Analysis of cereal extracts as conditioning solutes to suppress the initial attachment of Escherichia coli to abiotic surfaces


Modification of cell vulnerability to oxidative stress by N-(3-oxododecanoyl)-L-homoserine-lactone, a quorum sensing molecule, in rat thymocytes

N-(3-oxododecanoyl)-L-homoserine-lactone (ODHL), a quorum sensing molecule, affects intracellular Zn2+ concentration ([Zn2+]i) and cellular levels of nonprotein thiols ([NPT]i) of rat thymic lymphocytes, both of which are assumed to affect cell vulnerability to oxidative stress. Therefore, it is interesting to examine the effects of ODHL on the cells under oxidative stress. ODHL augmented the cytotoxicity of H2O2, but not calcium ionophore A23187. ODHL potentiated the H2O2-induced elevation of [Zn2+]i, wherein, it greatly attenuated the H2O2-induced increase in intracellular Ca2+ concentration. ODHL did not affect [NPT]i in the presence of H2O2. Therefore, we conclude that the elevation of [Zn2+]i is involved in the ODHL-induced potentiation of H2O2 cytotoxicity. Our findings suggest that ODHL modifies cell vulnerability to oxidative stress in host cells.


Zinc-dependent and independent actions of hydroxyhydroquinone on rat thymic lymphocytes

Coffee contains hydroxyhydroquinone (HHQ). HHQ is one of by-products released during bean roasting. Therefore, it is important to elucidate the bioactivity of HHQ to predict its beneficial or adverse effects on humans. We studied zinc-dependent and independent actions of commercially-procured synthetic HHQ in rat thymocytes using flow cytometric techniques with propidium iodide, FluoZin-3-AM, 5-chloromethylfluorescein diacetate, and annexin V-FITC. HHQ at 10¬50 µM elevated intracellular Zn2+ levels by releasing intracellular Zn2+. HHQ at 10 µM increased cellular thiol content in a Zinc-dependent manner. However, HHQ at 30 50 µM reduced cellular thiol content. Although the latter actions of HHQ (30 50 µM) were suggested to increase cell vulnerability to oxidative stress, HHQ at 0.3 100 µM significantly protected cells against oxidative stress induced by H2O2. The process of cell death induced by H2O2 was delayed by HHQ, although both H2O2 and HHQ increased the population of annexin V-positive living cells. However, HHQ at 10 30 µM promoted cell death induced by A23187, a calcium ionophore. HHQ at 10 30 µM exerted contrasting effects on cell death caused by oxidative stress and Ca2+ overload. Because HHQ is considered to possess diverse cellular actions, coffee with reduced amount of HHQ may be preferable to avoid potential adverse effects.


Hyperpolarization by N-(3-oxododecanoyl)-L-homoserine-lactone, a quorum sensing molecule, in rat thymic lymphocytes

To study the adverse effects of N-(3-oxododecanoyl)-L-homoserine-lactone (ODHL), a quorum sensing molecule, on mammalian host cells, its effect on membrane potential was examined in rat thymic lymphocytes using flow cytometric techniques with a voltage-sensitive fluorescent probe. As 3 300 µM ODHL elicited hyperpolarization, it is likely that it increases membrane K+ permeability because hyperpolarization is directly linked to changing K+ gradient across membranes, but not Na+ and Cl- gradients. ODHL did not increase intracellular Ca2+ concentration. ODHL also produced a response in the presence of an intracellular Zn2+ chelator. Thus, it is unlikely that intracellular Ca2+ and Zn2+ are attributed to the response. Quinine, a non-specific K+ channel blocker, greatly reduced hyperpolarization. However, because charybdotoxin, tetraethylammonium chloride, 4-aminopyridine, and glibenclamide did not affect it, it is pharmacologically hypothesized that Ca2+-activated K+ channels, voltage-gated K+ channels, and ATP-sensitive K+ channels are not involved in ODHL-induced hyperpolarization. Although the K+ channels responsible for ODHL-induced hyperpolarization have not been identified, it is suggested that ODHL can elicit hyperpolarization in mammalian host cells, disturbing cellular functions.


Determination of binding affinity of poly-gamma-glutamate to Shiga toxin


N-(3-oxododecanoyl)-L-homoserine-lactone, a quorum sensing molecule, affects cellular content of nonprotein thiol content in rat lymphocytes: Its relation with intracellular Zn2+

Cellular actions of N-(3-oxododecanoyl)-L-homoserine-lactone (ODHL), a quorum sensing molecule of bacteria, were studied on rat thymocytes using a flow cytometer with appropriate fluorescent dyes to elucidate the effects of ODHL on host cells. A bell-shaped concentration-response relation was observed in the ODHL-induced changes in cellular glutathione content ([GSH]i). ODHL concentration-dependently increased intracellular Zn2+ levels ([Zn2+]i) and cellular O2- content ([O2-]i). The bell-shaped relation induced by ODHL can be explained as follows: a low concentration of ODHL is expected to induce moderate oxidative stress that intracellularly releases Zn2+ by converting thiols to disulfides. A slight elevation of [Zn2+]i may increase the [GSH]i. On the other hand, it is likely that a high concentration of ODHL causes severe oxidative stress that further causes both the decrease in [GSH]i and the increase in [Zn2+]i. Excessive increase in [Zn2+]i may augment oxidative stress that further decreases the [GSH]i. Other notable actions induced by ODHL included the elevation of [Zn2+]i by Zn2+ influx and the increase in [GSH]i under Zn2+-free conditions. Therefore, it is suggested that ODHL elicits diverse actions on host cells.


