Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli, and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87.

We characterized the membrane vesicle fraction (RD MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD MVs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the RD MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patches cells following the addition of the RD MV fraction. In the presence of the RD MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial Toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

OBJECTIVE: Postmenopausal women are at increased risk of metabolic diseases such as obesity and diabetes. Therefore, the chemoprevention of postmenopausal changes in health via dietary supplements is important. Syringic acid (SA) is a phenolic compound present in the fruit of the assai palm, Euterpe oleracea, and in the mycelium of the shiitake mushroom, Lentinula edodes. This compound shows no affinity for estrogen receptors and may exert disease-preventive effects. Reportedly, dietary SA ameliorates high-fat diet-induced obesity in mice; however, its effects on estrogen deficiency-induced obesity are still unclear. Therefore, in this study, we investigated whether and how dietary SA affects these factors in ovariectomized (OVX) mice. METHODS: Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/d) for 12 weeks. Their body weights, food intake, and uterus weights as well as other parameters were measured and comparisons were made with mice in the control group. RESULTS: Dietary SA did not affect the body weight, food intake, or uterus weight of OVX mice over the study period; however, the SA-fed group showed lower fat mass (ie, visceral, subcutaneous, and total fat) than the OVX-control group (11.1 ± 3.3 vs. 8.3 ± 2.4, P < 0.05; 7.9 ± 1.1 vs. 5.9 ± 1.6, P < 0.05; 19.0 ± 4.2 vs. 14.1 ± 3.8, P < 0.05, respectively). Furthermore, blood analysis revealed that SA-treatment resulted in a dose-dependent decrease and increase in serum triglyceride (59.2 ± 8.3 vs. 43.9 ± 12.2 mg/dL P < 0.05) and adiponectin (7.7 ± 0.3 vs. 9.5 ± 0.6 μg/mL, P < 0.05) levels, respectively. CONCLUSIONS: These results suggest that the SA diet improves lipid metabolism without affecting the uterus in OVX mice. Therefore, dietary SA has potential applicability for the prevention of postmenopausal obesity and type 2 diabetes.

In recent years, the number of patients with osteoporosis has increased as population grows older. Therefore, the chemoprevention of osteoporosis by better nutrition is important. White-rot fungi degrades milled wood lignin for growth and development. This degradation results in the formation of phenolic compounds such as syringic acid (SA) and vanillic acid (VA). In the artificial culture of edible mushrooms using a mushroom bed, the disposal of waste beds after mushroom cultivation is an important issue. The present study investigated the presence and amount of both SA and VA in the discarded waste beds after mushroom cultivation. The extracts from waste beds after cultivation of shiitake mushrooms, Lentinula edodes; buna shimeji, Hypsizygus marmoreus; maitake, Grifola frondosa; king trumpet mushrooms, Pleurotus eryngii; and butterscotch mushrooms, Pholiota microspora were analyzed using high performance liquid chromatography. Although the content of SA and VA was considerably different among the mushrooms, SA and VA were present in extracts obtained from all the waste beds. We also demonstrated that SA and VA exert their anti-osteoporotic effect independently of the estrogen receptor-mediated pathway using murine monocytic RAW264.7 cells, ovariectomized mice, and human breast cancer MCF-7 cells. Thus, these results suggest that the extracts are effective sources of SA and VA, which are effective in preventing osteoporosis.

In recent years, the number of patients with osteoporosis has risen with the increase in average longevity. Therefore, the chemoprevention of osteoporosis using food materials or food components has become an increasingly important target. Syringic acid (SA) is a phenolic compound present in the fruit of the a double dagger ai palm Euterpe oleracea and the mycelium of the shiitake mushroom Lentinula edodes. This compound has no affinity for estrogen receptors and is potentially useful for disease prevention. However, little is known about the effects of a SA diet on bone metabolism, particularly bone resorption in vivo. Here, we demonstrated the effects of a SA diet on bone loss and uterine weight loss in ovariectomized (OVX) mice. Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/day) for 10 weeks. After 10 weeks of dietary SA, the body weight, food intake, and uterine weight of the OVX mice were unaffected; however, femoral bone mineral density (cortical bone density, cancellous bone density, and total bone density) was higher in the SA-fed groups than in the OVX-control group. Furthermore, histomorphometric analysis revealed that the number of osteoclasts and osteoblasts was decreased and increased, respectively, in the SA-fed groups. These results suggest that a SA diet suppresses bone loss by downregulating bone resorption and upregulating bone formation without affecting the uterus in OVX mice. Although further studies are needed, SA may be a compound that can be used to prevent or retard osteoporosis.

