The complex formation of zinc with constituents in instant coffee was studied by complexometry, using the modified tetramethyl murexide (TMM) method. The activity of Zn²⁺ binding was measured in a pH 5.0 hexamine buffer containing 1 mM ZnCl₂. Instant coffee to which Zn²⁺ had been added was separated into three color components by Sephadex G-25 gel permeation chromatography. Among these components, the second was the strongest in Zn²⁺ binding activity. Instant coffee with Zn²⁺ was also chromatographed on an anion exchange column of Amberlite IRA-410 (OH^-), the zinc adsorbed on the column being eluted with 0.01 M acetic acid. A mixture of zinc chloride and coffee was subjected to paper electrophoresis, and Zn²⁺ moved toward the anode at pH 8.5 and pH 6.4. Even at pH 3.2, some compounds carrying Zn²⁺ moved toward the anode. It follows that the ligands in instant coffee to bind Zn²⁺ are acidic in nature and that their molecular size is less than 5, 000.

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The soy-protein denaturation rates of defatted soy flour and isolated soy protein of varying moisture content were analyzed by a temperature-programmed heat-denaturation (TPHD) technique with a hand-press reactor. The degree of protein denaturation of the soy proteins, which is defined as the solubility in 0.1 M phosphate buffer (pH 8.0) containing 1% SDS and 1% 2-ME, could be estimated with the TPHD model as a first-order heat-denaturation process. A kinetic compensation effect was observed for the protein denaturation of both soy proteins, the activation energy of the denaturation rate decreasing linearly with a moisture content below 0.4.

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Oligosaccharides from the digestion of 34% N-acetylated chitosan by Aeromonas hydrophila chitinase were separated by CM-Sephadex C-25 column chromatography. Sugar compositions and the sequences of main oligosaccharides were identified through their N-acetylation, their cleavage with exo-glycosidases, and their degradation with nitrous acid. Hetero-chitooligosaccharides such as GlcN•GlcNAc, GlcN•GlcNAc•GlcNAc, GlcNAc•GlcN•GlcNAc, and GlcNAc•GlcN•GlcNAc•GlcNAc, together with GlcNAc and (GlcNAc)₂, were detected. The structure of GlcN•GlcNAc was confirmed by the analysis with proton and carbon NMR spectroscopy. These studies indicate that Aeromonas hydrophila chitinase is more specific toward the N-acetyl-β-D-glucosaminidic bonds in partially N-acetylated chitosan.

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We found a novel protopectin-solubilizing enzyme that had potent activity on the protopectin in sugar beet pulp, in the culture filtrate of Bacillus subtilis IFO 3134. The enzyme was purified by treatment with EDTA and chromatography on CM-Cellulofine CH, Butyl-Toyopearl 650, and Toyopearl HW-55S, and was isolated as a homogeneous protein. The enzyme had an apparent molecular weight of 30, 000 in SDS-polyacrylamide gel electrophoresis, and 27, 000 in permeation chromatography on Toyopearl HW-55S, with its isoelectric point around pH 9.0. The enzyme was stable from pH 5.0 to 9.0 and up to 60°C. The optimum pH for enzyme action was 6.0 at 37°C and pH 8.0 at 60°C. The enzyme catalyzed the release of pectin from protopectin of various citrus fruit peels as well as sugar beet pulp, but much less from that of lemon peel, without catalyzing the degradation of polygalacturonic acid. The enzyme catalyzed the degradation of arabinan in sugar beet pulp, and it seemed to release pectin by splitting the arabinan connecting the pection to cell wall polysaccharides.

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The succinate metabolizing pathway under low oxygen concentrations in soybean nodule bacteroids was surveyed by pulse-chase experiments using [2, 3-¹⁴C] succinate and by radio-respirometry. Oxidation of exogenous succinate through the complete citric acid cycle was strongly suggested. Amino acid and sugar formations from exogenous succinate were also observed in the bacteroids. Addition of succinate to the bacteroids increased both the NAD⁺/NADH ratio from 1.8 to 3.0 and the adenylate energy value from 0.25 to 0.53.