Diverse cellular actions of tert-butylhydroquinone, a food additive, on rat thymocytes

Tertiary butylhydroquinone (TBHQ) is a food additive that possesses antioxidant activity. Its alternative applications have been explored in recent studies. However, there is controversy regarding safety. In this study using rat thymocytes, the cellular actions of TBHQ at sublethal concentrations were examined. TBHQ at concentrations of 3 µM or more elevated intracellular Zn2+ concentration ([Zn2+]i) in a dose-dependent manner, by increasing membrane Zn2+ permeability and releasing Zn2+ from cellular stores. TBHQ at 30 µM significantly increased side scatter (cell density) and the exposure of phosphatidylserine (PS) on cell membrane surfaces. It also decreased cellular glutathione (GSH) content without affecting cell lethality. Forward scatter was attenuated by 100 µM TBHQ. Thus, it is considered that TBHQ at sublethal concentrations (30 µM or less) exerts some adverse actions on cells. TBHQ at 10 30 µM attenuated the increase in cell lethality induced by hydrogen peroxide (H2O2), while potentiation of H2O2 cytotoxicity by 100 µM TBHQ was observed. The range of concentrations of TBHQ from benefit to toxicity under in vitro conditions may be 10 30 µM. Although TBHQ exhibits antioxidative actions at concentrations that are lower than those which elicit adverse cellular effects, sublethal levels of TBHQ cause some adverse actions that may be clinically concerned.


Screening and analysis of edible seaweeds in the ability to adsorb Shiga toxin.


Change in plasma membrane potential of rat thymocytes by tert-butylhydroquinone, a food additive: Possible risk on lymphocytes

Tertiary butylhydroquinone (TBHQ) is a food additive and has various beneficial actions under in vitro and in vivo experimental conditions. Therefore, it is necessary to collect additional data on the toxicity of TBHQ in order to avoid adverse effects during clinical applications. Changes in plasma membrane potential are associated with changes in physiological functions even in non-excitable cells such as lymphocytes. Thus, compounds that affect membrane potential may modify some lymphocytic functions. The effect of TBHQ on plasma membrane potential was examined in rat thymocytes using flow cytometric techniques. Treatment of rat thymocytes with TBHQ caused hyperpolarization and then depolarization. The TBHQ-induced hyperpolarization was due to the activation of Ca2+-dependent K+ channels. TBHQ elevated intracellular Ca2+ levels. The depolarization by TBHQ was caused by a nonspecific increase in membrane ionic permeability. Both the sustained depolarization and elevation of intracellular Ca2+ level by TBHQ are thought to be adverse for thymocytes because such changes disturb membrane and intracellular signaling. The thymus is most active during neonatal and pre-adolescent periods. If TBHQ exerts adverse actions on thymocytes, it may result in an immunotoxic effect in neonates and adolescents.


Adsorption of Shiga Toxin to Poly-γ Glutamate Precipitated.


Zinc increases vulnerability of rat thymic lymphocytes to arachidonic acid under in vitro conditions

Previous studies on the cytotoxicity of arachidonic acid (ARA) elucidated the involvement of oxidative stress and Ca2+. In the present study, the Zn2+-related cytotoxicity of ARA was studied by a flow cytometric technique with appropriate fluorescent probes in rat thymocytes. Addition of 10 µM ZnCl2 enhanced the increase in cell lethality induced by 10 µM ARA. The removal of Zn2+ by Zn2+ chelators attenuated the ARA-induced increase in cell lethality. Thus, Zn2+ is suggested to be involved in ARA cytotoxicity. ARA at 3 10 µM elevated intracellular Zn2+ level. The Zn2+ chelators attenuated the ARA-induced increase in intracellular Zn2+ level while ARA significantly increased intracellular Zn2+ level in the presence of 3 µM ZnCl2, suggesting the involvement of external Zn2+. Zn2+ reportedly exerts cytotoxic action under oxidative stress induced by hydrogen peroxide, via an excessive increase in intracellular Zn2+ levels. Since ARA induces oxidative stress, the simultaneous administration of zinc and ARA may be harmful.


Methyl cinnamate increases cell vulnerability to oxidative stress induced by hydrogen peroxide in rat thymocytes

Methyl cinnamate (MC) and essential oils containing MC possess beneficial antimicrobial, antifungal, and insecticidal effects, among others. Such effects are related to the biocidal action of MC. The antioxidant activity of MC has also been reported elsewhere. It has been suggested that MC may be cytotoxic to cells exposed to oxidative stress. To test this possibility, the effect of MC on rat thymocytes was examined while the cells were subjected to oxidative stress induced by hydrogen peroxide (H2O2). Flow cytometric techniques with appropriate fluorescent probes were used for quantification. MC increased cell vulnerability to oxidative stress via acceleration of the cell death process and/or potentiation of oxidative stress. The use of MC is widespread because of its beneficial actions, and thus further attention should be paid to whether MC is effective under oxidative stress.