A total of 123 actinomycetes was isolated from 12 varieties of wild orchids and screened for potential antagonistic activity against Phytophthora, which causes black rot disease in orchids. In vitro and in vivo experimental results revealed that Streptomyces sp. strain 9X166 showed the highest antagonistic activity; its -1,3-glucanase production ability was a key mechanism for growth inhibition of the pathogen. PCR amplification and DNA sequencing of the 16S ribosomal RNA gene allowed the identification of this strain, with high similarity (99.93%) to the novel species Streptomyces similaensis. The glucanase enzyme, purified to homogeneity by anion exchange and gel filtration chromatography, showed a specific activity of 58Umg(-1) (a 3.9-fold increase) and yield of 6.4%. The molecular weight, as determined by SDS-PAGE and gel filtration, was approximately 99 and 80kDa, respectively, suggesting that the enzyme was a monomer. The purified enzyme showed the highest substrate specificity to laminarin, indicating that it was -1,3-glucanase. The hydrolyzed products of cello-oligosaccharides suggested that this enzyme was endo-type -1,3-glucanase. Streptomyces sp. 9X166 culture filtrate, possessing -1,3-glucanase activity, could degrade both freeze-dried and living mycelium. This is the first report on a -1,3-glucanase-producing Streptomyces sp. that could be an effective biocontrol agent for black rot disease in orchids.

Here, we report the draft genome sequence of Flammulina velutipes TR19, which was newly isolated from commercial strains in Japan. The genes related to fruiting body formation in the basidiomycete were identified by whole-genome analysis.

  Large amount of radioactive cesium was emitted from the TEPCO Fukushima Dai-ichi nuclear power plant by the accident into atmospheric air, and a part of the radioactivity was brought to the ground by rain and snowfall. The Yamakiya district in Kawamata-machi, Fukushima is specified as the prepared evacuation zone. The authors collected wild mushrooms in this district as samples in 2012, 2013 and 2014. The concentration of radioactive cesium was measured by means of the hyperpure germanium semiconductor detector. The concentrations were ranged from 35 to 600,000 Bq/kg, and were different with points of sampling and kinds. The transfer coefficient of radioactive cesium to most wild mushrooms are lower than 0.5, although some mushroom species concentrate environmental radioactive cesium more than twice.

The purification and characterization of a neutral protease (PE-4) produced in mycelia during fruit-body formation in Hypsizygus marmoreus was carried out in the present study. The enzyme was purified to homogeneity using ammonium fractionation, ion exchange chromatography on DEAE-Toyopearl, hydrophobic exchange chromatography on Butyl-Toyopearl, and gel chromatography on Superdex 200. Protease activity was enhanced by Co^<2+>, in particular, and was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF), aprotinin and Pefabloc SC, which are known serine protease inhibitors. Phosphoramidon did not inhibit the protease. The N-terminal amino acid sequence was identical with the serine protease from the fruit-body of H. marmoreus, hmsp. The same serine protease was present in both vegetative mycelia and the fruit-body in the growth stage. Protease activity activated in mycelia at fruit-body formation was inhibited by PMSF, EDTA, and phosphoramidon but not completely. These results also suggest that several neutral proteases besides PE-4 were present during kinkaki, which is removal of both the spawn and the uppermost layer of the medium, in the mature fruit-body in the mycelia.

type:Departmental Bulletin Paper [Abstract] Large amount of radioactive cesium was emitted from the TEPCO Fukushima Dai-ichi nuclear power plant by the accident into atmospheric air, and a part of the radioactivity was brought to the ground by rain and snowfall. The Yamakiya district in Kawamata-machi, Fukushima is specified as the prepared evacuation zone. The authors collected wild mushrooms in this district as samples with gentle guide of local mushroom lovers in October, 2012. The kinds of mushroom were specified by the mushroom specialist. 16 kinds of mushrooms have been extracted. The extracted mushroom was brought back to the university. The concentration of radioactive cesium was measured by means of the hyperpure germanium semiconductor detector. The concentrations were ranged from 0.5 to 2600 Bq/g, and were different with points of sampling and kinds. The concentrations were compared with before washing and after washing by means of ultrasonic cleaning. The amount of radioactive cesium reduced to the range from 30％ to 60％ of the before washing. HOHARA, Sin-ya ITOH, Tetsuo

Profiling of cellulases (endo-type cellulase, exo-type cellulase, and β-glucosidase) of Tremella fuciformis and Hypoxylon truncatum were carried out using crude enzymes from these two microorganisms cultured in sawdust-rice bran medium. Specific activity of the crude enzyme from T. fuciformis toward cellulose powder, Avicel, and p-nitrophenyl-β-D-glucopyranoside was 13.5, 35.9, and 47.6U/mg, respectively. The enzyme did not act on carboxymethyl cellulose (CMC). The crude enzyme from H. truncatum acted toward all substrates tested. Endo-type cellulase from H. truncatum was purified to homogeneity and its enzymatic properties were characterized. Following filtration and centrifugation, the enzyme solution was purified by ammonium sulfate fractionation, heat treatment, ion exchange chromatography, and gel chromatography. Its molecular weight was estimated as 46,000 by SDS-PAGE and 41,000 by gel chromatography, which suggested that the native enzyme is active as a monomer. The enzyme was stable at 60℃ and pH 5.0-6.0. The purified enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose, but did not degrade cellobiose or cellotriose. These results suggested that endo-type cellulase from H. truncatum can provide short cellooligosaccharides such as cellobiose, cellotriose, and cellotetraose to compensate for the slight or absent endo-cleavage of the T. fuciformis cellulase system during co-cultivation of these two fungi.