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β-N-Acetylhexosaminidase (EC 3.2.1.52) from the culture filtrate of Nocardia orientalis was purified to homogeneity by precipitation with ammonium sulfate followed by column chromatography on CM-Sephadex, Bio-Gel P-60, and phenyl-Sepharose CL-4B. The molecular weight of the enzyme was about 56, 000 by gel filtration and 54, 000 by SDS polyacrylamide gel electrophoresis. The enzyme showed about 1.6-fold higher β-N-acetylglucosaminidase activity than β-N-acetylgalactosaminidase activity. The optimum pH and temperature were 5.0 and around 70-75°C for PNP-GlcNAc, and 4.0 and 60°C for PNP-GalNAc. The enzyme was stable in the pH range from 4.0 to 8.0 and below 45°C. The enzyme hydrolyzed N-acetyl-chitooligosaccharides, di-N-acetyl-chitobiose through hexa-N-acetyl-chitohexaose. The enzyme showed glycosyl transferase activity during the hydrolysis of di-N-acetyl-cnitobiose. Two major transfer products were isolated and identified as the β(1→6)-linked disaccharide of N-acetylglucosamine and tri-N-acetyl-chitotriose.

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The distribution of phospholipids between the inside and outside of an emulsion droplet was studied in relation to the emulsion stability. The emulsion was prepared with various mixtures of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC), n-decane and water containing HC1, NaCl or NaOH. ¹³P-NMR spectra of the emulsion in the presence of lanthanide ion Pr³⁺ allowed quantitative measurement of the phospholipids in the internal and external aqueous phases, and in multilamellar vesicles. By a phospholipase A₂ treatment of PC facing the continuous aqueous phase, most of the internal phospholipids were suggested to be composed of PC. The ratio of the phospholipids inside and outside changed with the emulsifying conditions, and was well correlated with the emulsion stability. Thus, it is suggested that the phospholipid distribution in the emulsion of PC and LPC showed the change of their hydrophile-lipophile balance and of the interfacial absorption force.

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Arthrobacter sp. K-1, isolated from soil, produces β-fructofuranosidase. This enzyme preparation was separated into three fractions (I, II, and III) by butyl-Toyopearl 650 M column chromatography. The β-fructofuranosidase I was purified to homogeneity by disc-electrophoresis after consecutive column chromatographies. The enzyme had a molecular weight of 52, 000 by SDS-polyacrylamide gel electrophoresis and 51, 000 by gel filtration with Ultrogel Ac A 44, and an isoelectric point of 4.3. The enzyme was most active at pH 6.5-6.8 and at 55°C and stable up to 45°C at pH 6.5 for 30min of incubation, and from pH 5.5 to 10.0 at 40°C in 2hr of incubation. This enzyme hydrolyzed sucrose, erlose, raffinose, fructosylxyloside, neokestose, and stachyose at the relative velocities of 100, 75, 61, 59, 54, and 11, but hardly hydrolyzed 1-kestose and nystose (<0.02).

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An endo-1, 3-β-D-xyIanase (1, 3-β-D-xylan xylanohydrolase, EC 3.2.1.32) was purified from the culture fluid of Pseudomonas sp. PT-5 by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Toyopearl HW-50S, and Butyl-Toyopearl 650 M column chromatography. The purified enzyme gave a single band on poly aery lamide gel disc electrophoresis and its molecular weight was 35, 000 by SDS-polyacrylamide gel electrophoresis. The enzyme was stable from pH 5.5 to 8.0 and had its maximum activity at pH 7.5. The enzyme rapidly reduced the viscosity of glycol β-1, 3-xylan solutions and produced xylose and xylooligosaccharides from seaweed β-1, 3-xylan. The enzyme activity was greatly inhibited by Hg²⁺, SDS, ethylenediamine tetraacetic acid (EDTA), and N-bromosuccinimide (NBS).

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The effect of aging on the rheological properties of gluten gel was investigated through continuous dynamic viscoelastic measurements. The value of the storage modulus rapidly increased with time for the first several hours, settled down to a slow increase, and attained an equilibrium state after a long period. Such behavior could be approximated by the equation for an orthogonal hyperbola. The rate of storage modulus increase at the early stage of the aging process increased with increasing temperature, while the rate after a long period of aging decreased with increasing temperature. Both the rates at the early stage and after a long period of the aging processes appeared to be faster if the concentration was higher. In addition, the change of the average molecular weight between the crosslinks in gluten during aging was determined by the theory of entanglement coupling. These results suggest that each entanglement involved segments from at least several molecules, and that gluten gel is composed of a network of loosely crosslinked polymers.