A biosynthetic pathway for (E)-methyl cinnamate formation in the ectomycorrhizal fungus Tricholoma matsutake


Genotyping and characterisation of the secretory lipolytic enzymes of Malassezia pachydermatis isolates collected dogs.

Malassezia species are commensals of normal skin microbial flora of humans and animals. These may become pathogenic under certain conditions such as those associated with atopic dermatitis or otitis externa in dogs. Isolates of Malassezia pachydermatis were obtained from 27 dogs with healthy external ears and 32 dogs with otitis externa. Isolates were characterised on the basis of their first internal transcribed spacer (ITS) and internal spacer 1 (IGS1) sequences. Their extracellular lipase and phospholipase activity were also analysed. Three types of phospholipase inhibitor were used to identify the subclasses of phospholipase associated with otitis externa. The clinical isolates were classified into three ITS and three IGS1 sequence types. No significant differences in pathogenicity were detected among the ITS or IGS1 genotypes, and all of the isolates exhibited similar levels of lipase activity. The isolates derived from the dogs with otitis externa showed significantly higher phospholipase activity than those obtained from the dogs with healthy external ears. A phospholipase D inhibitor reduced the phospholipase activity of the isolates obtained from the dogs with otitis externa. This study did not show any significant differences in pathogenicity among the ITS or IGS1 genotypes but does suggest that phospholipase D might be one of the virulence factors involved in the inflammation of the external ear caused by M. pachydermatis.


Analysis of cumin in the ability to suppress verocytotoxin production by enterohemorrhagic Escherichia coli O157.

腸菅出血性大腸菌(EHEC)O157のべロ毒素(VT)産生に対するクミンの抑制能力を調べた．クミン抽出液は70%エチルアルコールを用いて調製した．0．02%クミン抽出液を添加したLB(Luria-Bertani)培地を用いて,37℃で定常期までEHECO157を培養したとき,ベロ毒素生産はVT1とVT2共に有意に低下した．また,この培養条件では,増殖抑制や誘導期の遅延は認められなかった．クミン抽出液の活性成分は,HPLC法によって精製され,4-イソプロピルベンズアルデヒド(IPBA)と同定された．EHEC O157のVT生産に対するIPBAの抑圧効果を調べた結果,細胞内および細胞外のべロ毒素量は,IPBA濃度の上昇とともに低下した．この結果は,IPBAがEHEC O157の有害性を低下させることに有用であることを示唆する．


Attachment of Escherichia coli O157:H7 to abiotic surfaces of cooking utensils.

We examined the attachment of enterohemorrhagic Escherichia coli O157:H7 to abiotic surfaces of cooking utensils. When the cell suspension in 0.85% NaCl (about 100 cells/mL, 10 mL) was contacted with various abiotic surfaces (square pieces, 25 cm²) at 25 °C for 20 min, the number of attached cells varied depending on the types of abiotic materials. The pathogen well attached to stainless steel (about 50 cells/25 cm²), pure titanium (35 to 45 cells/25 cm²), and glass (about 20 cells/25 cm²), but little attached to aluminum foil and plastics, irrespective of strains used. Fewer cells (below 10 cells/25 cm²) attached to stainless steel, pure titanium, and glass surfaces conditioned with aseptically sliced beef (sirloin) and autoclaved beef tallow at 25 °C for 20 min, but bovine serum albumin did not reduce the number of attached cells. The cells grown at 15 °C to the stationary phase (OD660 = about 2.8) less attached to the abiotic surfaces than those grown at 25 °C and 37 °C. When we pretreated the cells at 37 °C for 2 h with 50 μM N-hexanoyl-L-homoserine lactone (HHL), the number of cells attached to stainless steel was reduced by 70%. The number of cells attached to cooking utensils seemed to change depending on types of abiotic materials, adhesion of beef tallow to abiotic surfaces, growth temperature of the pathogen, and HHL-producing bacteria.


Acid resistance and verocytotoxin productivity of enterohemorrhagic Escherichia coli O157:H7 exposed to microwave

We examined the acid resistance and verocytotoxin (VT) productivity of enterohemorrhagic Escherichia coli O157:H7 irradiated by microwave with a domestic microwave oven and a commercial microwave radiator equipped with a thermo-regulator. When the cell suspension (5 mL) chilled at 0 °C was treated with a domestic microwave oven at weak power (2.45 GHz, 100 W) for 60 s, the living cell number was reduced by 2 orders (final temperature, about 65 °C). The surviving cells showed lower acid resistance and VT productivity than nonirradiated cells. To examine the nonthermal effect of microwave on acid resistance and VT productivity, the cells in Luria-Bertani medium were intermittently irradiated to keep the culture temperature at 37 °C with the microwave radiator (2.45 GHz, 0.6 W/mL). The intermittent radiation slightly reduced the acid resistance, but clearly suppressed the VT productivity. Microwave oven is probably useful for reducing not only the living cell number but also the acid resistance and VT productivity of EHEC O157:H7.