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography-size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50A degrees C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50A degrees C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.

We isolated yellow pigment from the fruit body of Pleurotus cornucopiae var. citrinopileatus and evaluated the properties of the pigment. The yellow pigment was extracted by water and purified by repeated gel filtration on Sephadex G-50 and G-25 column chromatography. The pigment was insoluble in usual organic solvents, slightly soluble in methanol and soluble in water. The yellow pigment was heat stable, as no color changes were observed after heating for 20 min at 100℃. The yellow pigment did not show a specific absorption peak within the visible spectral range (370 to 500 nm) in spite of its yellow color, although it had an absorption peak and trough at 286 nm and 257 nm, respectively. The pigment became bleached in strong oxidative solutions such as H_2O_2, KMnO_4, and NaOCl, and reduced Fe^<3+> to Fe^<2+>. These results suggest that the yellow pigment of P. cornucopiae var. citrinopileatus is a pheomelanin-like pigment generated from tyrosine and cysteine by tyrosinases. Furthermore, the molecular weight of the yellow pigment was estimated to be approximately 4,200 Da by HPLC-GPC.

Whisky exerts an inhibitory effect on melanogenesis in B16 cells, the anti-melanogenic activity being positively correlated with the aging period and anti-oxidative activity of whisky. We examined the correlation between the inhibition of melanogenesis and the concentration of each compound in various whiskies to evaluate the importance of 11 different whisky polyphenols, including ellagic acid, gallic acid and lyoniresinol, in the anti-melanogenic activity of whisky. The concentration of all the compounds was positively correlated with the anti-melanogenic activity of whisky. Ellagic acid, gallic acid and lyoniresinol were the predominant polyphenols in the whiskies measured by HPLC. These three compounds also significantly inhibited the melanogenesis and tyrosinase activity in B16 cells. Ellagic acid, gallic acid and lyoniresinol were confirmed as the major participants in the anti-melanogenic activity of whisky.

SOD (Superoxide dismutase)-like activities of 23 kinds of single malt whisky (Scotch and Japanese) were evaluated. There was a positive correlation between SOD-like activity and the maturation age of whisky that exceeded the difference resulting from the manufacturing region. The SOD-like activity of Yamazaki 18, a typical single malt whisky in Japan, was approximately 1333. U/ml and that of non-volatile components in the whisky was 388. U/mg, indicating that single malt whisky generally has a very strong SOD-like activity. To elucidate their contribution to SOD-like activity, the non-volatile components of whisky (Yamazaki 18) were ultrafiltered and separated with a Diaion HP20/water-EtOH system. Elution of the fraction less than 5000 molecular weight (< 5000 MW fraction) with 60% (v/v) EtOH contributed most to SOD-like activity of the whisky. As this elution contained a considerable amount of polyphenolics, the content and SOD-like specific activity of ellagic acid, gallic acid, and lyoniresinol - the main whisky polyphenolics - were evaluated. The contribution of these compounds to the SOD-like activity of whisky was approximately 15%. Polyphenolics in whisky were relatively distributed to a higher MW fraction compared to carbohydrates in whisky, and specific activity (SOD-like activity per weight) of the > 10,000. MW fraction was greater than that of the < 5000. MW fraction, although the content of this fraction was low. These results indicate that various polyphenolics with higher molecular weights also contribute to the SOD-like activity of whisky together with main whisky polyphenolics. © 2011 The Society for Biotechnology, Japan.

Aims: To determine the effects of food restriction (FR) on the expression of Sirt1 and its down-stream factors related to lipid and glucose metabolism in obese and hypertensive rats (SHRSP/IDmcr-fa), as a model of human metabolic syndrome. Main methods: Male, 10-week-old SHRSP/IDmcr-fa rats were treated with 85% FR for 2 weeks. Metabolic parameters, serum adipocytokines and distribution of serum adiponectin multimers were investigated. Sirt1 expression was determined in epididymal adipose tissue, liver and skeletal muscle. We also determined the expression of PPAR alpha, gamma and other adipocyte-related genes in epididymal adipose tissue, and glucose transporters (GLUT2 and GLUT4) in the liver and skeletal muscle. Key findings: FR improved the general conditions as well as blood chemistry of SHRSP/IDmcr-fa rats. In the epididymal adipose tissue of the FR rats, Sirt1 expression was enhanced, as was adiponectin, whereas leptin was downregulation, findings that were paralleled by the serum protein levels. Furthermore, the serum ratio of high to total adiponectin was increased in the FR group. The mRNA expression of Sirt1 was upregulated in the adipose tissue in the FR group. Sirt1 mRNA expression was downregulated, while PPAR alpha and GLUT2 expression was enhanced in the liver. No differences were found in terms of Sirt1, PPAR or GLUM expression in skeletal muscle. Significance: These results indicate that FR corrects adipokine dysfunction by activating PPAR gamma via Sirt1 in adipose tissue. Furthermore, glucose and lipid metabolism are activated by upregulation of GLUT2 via the activation of PPAR alpha in the liver. (C) 2011 Elsevier Inc. All rights reserved.