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Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50 % PEG 4, 000, 15μg protamine sulfate/ml, 0.15M sucrose, and 10 mM MgSO4 in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 × 10⁵ PFU/Μg DNA. The eclipse period of growth of progeny phages in the transfectants was 3hr and the average burst size was 200.

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Changes in the dolichyl fatty ester (Dol-FA) compositions in soybean embryos were investigated during germination. After 3 days of germination, the fatty acid composition of Dol-FA changed; the percentage of palmitic acid decreased and that of linoleic acid increased. There were also significant amounts of behenic and lignoceric acids in the Dol-FA. However, the composition of dolichol moieties of Dol-FA (which contained 16 to 21 isoprene units) did not change. Percentages of linoleic acid in phospholipids decreased with increases in linolenic acid. However, behenoic and lignoceric acids found in Dol-FA were not present in glycerolipids.

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Bovine β-lactoglobulin A was expressed in Escherichia coli in its mature form. The gene was constructed using a cDNA clone which coded for amino acid residues Leu-11 to He-162 and a synthetic oligonucleotide coding for the initial 10 amino acids preceded by a translational start. The met-β-lactoglobulin was expressed using a tac promoter vector, pTTQ18, and accounted for approximately 15% of the total cellular protein. The recombinant met-β-lactoglobulin migrated with the same molecular weight as native β-actoglobulin A on SDS-PAGE. The majority of the met-β-lactoglobulin produced was found in an insoluble form but could be solubilized using guanidine-HCI. The renatured preparation was > 80 % pure and migrated similarly to purified β-lactoglobulin A under nondenaturing conditions.

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Finely powdered cellulose was pyrolysed in a Curie-point pyrolyser, which was directly attached to a gas chromatograph and mass spectrometer (CPP-GC/MS), at 358°C for 5 sec in He (gas) without any additives. The pyrolysis products were levoglucosenone as the main component, 2-furaldehyde, l, 4:3, 6-dianhydro-a-D-glucopyranose and 5-hydroxymethyl-2-furaldehyde. However, CPP-GC/MS of cellulose in the presence of potassium malate (5 % as potassium) gave 2-furanmethanol as the main component, and a number of low molecular compounds were formed; e.g., lactones, ketones and furans. A possible new reaction path for cellulose to 2-furanmethanol via D-xylulose is presented.

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Palm oil (PA) contains about 50% saturated fatty acid and is rich in tocotrienol (T-3). Plasma lipid and eicosanoid levels, and fatty acid compositions of tissue lipids in rats given refined PA were compared to those given lard (LA) rich in stearate, olive oil (OL), palm olein rich in oleate, and safflower oil (SA) rich in linoleate. The effects of supplementation with T-3 was also investigated. An elevation of the plasma cholesterol level due to cholesterol feeding was moderate in rats given PA or LA compared to rats given OL, although it was higher than in the SA group. The proportion of linoleate in dietary PA, LA and OL was similar, but that of arachidonate in plasma phosphatidylcholine and platelet lipids was slightly but significantly higher in the PA group, when compared to the SA group, but the proportion of docosahexaenoate in plasma and liver phosphatidylcholine was significantly lower in the PA group than that in the LA and OL groups. Although aortic production of prostacyclin and the plasma level of thromboxane A₂ did not differ significantly among rats fed various fats, the ratio of prostacylin/thromboxane A₂ was in the following order: SA > PA = LA > OL. The addition of T-3 to PA did not affect plasma total cholesterol, eicosanoids, or tissue fatty acid compositions, but plasma high density lipoprotein-cholesterol increased dose-dependently. This study, therefore, suggests that both the fatty acid composition and T-3 affect the metabolic effect of dietary fat on plasma cholesterol, tissue polyunsaturated fatty acids, and probably eicosanoid balance.Α

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Chitinase (EC 3.2.1.14) was purified from the stomach of Japanese eel, Anguilla japonica, by fractionations with ammonium sulfate, Sephadex G-100 gel filtration, and DEAE-cellulose, CMcellulose, and hydroxylapatite column chromatography. The molecular weight of this enzyme was 50, 000 by SDS-PAGE, and the optimum pH was 4.4. The activity was strongly inhibited by Hg²⁺ and slightly activated by EDTA. The hydrolysis products of colloidal chitin by the enzyme were GlcNAc and GlcNAc₂. When GlcNAc_3-6 was used as substrate, GlcNAc and GlcNAc₂ were recognized as final products with various ratios. GlcNAc₂ was not hydrolyzed by this chitinase.