A novel cytotoxic protein, Karatoxin, from the dorsal spines of the the redfin velvetfihi, Hypodytes rubripinnis


Screening and Analysis of Spices with Ability to Suppress Verocytotoxin Production by Escherichia coli O157

To reduce the amounts of verocytotoxin (VT) produced by Escherichia coli O157:H7, various spices were screened for their ability to suppress VT production. Extracts of these spices were prepared with 70% ethyl alcohol. When E. coli O157:H7 cells were grown to the stationary phase at 37 degrees C in Luria-Bertani medium supplemented with 0.02% allspice extract, the production of both VT1 and VT2 was significantly reduced. Neither growth inhibition nor a delay in the lag phase was observed when the cells were cultured in the presence of 0.02% allspice extract. An active component of the allspice extract was purified by HPLC and was identified as eugenol. When we examined the suppressive effect of eugenol on VT production by E. coli O157:H7, the amounts of both intracellular and extracellular VTs were found to decrease with an increase in eugenol concentration. Our results suggest that eugenol is useful for reducing the virulence of E. coli O157:H7.


Suppressive Effect of Poly-γ-glutamate on SOS Response of Salmonella typhimurium Induced by Chemical Mutagens

納豆の主要粘性物質であるポリγ-グルタミン酸(PGA)の抗変異原性を検討した．PGAは食品添加物として使用されている酸性多糖類よりも高い抗変異原性を示した．またPGAの分子量によって，抗変異原活性が異なることを明らかにした．

Journal of Bioscience and Bioengineering


Simple improvement in freeze-tolerance of bakers' yeast with poly-γ-glutamate

納豆の主要粘性物質であるポリγ-グルタミン酸は無味，無臭であり，高い保水性と陽イオン結合性等の特徴を有することから，食品分野での利用が期待されている．本研究では，ポリγ-グルタミン酸を用いて，パン酵母の冷凍耐性を簡便に改善する方法を確立した．


Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of the gene products expressed in Escherichia coli.

細菌細胞壁の生合成に必須の役割を果たすアラニンラセマーゼの酵素化学的特徴を明らかにするため，Pseudomonas fluorescens由来の本酵素遺伝子をクローニングして大腸菌細胞内で発現させた．生成した酵素は，サブユニット間の相互作用により，単量体と二量体の動的平衡状態であることが明らかとなった．


Intracellular glycerol influences resistance to freeze stress in Saccharomyces cerevisiae: analysis of a quadruple mutant in glycerol-dehydrogenase genes and glycerol-enriched cells.

酵母の冷凍耐性における細胞内グリセロールの役割を解明するために，グリセロール代謝にかかわる4つの遺伝子を破壊した変異株を作製した．変異株の細胞内グリセロール含量は野生株よりも上昇し，冷凍耐性も増加した．以上のことから，酵母の冷凍耐性にグリセロールは重要な役割を果たすことが明らかとなった．


Improvement in Thermostability and Psychrophilicity of Psychrophilic Alanine Racemase by Site-directed Mutagenesis.

これまでの低温性アラニンラセマーゼに関する構造と機能に関する研究で得られた知見に基づき，本酵素に部位特異的変異を導入した．得られた変異酵素は，低温での触媒活性と熱安定性が共に向上したことから，耐熱性と低温性は表裏一体の性質ではないことが判明した．

Journal of Molecular Catalysis B: Enzymatic


Catalase is the Bacteria-derived Detoxifying Substance against Paramecium-killing Toxin in Wheat Grass Powder Infusion.

小麦若葉粉末の抽出液に含まれるゾウリムシに対する毒物質を無毒化できる細菌由来タンパク質はカタラーゼであることを証明し，かつ，毒物質は微量(約30 μM)の過酸化水素であることを解明した．ゾウリムシ由来カタラーゼは細胞内で生成する過酸化水素には作用するが細胞外の過酸化水素は無毒化できないことが判明した．


Acid Tolerance and gad mRNA Levels of Escherichia coli O157:H7 Grown in Foods.

各種食品中で増殖した大腸菌O157の酸耐性の変動とグルタミン酸脱炭酸酵素の転写レベルの変動を明らかにし，大腸菌O157の酸耐性にはグルタミン酸脱炭酸酵素の活性が深く関与することを明らかにした．

International Journal of Food Microbiology


Subunit Interaction of Monomeric Alanine Racemases from Four Shigella Species in Catalytic Reaction.

赤痢菌由来アラニンラセマーゼは，単量体と二量体の動的平衡状態にあることを見いだし，活性発現に二量体形成が必須であることから，サブユニット相互作用を阻害する新しいタイプの酵素阻害剤が開発可能であることを示した．


Antimicrobial Activity of Nutmeg against Escherichia coli O157.

各種香辛料の大腸菌O157に対する抗菌活性を，非病原性大腸菌との比較において検討し，ナツメグが大腸菌O157の特異的殺菌に有効であることを見い出した．抗菌成分の精製も行い，β-ピネンが大腸菌O157の殺菌に有効であることを明らかにした．

Journal of Bioscience and Bioengineering


Immunohistochemical study of the localization of glutamate decarboxylase in rodent's submandibular gland

グルタミン酸脱炭酸酵素が顎下腺に局在していることを示し，唾液のpH調整に機能している可能性を示唆した．


Gene Cloning and Characterization of Alanine Racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.

赤痢菌の全亜族由来のアラニンラセマーゼ遺伝子をクローニングし，その塩基配列を決定すると共に，クローン株から本酵素を均一に精製し，赤痢菌由来本酵素の特性を明らかにした．また，本酵素の触媒機構から，新しい酵素阻害剤開発の可能性を示した．

Biochemical and Biophysical Research Communications


Improving the Freeze-tolerance of Bakers' Yeast by Loading with Trehalose.