We purified and characterized extracellular β-glucosidase of the Tricholoma matsutake J-1 strain isolated from hardwood forest (Quercus sp.). The purified enzyme was obtained from about 0.76l static culture filtrate, with 17.7% recovery and a single band on SDS-PAGE. The enzyme displayed the most activity around 60℃ and pH4.0. The activity of this enzyme against cello-oligosaccharides as a substrate was higher than that of the T. matsutake Z-1 strain isolated from softwood (Pinus densiflora). Furthermore, the enzyme activity increased with an increase in the number of β-1,4 glucosidic bonds.

Yield, pileus size, freshness preservation, trehalose content and withdrawal rate of Lentinula edodes fruit bodies grown on substrates treated with trehalose by different methods were analyzed. Either 15 or 30g trehalose was injected into substrate 7 or 3 days before fruit body emergence at the first through third flushes. In the control group, fruit bodies were grown on substrate mixed with 30g trehalose. Injection of 15 or 30g trehalose 3 days before fruiting resulted in a greater fruit body yield and more fruit bodies above M-size compared to the control. Freshness of the fruit bodies at the first flush was evaluated based on the L^* value of the underside of the pileus and the visual commercial freshness score. Seven days after injection of 15 or 30g trehalose, freshness preservation was improved as compared with the control. Taken together, these results suggest that injection of 15 or 30g trehalose into substrates 3 days before fruit body emergence improves the yield, the percentage of M-sized or above fruit bodies and freshness preservation compared to the mixed treatment. Moreover, the trehalose content of air-dried fruit bodies at any flush after 15 or 30g injection was greater than that after mixing, while the withdrawal rate for injection of 15g trehalose was higher than that for the 30-g injection.

Pactamycin is an aminocyclopentitol-derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456,revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe-S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6-methylsalicylic acid (6-MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de-6-MSA-pactamycin and de-6-MSA-pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure-activity relationship of pactamycin.

The fatty acid desaturation and elongation reactions catalyzed by Trichoderma sp. 1-OH-2-3 were investigated. This strain converted palmitic acid (16: 0) mainly to stearic acid ( 18: 0), and further to oleic acid (c9-18:1), linoleic acid (c9, c12-18:2), and a-linolenic acid (c9, c12, c15-18:3) through elongation, and Delta 9, Delta 12, and Delta 15 desaturation reactions, respectively. Palmitoleic acid (c9-16:1) and cis-9, cis-12-hexadecadienoic acid were also produced from 16: 0 by the strain. This strain converted n-tridecanoic acid (13: 0) to cis-9-heptadecenoic acid and further to cis-9, cis-12-heptadecadienoic acid through elongation, and D9 and D12 desaturation reactions, respectively. trans-Vaccenic acid (t11-18:1) and trans-12-octadecenoic acid (t12-18:1) were desaturated by the strain through Delta 9 desaturation. The products derived from t11-18:1 were identified as the conjugated linoleic acids (CLAs) of cis-9, trans-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. The product derived from t12-18:1 was identified as cis-9, trans-12-octadecadienoic acid. cis-6, cis-9-Octadecadienoic acid was desaturated to cis-6, cis-9, cis-12-octadecatrienoic acid by this strain through Delta 12 desaturation. The broad substrate specificity of the elongation, and Delta 9 and Delta 12 desaturation reactions of the strain is useful for fatty acid biotransformation.

Effect of trehalose on yield, trehalose content. freshness preservation and taste evaluation of Lentinula edodes fruit bodies were examined after 0.5, 1, 2, 3 or 4% was added to the cultivation substrate. There was no significant difference between the fruit body yields from the substrates with 0.5-4% trehalose and the yield from the substrate without. The percentages of fruit body number above M size which emerged in the first flush from the substrates with 0.5, 1, 2 and 3% additions were greater than the ratio from the substrate without. Comparing to the substrate without addition, the trehalose contents of the fruit bodies from the substrates with 2, 3 and 4% additions were greater in any of the three flushes. The addition of 2% was effective in the freshness preservation of the fruit bodies in any of the flushes and the addition of had a significantly large effect. The taste evaluation with 2 and 3% additions resulted in higher scores of the three items after tasting for odor, taste and texture, and of total evaluation as compared to no addition.

