-HMA, like iron from FeSO₄, was effectively incorporated into tissues and metabolized, therefore implying that ferric complexes of analogues of mugineic acid could be useful as a new iron fortificant for foods.

View full abstract

Asp. niger α-glucosidase was crystallized in ammonium sulfate solution after chromatographies on DEAE-Sepharose CL-6B and TOYOPEARL HW-55 columns. The crystalline α-glucosidase, which was a glycoprotein containing 25.5% carbohydrate as glucose, gave a single band on polyacrylamide disc gel electrophoresis. The molecular weight was estimated to be about 12.5 × 10⁴ by SDS-disc gel electrophoresis. However, the crystalline enzyme consists of two components (M.W. about 3.3 × 10⁴ and 9.8 × 10⁴, respectively) separable only by reverse-phase HPLC causing irreversible inactivation. The optimum pH was 4.3. The enzyme also hydrolyzed α-glucans such as soluble starch and amylose. The ratios of V values (Km values, in parentheses, mM of nonreducing terminal) for maltose, kojibiose, nigerose, isomaltose, phenyl α-glucoside, phenyl α-maltoside, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, maltodextrin (DP= 17), and soluble starch were estimated to be 100 (0.75): 23 (4.6): 62 (12): 35 (8.0): 1.3 (0.34): 126 (0.87): 126 (0.69): 135 (1.1): 102 (1.9): 119 (3.2): 102 (4.9):89 (5.3):89 (11): 114 (4.3). The subsite affinities in the active site were 0.790, 5.93 and 0.191 kcal/mol for subsites 1, 2, and 3, respectively. The three subsites were considered to be effective for the binding of substrate.

View full abstract

(-)-Capsaicinol, a natural antioxidant and an analog of capsaicin with no pungency, was synthesized from δ-valerolactone. The absolute stereochemistry of natural (-)-capsaicinol was derermined.

View full abstract

The adsorption isotherms for CO₂ on several proteins were measured over the wide range of pressures by the gravimetric method. The apparent amount of isothermal adsorption was a maximum at just above the critical pressure, and remained constant at higher pressures, the amount differing among casein, gelatin, gluten and ovalbumin. A sharp peak was also observed in the adsorption isotherm for SCF on an inorganic adsorbent with a smooth surface, while finite adsorption at higher pressures seems to be characteristic of physical adsorption on porous adsorbents and of chemical adsorption. The adsorption isotherm for soybean proteins differed from those of the other proteins, exhibiting a gradual increase after falling from the maximum value and showing hysteresis over the range of sub- and super-critical pressures. This hysteresis was insignificant after denaturation of the proteins.

View full abstract

A lipase gene (lip) and its activator gene (act) on a 2.9kb BglII-EcoRI fragment from Pseudomonas sp. KWI-56 were cloned in Escherichia coli using pUC19 as a vector plasmid. From the sequencing results, the open reading frames of the lip and the act were found to contain 1092 and 1032 nucleotides, respectively. The act existed downstream of the lip with the same orientation. When the lip was expressed in E. coli using the lac promoter on the pUC plasmid vector, the lipase activity of E. coli carrying both the lip and the act was 200-fold greater than that carrying only the lip. This result suggested the act was important in the expression of the lip in E. coli.

View full abstract

A gene coding for a thermostable lipase of Pseudomonas fluorescens SIK W1 was cloned into Escherichia coli JM83 by inserting Sau3AI-generated DNA fragments into the BamHI site of pUC19. Twenty colonies with esterase activity on the tributyrin agar plate were isolated by screening the constructed Pseudomonas fluorescens genomic library. Only one out of the esterase positive 20 colonies had lipase activity on the agar plate containing olive oil and Rhodamine-B.
The complete nucleotide sequence of the lipase gene was identified. The lipase gene consists of an open reading frame, 1347 bp long, commencing with an ATG start codon encoding a polypeptide of 449 amino acid residues and a TGA stop codon. Comparison of this lipase amino acid sequence with those from another organisms sequenced to data showed the presence of the short homologous region Gly-X-Ser-X-Gly.

View full abstract

The mature portion of the TEM β-lactamase (BLA) gene (bla) derived from pBR322 was fused with the promoter and signal region of Bacillus subtilis cellulase (BSC) gene (bsc), and the productivity was compared with that of the cloned native bsc gene, using a wild-type B. subtilis strain and a strain deficient in three proteases (i.e., extracellular serine protease, extracellular neutral protease, and the major intracellular serine protease) as hosts. The effects of the sen, sacQ, and prtR genes, carried on the same plasmids, were tested as for the productivities of BSC and BLA. The production of BSC was increased 9-fold by using the combination of the triple-protease deficient strain and the sacQ gene, compared with that by using the wild-type strain as a host, and no degradation of BSC was observed in the triple-protease deficient strain. On the other hand, though the production of the BSC-BLA fusion protein increased 2.5-fold in the triple-protease deficient strain, the BLA activity was decreased after the cell reached the stationary phase of growth, possibly due to some proteolysis. These observations show different sensitivity of secretary proteins to cellular proteases, and suggest that BLA is decomposed by remaining minor proteases.

View full abstract

Changes in the crystallinity and polymorph of chitosan, which may affect its functionality, by heating (up to 200°C) its water suspension were studied by X-ray diffraction measurements, using tendon chitosan prepared by N-deacetylation of a crab tendon chitin, and chitosan powders with various degrees of polymerization (DPv=1, 720-12, 600) and N-acetylation (zero to 26%). It was found that the presence of hydrated polymorphs or anhydrous crystals in a chitosan sample could be examined easily by measuring the powder diffraction pattern of a sample. Chitosan with a low molecular weight or low degree of N-acetylation was highly crystallized, especially in the anhydrous form that is considered to spoil chitosan's functionality, by heating.

View full abstract

The lower precipitated layer of a methanol extract of Citrus natsudaidai seeds, when applied to paper discs at a dosage ranging from 500 to 2000 μg/disc, significantly deterred feeding by Reticulitermes speratus nymphs. Three termite antifeedant limonoids (i.e., obacunone, nomilin and limonin) were identified as the active principles. Obacunone (with a minimum effective dosage of 150 μg/disc) was about two-fold more active than nomilin. Limonin was weak even at 1000 μg/disc.

View full abstract

A new mutagenic method for increasing the productivity of extracellular enzymes, such as cellulases, proteases, and amylases, has been developed, using vancomycin-and ristocetin-resistance as indicators. Among the mutants of Bacillus that were resistant to these atibiotics, strains with improved productivity of the extracellular enzymes were found at high frequency. The use of these antibiotics seems to provide a very effective method for the improvement of industrially important strains of Bacillus, regardless of the specific strains used and the particular extracellular enzymes produced by them.

View full abstract