We examined the freeze tolerance of bakers' yeast loaded with exogenous trehalose. Freeze-tolerant and freeze-sensitive compressed bakers' yeast samples were soaked at several temperatures in 0.5 M and 1 M trehalose and analyzed. The intracellular trehalose contents in both types of bakers' yeast increased with increasing soaking period. The initial trehalose-accumulation rate increased with increasing exogenous trehalose concentration and soaking temperature. The maximum trehalose content was almost identical (200-250 mg/g of dry cells) irrespective of the soaking temperature and the type of bakers' yeast, but depended on the exogenous trehalose concentration. The leavening ability of both types of bakers' yeast loaded with trehalose was almost identical to that of the respective original cells, irrespective of the soaking conditions. The freeze-tolerant ratio (FTR) of both types of bakers' yeast increased with increasing intracellular trehalose content. However, FTR decreased during over-soaking after the maximum amount of trehalose had accumulated. FTR of the freeze-sensitive bakers' yeast was more efficiently improved than that of the freeze-tolerant type.

ストレス保護物質であるトレハロースを，パン酵母の細胞内に簡便に導入する方法を確立し，冷凍耐性を著しく改善させることに成功した．

Bioscience, Biotechnology, and Biochemistry


Structure and function of Psychrophilic Alanine Racemase.

低温性アラニンラセマーゼの低温での高い触媒活性と低い耐熱性に関与するアミノ酸残基を特定し，低温性酵素の構造と機能の関係を明らかにした．

Journal of Molecular Catalysis B: Enzymatic


Suppressive Effects of the Extract from Sake Cake on Chemical Mutagen-Induced SOS Response in Salmonella typhimurium.

The suppressive effects of the extract from sake cake on the SOS response of Salmonella typhimurium TA 1535/pSK1002 induced by AF-2, 4NQO, Trp-P-1, Trp-P-2, IQ and MeIQx were investigated for the purpose of finding antimutagenic substances in sake cake. The water-extract and methanol-extract from sake cake strongly suppressed SOS response induced by IQ in the presence of S9 mix in a dose dependent manner. The molecular weight of a suppressive compound in the water-extract was less than 3000 in an analysis of gel filtration. Several suppressive compounds also seemed to exist in the methanol-extract from sake cake.

サルモネラ菌のSOS応答を利用して，酒かすの抗変異原性を見い出した最初の論文である．


One-pot chemo-enzymatic enantiomerization of racemates.

DL-アミノ酸およびDL-乳酸を，それぞれ1つの反応器内で酸化反応と還元反応を効率的に進めることにより，高収率で光学分割できることを示した．


Leavening Ability of Bakers' Yeast Exposed to Hyperosmotic Media.

高浸透圧を負荷したパン酵母は，高い発酵力を有することを見い出し，その生理学的変化を細胞内物質の消長から解析して，発酵力上昇の機構を解明した．


Microanalysis of GABA: an application for evaluating GABA production in yeast strains and the effect of spice extracts on glutamate decarboxylase activity.

機能性物質として有用なガンマーアミノ酪酸の種々の酵母における含量を測定すると共に，細胞内ガンマーアミノ酪酸含量に対するスパイスの影響を明らかにした．


Improvable effect of sodium chloride on leavening activity of Sakadane.

酒種の発酵力に対する浸透圧の影響を検討し，1ー3%塩化ナトリウム存在下で培養した酒種は，高い発酵力を有することを明らかにした．


High Catalytic Activity of Alanine Racemase from Psychrophilic Bacillus psychrosaccharolyticus at High Temperatures in the Presence of Pyridoxal 5'-phosphate.

補酵素の存在下でBacilus psychrosaccharolyticus 由来の低温性アラニンラセマーゼは好熱菌由来酵素より高い触媒活性を有することを明らかにした．低温で高い触媒活性を有する低温性酵素は，触媒として高い潜在能力を有することを示す最初の論文である．


Difference between Escherichia coli O157:H7 and Non-pathogenic E. coli: Survival and Growth in Seasonings.

We examined the survival and growth of Escherichia coli O157:H7 cells incubated with several seasonings, in comparison with those of non-pathogenic E.coli. The cells were incubated at 25℃ for 24 h with several concentrations of NaCl, sucrose, soy sause, worcester sause and tomato ketchup, and their survival ratios were determined. The E.coli O157:H7 strains showed relatively higher survival ratios in 0.5-1.0 M sucrose, 25% soy sauce and 12.5-50% worcester sauce than the non-pathogenic strains, but slightly lower survival ratios in 0.5-2.0 M NaCl. A noteworthy difference between E.coli O157:H7 and the non-pathogenic strains was that incubation in the presence of 12.5% soy sauce allowed the growth of E.coli O157:H7 strains but reduced the viable cell numbers of non-pathogenic E.coli strains.

食品中に混入した大腸菌 O157:H7を非病原性大腸菌と区別して，簡便に分離同定するために，各種調味料中での両株の増殖と生存における違いを調べた．その結果，12.5%醤油中において，大腸菌 O157:H7は増殖するが，非病原性大腸菌の生菌数は低下することを見い出した．

Journal of Bioscience and Bioengineering


Effect of Salt Stress on the Leavening Ability of Bakers' Yeast.