The quality of whisky is known to improve remarkably by its storage over many years. This process is commonly termed "maturing." In this process, polyphenols derived from lignin and tannin of the barrel have an important role in not only forming the matured flavor and taste but also contributing to the advance of clustering ethanol and water in whiskey. It is also likely that polyphenols generally possess reactive oxygen (RO) scavenging activity. The present study evaluated the RO scavenging activity (free-radical scavenging activity, H2O2 reduction activity under peroxidase coculture and H2O2 scavenging activity) of 24 single malt whiskeys with a maturation age of 10 to 30 y produced in Japanese, Scotch (Islay), or Scotch (Speyside and Highland) regions. Single malt whiskey not only showed RO scavenging activity but there was also a positive correlation between this activity and the maturation age of whiskey exceeding the difference resulting from the manufacturing region. A nonvolatile fraction derived from the barrel was responsible for RO scavenging activity. In particular, the contents of ellagic and gallic acids and lyoniresinol, the main polyphenolic compounds in whiskey, increased with maturation age. For the free radical scavenging activity per molecule each compound was 1.68 to 3.14 times that of trolox (a water-soluble vitamin E). The activities of ellagic acid, gallic acid, and lyoniresinol in the whiskey (Yamazaki 18) were equivalent to that of 80.3, 31.2, and 11.1 ppm trolox, respectively. Accordingly, the total activity of these 2 compounds accounted for about 20% of the activity of the whiskey (630.7 ppm trolox).

The growth behavior of Zygosaccharomyces yeasts in commercial beverages was investigated. In beverages with high sugar contents, such as high-sugar tea, fruit juices, beverages containing lactic acids, and sports drinks, excellent cell growth and generation of large amounts of CO2 gas were observed. In contrast, cell growth was very slow and little gas generation was observed when Zygosaccharomyces yeasts were grown in beverages with low sugar contents, such as low-sugar tea and green tea. Cell growth in high-sugar tea was found to increase in parallel with increasing growth temperature. These results suggest that yeast growth and gas generation in beverages contained in previously opened PET bottles may result in explosions.

The growth behavior of Zygosaccharomyces yeasts in commercial beverages was investigated. In beverages with high sugar contents, such as high-sugar tea, fruit juices, beverages containing lactic acids, and sports drinks, excellent cell growth and generation of large amounts of CO2 gas were observed. In contrast, cell growth was very slow and little gas generation was observed when Zygosaccharomyces yeasts were grown in beverages with low sugar contents, such as low-sugar tea and green tea. Cell growth in high-sugar tea was found to increase in parallel with increasing growth temperature. These results suggest that yeast growth and gas generation in beverages contained in previously opened PET bottles may result in explosions.

Linoleic acid hydroperoxide (LAOOH) was effectively degraded by horseradish peroxidase (HRP) in the presence of quercetin. Several natural phenolic antioxidants, such as quercetin, capsaicin, and alpha-tocopherol, acted as good hydrogen donors in the peroxidase reaction that occurs during lipid hydroperoxide degradation. However, glutathione, which is a non-phenolic antioxidant that acts as a hydrogen donor for glutathione peroxidase, could not suppress lipid peroxidation in the presence of HRP. Lipid hydroperoxides generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also degraded with HRP in the presence of quercetin, and oxidative decomposition of DHA was suppressed by this reaction.

Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA) have been shown to modulate a number of inflammatory disorders. Mast cells play a critical role in the initiation and maintenance of inflammatory responses. However, the effects of PUFAs on mast cell functions have not been fully addressed. We here-in examined the effects of PUFAs on the high affinity IgE receptor (Fc epsilon RI)-mediated mast cell activation using RBL-2H3 cells, a rat mast cell line, that were cultured in the medium containing palmitic acid (PA), AA, or the AA analogs mead acid (MA) and eicosapentaenoic acid (EPA). In AA-supplemented cells, the Fc epsilon RI-mediated beta-hexosamidase and TNF-alpha release, calcium (Ca2+) influx, and some protein tyrosine phosphorylations including Syk and linker for activation of T cells (LAT) were enhanced, whereas, in MA- or PA-supplemented cells, they were not changed when compared with cells cultured in control medium. In EPA-supplemented cells, the enhancements of beta-hexosamidase release and protein tyrosine phosphorylations were observed. Furthermore, in AA- or EPA-supplemented cells, Fc epsilon RI-mediated intracellular production of reactive oxygen species (ROS) that is required for the tyrosine phosphorylation of LAT and Ca2+ influx were enhanced when compared with the other cells. Thus, preincubation of AA or EPA augmented F epsilon FRI-mediated degranulation in mast cells by affecting early events of Fc epsilon RI signal transduction, which might be associated with the change of fatty acid composition of the cell membrane and enhanced production of ROS. The results suggest that some PUFAs can modulate Fc epsilon RI-mediated mast cell activation and might affect Fc epsilon RI/mast cell-mediated inflammation, such as allergic reaction. (c) 2005 Elsevier Inc. All rights reserved.

Changes in the fatty acid composition of docosahexaenoic acid (DHA)-producing Schizochytrium limacinum SR21 were investigated. The addition of cyanocobalamin, which is an active component of vitamin B12, decreased the content of odd-chain fatty acids such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). Cyanocobalamin may upregulate the cobalamin-dependent methylmalonyl-CoA mutase, which converts propionic acid to succinic acid, thereby decreasing the content of odd-chain fatty acids. The addition of p-toluic acid resulted in a decrease in docosapentaenoic acid (DPA, 22:5n-6) content and an increase in eicosapentaenoic acid (EPA, 20:5n-3) content in a dose-dependent manner. Two additional peaks of fatty acids, characterized as Δ4,7,10,14-eicosatetraenoic acid (20:4n-7) and Δ4,7,10,14- docosatetraenoic acid (22:4n-9), were detected. © The Mycological Society of Japan and Springer-Verlag 2005.