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The KHR gene cloned from a genomic library was on 4.7-kbp DNA fragment and was inserted into YCpGll vector (KHR-YCp) and YEp vector (KHP-YEp). Transformants with KHR-YEp could secrete 3-4 times as much killer toxin into the media as the donor strain. The complete nucleotide sequence of the KHR gene was analyzed. It was found that the KHR gene consisted of 888 bp. It was suggested that this protein was processed before being secreted into the media, because its molecular mass presumed from the nucleotide sequence was larger than that of the mature killer toxin.

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The molecular size distribution of unreduced glutenin was studied by gel filtration on a Toyopearl HW-75F column, with 0.05 M Tris-HCl buffer (pH 8.3) containing 0.2% sodium dodecyl sulfate (SDS) as the elution solvent. The unreduced glutenin was extracted with 0.5% SDS (pH 7.0) from the flour residue after exhaustive extraction of the gliadin and water-soluble proteins. The yield of glutenin with a little gliadin as a contaminant was about 20% of the total flour proteins, and the flour protein was almost completely extracted by this procedure. The gel filtration patterns gave two peaks, a small one at the void volume of the column and a broad peak eluting from near the void volume to the elution volume for gliadin. The proteins eluted from the column were analyzed by SDS-polyacrylamide gel electrophoresis with or without prior reduction of their disulfide bonds. Only a slight difference among the glutenin samples in the fractions eluted from the column was observed in their subunit patterns. The peak eluted at the void volume contained no glutenin. However, a markedly increased proportion in the first peak was observed when the glutenin sample was prepared by an ethanol-fractionation method. The results suggest polymerization of the glutenin proteins during the purification procedure.

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The effects of twelve novel triterpenoid acids isolated from the ethanolic extract of the stems of Kadsura heteroclita and k. longipedunculata, Chinese folk medicines, and three other novel triterpenoid acids semi-synthesized on cholesterol biosynthesis were examined. (24Z)-3-Oxo-lanosta8, 24-dien-26-oic acid (3-oxo-LA) was most active in depressing cholesterol biosynthesis from [2-¹⁴C]mevalonate in a 10, 000 × g supernatant fraction of rat liver homogenate. It was suggested from the TLC analysis of radio-labeled products that these compounds inhibit the demethylation of lanosterol. Some triterpenoid acids including 3-oxo-LA also inhibited cholesterol biosynthesis from [1-¹⁴C]acetate in primary cultures of rat liver cells.

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An intracellular protease from a bacterium, Bacillus pumilus HL721, was purified about 5000-fold by chromatography with a Q-Sepharose Fast Flow column, TSK-gel HA-1000 glass column, and TSK-gel G3000SWXL column using Bz-Gly-Ala-Pro as a substrate. The enzyme was the most active at pH around 7.5 and stable from 4.5 and 8.0. The enzyme activity was inhibited by Cu²⁺, EDTA, N-ethylmaleimide, o-phenanthroline, and p-chloromercuribenzoic acid. The molecular weight of the enzyme was 155, 000 by gel filtration. The enzyme removed dipeptide from the carboxyl end of some peptides used as substrates. From these results the enzyme seems to be a dipeptidyl carboxypeptidase.

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An actinomycete identified as Streptomyces flavidovirens was found to produce a new cyclic hexadepsipeptide antibiotic, designated citropeptin. Citropeptin showed antitumor activity against P388 murinc leukemia.