The leavening ability of bakers' yeast cells grown in several media containing NaCl was evaluated in several types of dough. Three types of growth media were used: YPG medium containing 5% glucose, 1% peptone, 0.5% yeast extract, 0.1% KH_2PO_4 and 0.1% MgSo_4·7H_2O (pH 5.6); YPS medium containing 2% sucrose, 4% peptone, 2% yeast extract, 0.2% KH_2PO_4 and 0.1% MgSO_4·7H_2O (pH 5.6); and MO medium containing 1% urea, 0.2% KH_2PO_4, and 5% total sugar as cane molasses (pH 5.6). The addition of 3% NaCl to each of these growth media clearly suppressed the growth of the yeast cells, irrespective of the types of media; the cell yield was decreased by 15-60% in the stationary phase. When we analyzed the leavening ability of these cells that had been exposed to salt stress, the cells showed higher leavening ability than those grown in the absence of NaCl, irrespective of the types of growth media and dough. The increase ratio of leavening ability in the presence of NaCl was highest at 45% when we used the YPS of YPG medium containing 3% NaCl and dough supplemented with 5% glucose. However, the increase was at a much lower level (2-7%) in sugar-less dough, irrespective of the types of growth media. Since the leavening ability of yeast is well known to be primarily limited by the rate of sugar uptake, the high leavening ability of bakers' yeast grown in the presence of NaCl may be due to an increase in sugar-uptake rate. The presence of NaCl in each growth medium also enhanced the leavening ability of various Saccharomyces yeast strains. (Received July 8, 1998; Accepted in revised form February 17, 1999)

塩化ナトリウムによる浸透圧ストレスを与えたパン酵母は，高い発酵力を有することを見いだした．また，塩化ナトリウムを添加する増殖用培地の酒類とパン生地組成の関係を明らかにした．


Primers for Amplifying an Alanine Racemase Gene Fragment to Detect E. coli strains in Food.

ポリメラーゼ連鎖反応(PCR)法により，種々の食品中の大腸菌を迅速かつ特異的に検出する方法を検討した．大腸菌アラニンラセマーゼ遺伝子の配列における大腸菌に特異的な領域を検索し，その領域に対するオリゴヌクレオチドプライマーを用いるPCR法を検討したところ，種々の食品中の大腸菌を5時間以内に特異的に検出できることが明らかとなった．


Characterization of Psychrophilic Alanine Racemase from Bacillus psychrosaccharolyticus,

低温細菌 Bacilus psychrosaccharolyticus 由来の低温性アラニンラセマーゼ遺伝子をクローニングし，その塩基配列を決定すると共に，クローン株から本酵素を均一に精製し，低温性酵素の特性を明らかにした．また，本酵素のアミノ酸配列から，高次構造を予測し，構造と機能の関係を考察した．

Biochemical and Biophysical Research Communications


Effect of Ethyl Alcohol on Growth and Intracellular Alanine Racemase of Psychrotrophs.

The psychrotrophic alanine racemase from Pseudomonas fluorescens, a typical psychrotroph, is less resistant to organic solvents than the enzymes from thermophilic and mesophilic bacteria (Okubo et al., J. Home Econ. Jpn., 46: 1135-1140, 1995). To further elucidate this difference, we examined the effect of ethyl alcohol on the growth and intracellular alanine racemase activity of three typical psychrotrophs-P. fluorescens, Bacillus psychrosaccharolyticus and B. psychrophilus-in comparison with two mesophiles, Escherichia coli and B. subtilis. Although all the bacteria grew to the early stationary phase when cultivated at 22 degrees C for 36 h in the absence of ethyl alcohol, the growth of the psychrotrophs was more effectively suppressed by the addition of 3 and 5% ethyl alcohol to the medium than that of the mesophiles. The intracellular alanine racemase activity of the psychrotrophs was also more markedly reduced in the presence of ethyl alcohol than that of the mesophiles. When bacterial cells of each strain grown at 22 degrees C for 36 h in the absence of alcohol were suspended in 0-5 % ethyl alcohol solution and incubated at 30 degrees C for 1 h, both the survival ratio and intracellular alanine racemase activity of the psychrotrophs were lower than those of the mesophiles. Thus, ethyl alcohol effectively reduced both the growth of the psychrotrophs and their intracellular alanine racemase activity. Low concentrations of various other alcohols also repressed the growth of the psychrotrophs at 10 degrees C.

低温菌の増殖と細胞内アラニンラセマーゼに対するエチルアルコールの影響を常温細菌 との比較において検討した．低温菌の増殖は，低濃度のエタノールにより，常温菌よりも効果的に抑制された．低温菌の細胞内アラニンラセマーゼ活性もまた，培地中にエチルアルコールを添加することによって，常温菌より著しく低下した．他の種々のアルコールも，10℃での低温菌の増殖を低濃度で抑制した．


Effect of Sodium Chloride on Growth and Fermentative Activity of Saccharomyces Yeast,

塩化ナトリウム存在下で生育したパン酵母の発酵力を検討した．2ー3%の塩化ナトリウム存在下で生育したパン酵母は，高い発酵力を示し，この効果は種々のサッカロミセス酵母においても見られた．


A New Sterility Test for Cow's Milk Using Alanine Racemase Gene as the Index.