We investigated the characteristics of desaturation in Trichoderma sp. AM076. Although 6,9,12-octadecatrienoic acid (18:3ω6) was detected when Trichoderma sp. AM076 was cultivated in the presence of 6,9-octadecadienoic acid (18:2ω9), the desaturation products of 6,9,12-octadecatrienoic acid (18:3ω6) and 6-octadecenoic acid (18:1Δ6) were not detected. These results suggest that the double bonds at the Δ6 position of 18:3ω6 and 18:1Δ6 disturb their Δ15 and Δ9 desaturation, respectively. This fungus also introduced a double bond at the Δ15 position of 9,12-hexadecadienoic acid (16:2ω4), thereby yielding a novel C16 polyunsaturated fatty acid (PUFA) identified as 9,12,15-hexadecatrienoic acid (16:3ω1). Further investigations revealed that the mutant having enhanced accumulation of linolenic aid (18:3ω3) accumulates 16:3ω1 as one of the major PUFAs, together with 9,12-octadecadienoic acid (16:2ω4), when grown with palmitoleic acid (16:1ω7). These results suggest that, in this strain, the reaction that catalyzes the conversion of linoleic acid to linolenic acid, similar to the conversion of 16:2ω4 to 16:3ω1, is not ω3 desaturation but Δ15 desaturation. © The Mycological Society of Japan and Springer-Verlag 2005.

Corn fiber (CNF) is an abundant by-product of the wet corn milling process in the production of cornstarch. We have shown that the hot water-soluble fraction (HWSF) from CNF has a promoting effect on the mycelial growth of various edible mushrooms, including mycorrhizal fungi. To reveal the promoting mechanisms, the effect of CNF-HWSF on the stimulation of extracellular enzymes was examined. The production of extracellular carbohydrases such as amylase, CMCase, and xylanase was markedly enhanced by the addition of low molecular weight fractions (less than MW 500) prepared from CNF-HWSF. The enzymatic stimulation and enhancement of mycelial growth appeared during 3-15 days after inoculation. Furthermore, a fraction of less than MW 500 was separated by gel filtrate chromatography into five fractions (A-E), and the effect of each fraction was investigated. Promoting effects were shown from C and D fractions mycelial growth and enzyme production of Lentinula edodes were indicated although fraction D has no sugars and amino acids in CNF-HWSF. From these results, the promoting effect of CNF-HWSF seems to be a two-step reaction. The first step could be achieved by rich nutrients such as free amino acids and monosaccharides from CNF-HWSF. The second step (during 3-15 days) is considered to be that the marked promoting effect was caused by the stimulation of extracellular enzymes. © The Mycological Society of Japan and Springer-Verlag 2005.

Lvoniresinol content and antioxidative activity of commercially available seasoning pickled Ume was investigated. The seasoning pickled Ume with low salt concentration showed relatively low Iyoniresinol content and antioxidative activity compared to products with high salt concentrations. This result indicates that excessive de-salt operation leads to decrease of a Iyoniresinol content and antioxidative activity of flesh. Although remarkable decrease of Ivoniresinol content and antioxidative activity of pickled Ume was observed in the early stage of seasoning process, re-extraction of Ivoniresinol and antioxidative activity by osmotic pressure of seasoning liquid was thought to occur after two weeks and later. From the results obtained, we concluded that enough period seasoning by the seasoning liquid with enough osmotic pressure is necessary to produce the seasoning pickled Ume with rich nutritional function.

To investigate the function of amylases in the fruit-body formation of an ectomycorrhizal fungus, Lyophyllum shimeji, we purified the extracellular amylase in the medium of this fungus. The purified enzyme was obtained from 1.7l stationary culture filtrate, with 4.2% recovery, and showed a single protein band on SDS-PAGE. The molecular mass was about 25kDa. The enzyme was most active at around 40°C and pH 5.0 and stable over pH 4.5-6.5 for 30min at 37°C. This amylase was remarkably activated by the presence of Ca2+ ion (7.7 times that of the control), but Ba2+ and Ag+ completely inhibited the activity. The amylase readily hydrolyzed the α-1,4 glucosidic linkage such as dextrin and amylose A (MW, 2900), converting into glucose, and hydrolyzed the α-1,6 glucosidic linkage of isomaltohexaose and amylopectin. However, the enzyme did not hydrolyze the cyclic polysaccharides. On the other hand, when a low molecular mass amylose A was hydrolyzed by this amylase, β-anomer glucose was produced. From these results, we concluded that the amylase from L. shimeji seems to be a glucoamylase. © The Mycological Society of Japan and Springer-Verlag 2004.