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Dietary (R)(+)-salithion was a more potent insecticide (LC₅₀=9.3 ppm) than the (S)(-)-enantiomer (LC₅₀=63 ppm), correlating acetylcholinesterase (AChE) inhibition in vivo (I₅₀=7.7 and 63 ppm) against Tribolium castaneum larvae. (S)(-)-Salioxon derived from (R)(+)-salithion had a stronger inhibitory activity (I₅₀=8.8 μM) than the (R)(+)-enantiomer (I₅₀=22 μm) in vitro against larval AChE. However, against Musca domestica female adults, a reversed tendency was observed: (S)(-)-salithion and (R)(+)-salioxon were more potent than (R)(+)-salithion and (S)(-)-salioxon in topical insecticidal and anticholinesterase activities in vivo and in vitro, respectively. Hence, the reversed stereospecificity between the T. castaneum larvicidal and M. domestica insecticidal activities of salithion enantiomers is due to a stereospecific difference in the intrincic potency of salioxon enantiomers as an AChE inhibitor with the two insects. A suppression of M. domestica larval growth, gut amylase and invertase activities by dietary 0.5, 1.0 and 1.25 ppm of (S)(-)-salithion was observed 2 days after treatment. A similar tendency was recognized when 1.25 and 2.50 ppm of (R)(+)-salithion was used. However, against M. domestica female adults, no appreciable effect on these enzymes was recognized 1-72 hr after topical application of 0.05 and 0.10 μg/fly of (S)(-)-sa!ithion, and 0.01 and 0.05 μg/fly of (R)(+)-salithion. The whole-body levels of cyclic adenosine 3', 5'-monophosphate (cAMP) were slightly increased in T. castaneum larvae 3 days after a dietary 60 and 80 ppm of (S)(-)-salithion treatment, and in M. domestica larvae 2 days after treating by dietary 1.25 and 2.50 ppm of (R)(+)-salithion, respectively. Neither of the enantiomers of salithion activated adenylate cyclase prepared from Periplaneta americana ventral nerve cords, but suppressed octopamine potency, suggesting that salithion could act as an antagonist to the octopamine-receptor and that the increased level of whole-body cAMP could be due to the inhibition of phosphodiesterase.

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A crude enzyme was extracted from fresh tea leaves and it showed β-glucosidase activity, using p-nitrophenyl-β-D-glucoside as a substrate. The non-volatile fraction of a hot-water extract from green tea was incubated with the crude enzyme extract at 37°C for 30 min. After this enzymatic hydrolysis, the formation of (Z)-3-hexenol, linalool oxide I, linalool, methyl salicylate, geraniol, benzyl alcohol and 2-phenylethanol was observed. Geranyl-β-D-glucoside was synthesized by the Koenigs-Knorr reaction. When it was reacted with the crude enzyme extract of tea leaves, geraniol was identified on GC as an enzymatic hydrolysis product. Thus, geranyl-β-D-glucoside seems to be the precursor of geraniol in tea leaves.

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Volatile components in the headspace vapors of Cymbidium faberi 'Dawipin' and Cymbidium virescens 'Songme' were adsorbed on Tenax TA in a column, then flushed out by heating the column, and finally collected in a cold trap. They were directly analyzed by gas chromatography and mass spectrometry combined with gas chromatography. Thirty three compounds and fifty two compounds were identified as volatile components in the headspace vapors of 'Dawipin' flowers and 'Songmei' flowers, respectively. Methyl jasmonate and methyl epijasmonate were found to be indispensable for the beautiful scent of these flowers.

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Total lipids were extracted from five varieties of grape seeds and systematically analyzed for their chemical compositions. The yields of the total lipids were 10-16%, and triacylglycerol (TG) usually amounted to c. 90% of the whole. From a reversed-phase high-performance liquid chromatographic analysis, the major molecular species of TG were shown to be trilinolein (40%), oleoyldilinolein (21%) and palmitoyldilinolein (18%). The component fatty acids were asymmetrically distributed at C-1 and C-3 of the TG molecule. Palmitic acid was exclusively located at the C-1 position, although unsaturated fatty acids, especially linoleic acid, were predominant at the C-1 position, as at the C-2 and C-3 positions. Compared with TG, higher proportions of palmitic and linolenic acids were generally observed in thirteen other lipid classes isolated from grape seeds, although the fatty acid compositions of the diacylglycerol and free fatty acids were roughly identical with that of TG. As component sterols, sitosterol, campesterol and stigmasterol, especially the former, were predominant. Their relative proportions were somewhat different from each other between the neutral and polar sterol lipids.

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