Oligonucleotide primers designed from consensus sequences of alanine racemase genes were used for a sterility test of cow's milk by polymerase chain reaction (PCR). Commercial cow's milk in two 250 ml packages was separately centrifuged at 5000 x g for 10 min, bacterial cells in each precipitate were cultivated at 30 and 55 degrees C for 5 h in Luria-Bertani medium, and the cells from each culture were mixed and used for the PCR after being treated with 0.1 N NaOH at 60 degrees C for 10 min. When we performed the PCR using DNAs from various bacteria and eukaryotes as the templates, a unique PCR product of about 390 bp was amplified only from the bacteria. The sensitivity of the PCR method was such that an initial inoculum of 1 CFU of Bacillus stearothermophilus, Escherichia coli, and Pseudomonas fluorescens per 250 ml of cow's milk could be detected. When we analyzed 14 types of commercial cow's milk, all samples which were positive by the standard sterility test at 30 or 55 degrees C were also found positive by the PCR method.

迅速かつ正確な牛乳の無菌試験法を開発した．細菌に広く分布するアラニンラセマーゼ遺伝子の保存領域から設計したアラニンラセマーゼ遺伝子をプライマーとするPCR法は，培養法による無菌試験法よりも正確であることを明らかにした．培養法による無菌試験法は7日間以上を要するが，本法では1日間で検査が終了した．


Alanine Racemase Gene Fragments as Probes for Detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in Foods.

Alanine recemase gene fragments containing non-conserved regions of the gene were evaluated as probes for detecting Bacillus stearothermophilus and Bacillus psychrosaccharolyticus in foods. The gene fragments were amplified from genomic DNA of each bacterium by polymerase chain reaction with degenerate oligonucleotide primers, and labeled with digoxigenin as probes for detecting each bacterium. Foods and bacteria were treated at 25℃ for 10 min in 0.1 N NaOH containing 0.5% SDS before being directly spotted onto nylon membranes for DNA hybridization. When the specificities of the probes were analyzed using a total of 86 strains(23 genera and 65 species)of bacteria including 29 Bacillus strains(20 species), each probe was specific for the respective target bacteria. A variety of foods inoculated with B. stearothermophilus or B. psychrosaccharolyticus were positive as determined by hybridization with the respective probe, whereas uninoculated foods were negative. The alanine recemase gene fragments could be used as specific probes for detecting B. stearothermophilus and B. psychrosaccharolyticus in foods.

食品の低温保存や保温状態において，品質の低下をもたらす低温細菌 および 好熱細菌を迅速かつ正確に検出するため，アラニンラセマーゼ遺伝子断片をプローブとするDNAハイブリダイゼーション法を検討した．その結果，本プローブはそれぞれの細菌の検出にきわめて有効であった．

Journal of fermentation and bioengineering


Trehalose 6-phosphate Production with Energy Coupling Fermentation by Yeast Cells.

パン酵母のエネルギー共役発酵によってトレハロース-6-リン酸(T6P)を生産する方法を検討した．種々の条件検討によって，対糖収率11%でT6Pが生産された．

Bioscience, Biotechnology, and Biochemistry


Suppressing Effects of Polysaccharide Produced by Bacillus circulans on Chemical Mutagens-Induced SOS Responce in Salmonella typhimurium.

Salmonella typhimurium における化学的変異原誘導性のSOS反応に対する，Bacillus circulans 由来多糖の抑制効果を検討した．その結果，本多糖は，IQ，MelQxによって誘導されるSOS反応を強く抑制し，抗変異原性を有することが明らかとなり，食品添加物としての有用性が示唆された．


Trehalose Metabolism and Leavening Ability of Bakers' Yeast Grown in the Presence of Sodium Chloride.

塩化ナトリウム存在下で生育したパン酵母の細胞内トレハロースは，一時的に増加しても蓄積されず，浸透圧ストレスを受けた酵母の高い発酵力は，トレハロース含量と相関しないことが明らかとなった．

Journal of Fermentation and Bioengineering


Electrophoretic Karyotypes of Commercial Freeze-tolerant and Freeze-sensitive Bakers' Yeast.

市販の冷凍耐性パン酵母と冷凍感受性パン酵母の電気泳動核型をパルスフィールドゲル電気泳動により分析した．パン酵母には16本の染色体DNAが存在するが，冷凍耐性酵母と冷凍感受性酵母の電気泳動核型において，約500kpbのサイズを有する染色体は，冷凍感受性株にのみ認められた．このことは，冷凍耐性株の同定や汚染感受性酵母の検出において，迅速分析を可能にするものと考えられる．


Structural Study of an Exocellular Polysaccharide of Bacillus circulans.

Bacillus circulans の生産する高粘性多糖を均一に精製し，その構造解析を行った．本多糖は，ガラクトース，マンノース，ラムノース，グルクロン酸を含むオリゴ糖の繰り返し構造を有することが明らかとなった．


Evaluation of an Alanine Racemase Gene as an Indicator for the Detection of Various Escherichia coli: Reactivity of the Gene Fragment with Various E. coli O157:H7 Isolates.