Umezu (plum vinegar) is an exudate obtained from the pickled fruit of Prunus mume Sieb. et Zucc., Rosaceae. Umezu was passed through a Diaion HP20 adsorption column, which was washed with distilled water to remove salt and eluted with 90% ethanol solution. The eluate was concentrated in vacuo to obtain Umezu extract. In this study, the effects of dietary Umezu extract on blood pressure and lipid metabolism were examined in stroke-prone spontaneously hypertensive rats (SHRSP) fed a high-cholesterol diet. The high-cholesterol diet (control diet) was prepared by addition of 1% cholesterol and 0.25% sodium cholate to the purified diet according to the AIN-93G formula. Two groups of 6 male rats were used at the age of 6 weeks. One was fed the control diet and the other was fed the experimental diet (control diet containing 1% Umezu extract) for 6 weeks with free access to the diet and water. Elevation of systolic blood pressure was significantly suppressed with time in the experimental group (p<0.001 by two-way repeated ANOVA) after 4 weeks on the diet. Serum lipid hydroperoxide concentration was significantly decreased in the experimental group (p<0.05). Moreover, hepatic triglyceride content in the experimental group was significantly lower than that in the control group (p<0.05). These results indicate that Umezu extract suppresses the elevation of blood pressure, and the increases in serum lipid hydroperoxide concentration and hepatic triglyceride accumulation in SHRSP fed a high-cholesterol diet.

The effect of maternal protein restriction during pregnancy on the offspring's blood pressure was assessed in stroke-prone spontaneously hypertensive rats (SHRSP) which are genetically predisposed to hypertension and stroke. After the confirmation of pregnancy, the control group was given a 20% casein diet, and the low-protein group was fed a 9% casein diet. After the confirmation of delivery, commercial feed was given to both of the groups. No differences were seen between the control and low-protein offspring in regard to body weight, blood pressure elevation, or life span. One percent saline solution was put in the control and low-protein groups after the age of 11 weeks. Blood pressure increased markedly in the low-protein group, on the blood pressure level in the low-protein group on week 2 after salt loading (242 +/- 6 mmHg) was significantly higher than that in the control group (223 +/- 9 mmHg; p < 0.05). The survival duration was significantly shorter in the low-protein group (113 +/- 4 days) than in the control group (135 +/- 22 days; p < 0.05). These results suggest that maternal protein malnutrition in SHRSP exerted a high salt sensitivity and a malignant influence on stroke incidence on offspring.

The ethyl acetate extracted fraction of Ume vinegar (AT fraction) contained aryltetralin lignan, lyoniresinol. Lyoniresinol showed various antioxidative activities, such as not only suppressing autooxidation of linoleic acid, but also scavenge of DPPH radical and superoxide anion. Purified lyoniresinol and AT fraction suppressed mutagenesis caused by Trp-P-2 and ethylmethane sulfonate. Lyoniresinol indicated remarkable mutagen scavenging activity, which was based on the antioxidative activity, for Trp-P-2. And the existence of antimutagenetic compounds other than lyoniresinol were also suggested from the result of antimutagenetic activity for ethylmethane sulfonate.

Nicardipine and nifedipine, Ca channel blockers, inhibited rat liver microsomal desaturases, though verapamil, methoxyverapamil, cinnarizine, flunarizine, and diltiazem did not, However, nicardipine and nifedipine apparently did not inhibit the fungal desaturation in Mortierella alpina 1S-4. Nicardipine inhibited rat liver microsomal Delta 5 desaturase specifically (50% inhibitory concentration, 170 mu M), and nifedipine inhibited Delta 6 desaturase specifically (78 mu M). The inhibition of nicardipine and nifedipine is uncompetitive,the K-i values for Delta 5 and Delta 6 desaturases being 62 and 44 mu M, respectively.

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) inhibited noncompetitively rat liver microsomal Delta 5 desaturase (K-i = 36 mu M) and Delta 6 desaturase (K-i = 28 mu M). Although curcumin has a symmetrical structure with a methylene group as the center, only half the structure is essential for the desaturase inhibition. The structure necessary for the inhibition is similar to that of alkyl gallate (H. Kawashima et al., Biochim. Biophys. Acta, in press), i.e., a 3-hydroxy group of the aromatic ring is essential for the inhibition and a free carboxyl group at the end opposite to the aromatic ring interferes with the inhibitory effect. The following structural features of curcumin are necessary for the desaturase inhibition: (i) the aromatic ring conjugated with the double bond between the 1 and 2 (or 6 and 7) positions; (ii) both 4-hydroxy and 3-methoxy groups (for both desaturase inhibitions); and() only a 4-hydroxy group (for Delta 6 desaturase inhibition).

Alkyl gallate, which is known as an antioxidant, intensively inhibited Delta 5 and Delta 6 desaturation in both rat liver microsomes and an arachidonic acid-producing fungus Mortierella alpina 1S-4. The rat liver microsomal Delta 5 and Delta 6 desaturases were inhibited by gallic acid esterified with alcohols with various numbers of carbons, suggesting that the necessary structure in an esterified alcohol for the inhibition is not so strict. Among the three hydroxy groups in gallic acid, the m-hydroxy group was shown to be the necessary structure. Kinetic analyses revealed that propyl gallate is a noncompetitive inhibitor of Delta 5 desaturase (K-i = 2.6 . 10(-5) M) and Delta 6 desaturase (K-i = 1.7 . 10(-4) M). These data indicate that alkyl gallate is a new type of desaturase inhibitor and different from known natural. inhibitors, i.e., sesamin and curcumin.

まいたけの子実体中のD. グルカンに顕著な抗変異原作用があることが示唆された。

In order to evaluate the mechanisms for gamma-aminobutyric acid (GABA) accumulation in edible mushroom, we purified and characterized the enzymes for GABA formation from Grifola frondosa, Lentinula edodes and Flammulina velutipes. The enzymes from G. frondosa, and F. velutipes enzyme used only L-glutamic acid as substrate for decarboxylation reaction so that these enzymes should belong to glutamic acid decarboxylase. However, the enzyme from L. edodes can use aspartic acid, other than glutamic acid, as substrate for the reaction. The internal amino acid sequences of this enzyme showed that the enzyme should be phosphatidylserine decarboxylase (PSD) of L. edodes. However, the reaction of PSD couldn’t be detected.

食品より抗酸化性、抗アレルギー性、またはGABAなどの機能性化合物の生産能を有する新規の微生物を食品などから分離し、酒類やその他の食品素材への機能性の強化法の開発を目指す

Corn fiber (CNF) is an abundant by-product of the wet corn milling process in the production of corn starch. We have shown that the hot water soluble fraction (HWSF) from CNF has a promoting effect on the mycelial growth of various edible mushrooms including mycorrhizal mushroom such as Tricholoma matsutake and so on. Moreover, fruit-body yield increased at 1.2-1.6 times by using CNF, and the cultivation period was dramatically shortened compared to that of the control without CNF. To reveal the promoting mechanisms, the effect of CNF-HWSF on the stimulation of extracellular enzymes was examined. As a result, the production of carbohydrases such as amylase, CMCase and xylanase were markedly enhanced by the addition of low molecular weight fractions (less than M.W.500) prepared from CNF-HWSF. The enzymatic stimulations and enhancement of mycelial growth appeared during the about same time on the culture period. Furthermore, fraction less than the M.W.500 was separated by gel filtration into 5 fractions, and the effect of each fraction was investigated. The promoting effects were indicated even on a fraction D has no sugars and amino acids in CNF-HWSF, From these results, the promoting effect by CNF-HWSF seems to be a 2 step reactions. The first step could be achieve by rich nutrients such as mono- and ologo-saccharides and free amino acids from CNF-HWSF. The second step considered that the marked promoting effect was caused by the stimulation of extracellular enzymes. Now, we are in the improvement stage for practical application of CNF as a growth substrate using sawdust-based cultivation. Further attempts will be made to investigate the isolation and the characterization of promoting substances in CNF-HWSF to clarify the promotion mechanisms on edible mushrooms.

昨年までの研究において肥満細胞をアラキドン酸およびアラキドン酸アナログであるEPAやミード酸の存在下で培養し、抗原抗体反応により刺激を加えたところ、アラキドン酸を添加して培養した細胞においてのみ、添加濃度に依存してエイコサノイド(PGD_2)の産生、脱顆粒やサイトカイン(TNF-α)の産生量が上昇すること明らかにした。この現象は、細胞表面に存在する高親和性IgE受容体を介した細胞内情報伝達に関与するZAP-70/Syk FamilyプロテインキナーゼであるSykの発現およびリン酸化の亢進および、カルシウムの細胞内への流入増加によることが確認され、膜脂肪酸組成が肥満細胞におけるチロシンリン酸化を介した細胞内情報伝達に強く関与することが明らかとなった。そこで、類似の構造を持った脂肪酸によりアラキドン酸取り込みを調節することによって細胞の機能をコントロールできる可能性が示されたことより、新たなアラキドン酸アナログとして、アラキドン酸のΔ8位の二重結合が欠損した5,11,14-20:3をアラキドン酸生産菌MortierellaalpinaのΔ6不飽和化酵素欠損変異株を用いて調製し、細胞機能へ与える影響について評価した。5,11,14-20:3は細胞膜への取り込みが、これまでに検討を行ったアラキドン酸、EPA、DGLA、ミード酸に比べ極端に低く、アラキドン酸との膜中への取り込みにおける競合は認められなかった。しかし、アラキドン酸による脱顆粒亢進を効果的に抑制する傾向が認められたことより、膜中のアラキドン酸量の抑制以外の別の機序で脱顆粒亢進を抑制していることが考えられた。 一方、より効果的に細胞内のアラキドン酸レベルを制御することを目的として、部分的に構造を変化させた脂肪酸の合成についても更に検討を行った。α-リノレン酸の部分水添反応により生ずるモノエン酸を硝酸-亜硝酸系を用いて二重結合を異性化し、トランス型モノエン酸混合物(Δ9、Δ12およびΔ15-18:1)を調製した。トランス型モノエン酸混合物を基質としてMortierella alpinaによるアラキドン酸生合成経路を利用した微生物変換により、5c,8c,11c,14t-20:4、5c,8c,11c,14c,17t-20:5を合成した。