自然界から多様な大腸菌O157を分離し，それらの遺伝的多様性を明らかにすると共に，典型的細菌酵素であるアラニンラセマーゼの遺伝子断片との反応性を明らかにし，アラニンラセマーゼを指標とした大腸菌検査法が多様な大腸菌O157を検出できることを示した．


Comparison of Physicochemical Properties of a Novel Polysaccharide Produced by Bacillus circulans with Commercial Polysaccharides.

Bacillus circulans の生産する高粘性多糖の物理化学的性質を各種既知多糖と比較検討した．本菌の多糖は，親水性にすぐれ，また，高い乳化安定性を示した．粘性は，pHの変化に対しても安定であった．


An Alanine Racemase Gene as a New Index for Detecting Escherichia coli in Foods.

食品中の大腸菌を迅速かつ正確に検出するための指標として，細胞壁生合成に必須のアラニンラセマーゼ遺伝子が有用であることを明らかにした．本酵素の遺伝子断片をプローブとしたDNAハイブリダイゼーション法において，本プローブは大腸菌以外の細菌群とはほとんど反応性を示さず，病原性大腸菌を含む多様な大腸菌にきわめて特異的であった．大腸菌食品中でも，その特異性は保持されることが確認された．


Lipid Composition of Commercial Bakers' Yeasts Having Different Freeze-tolerance in Frozen Dough.

酵母の冷凍耐性と脂質組成の関係を明らかにするため，冷凍耐性率の異なる各種市販パン酵母の脂質組成を分析した．総脂質量や各種リン脂質含量，脂肪酸組成は，各酵母で違いが見られなかったが，リン脂質に対するステロールの比率は，耐性株と感受性株で違いが見られ，冷凍耐性と膜流動性は相関するものと考えられた．

Bioscience, Biotechnology, and Biochemistry


Lipid Composition of a Freeze-tolerant Yeast, Torulaspora delbrueckii, and Its Freeze-sensitive Mutant.

冷凍耐性酵母と冷凍感受性酵母の脂質組成を比較分析し，両株の違いを明らかにした．特に，リン脂質に対するステロールの比率の違いから，冷凍耐性酵母は冷凍感受性酵母に比べ，膜の流動性が高いことが明らかとなった．


Trehalase Activity and Trehalose content in a Freeze-tolerant Yeast, Torulaspora delbrueckii and Its Freeze-sensitive Mutant.

酵母の冷凍耐性に関与するトレハロースとその加水分解酵素トレハラーゼについて，冷凍耐性株とその冷凍感受性変異株を用いて比較分析した．冷凍感受性酵母は，冷凍耐性酵母に比べ，トレハラーゼ活性が高く，細胞内トレハロース含量も少ないことが明らかとなった．また，トレハラーゼには至適反応pHの異なる3種類の酵素が存在することが判明した．


Lability of Alanine Racemase from a Psychrotroph.

増殖に必須な酵素の不活性化を通して低温菌の増殖を抑制することを目的として，典型的低温細菌である Pseudomonas fluorescens 由来アラニンラセマーゼの不安定性を，好熱菌，常温菌由来の本酵素と比較分析し，低温菌酵素はアルコール等の有機溶媒や界面活性剤により容易に不活化されることを明らかにした．


Induction of Freeze-sensitive Mutants from a Freeze-tolerant Yeast, Torulaspora delbrueckii.

冷凍生地製パン法に不可欠な酵母の性質である冷凍耐性について，その機構を明らかにするため，冷凍耐性酵母から冷凍感受性酵母を変異誘導して，その冷凍感受性株を分離し諸性質を明らかにした．


Enantioselective Synthesis of N-Acetyl-L-methionine with Aminoacylase in Organic Solvent.

NーアシルL-アミノ酸やD-アミノ酸は，抗生物質の重要な成分として用いられている．本論文は，NーアシルL-アミノ酸とD-アミノ酸の両者を，ラセミ化アミノ酸から合成する効率的な方法を確立したものである．


Thermolabile Alanine Racemase from a Psychrotroph, Pseudomonas fluorescens: Purification and Properties.

低温保存食品の品質低下を招く低温菌の熱に不安定な酵素を均一に精製し，その酵素化学的諸性質を明らかにした．本酵素は低温できわめて高い活性を有し，30℃以上の温度で容易に変性する典型的な低温性酵素であった．


A New Amino Acid Racemase with Threonine α-Epimerase Activity from Pseudomonas putida: Purification and Characterization.

うま味を有するトレオニンに対してα-エピメラーゼ活性を有する新規酵素を発見し，その酵素を均一に精製して，酵素化学的諸性質を明らかにした．本酵素は，他種類のアミノ酸をもラセミ化するユニークな酵素であった．


Synthesis of L-Proline from the Racemate by Coupling of Enzymatic Enantiospecific Oxidation and Chemical Non-enantiospecific reduction.

酵素的酸化反応と化学的還元反応の共役反応により，アミノ酸(プロリン)のラセミ体を特異的にL体に変換する方法を確立した．本法を用いれば，安価なラセミ体から，高収率でL-体を生産することが可能である．

Journal of Fermentation and Bioengineering


Total Conversion of Racemic Pipecolic Acid into the L-Enantiomer by a Combination of Enantiospecific Oxidation with D-Amino Acid Oxidase and Reduction with Sodium Borohydride.

抗菌物質の側鎖修飾剤として有用なL-ピペコリン酸の合成に，ラセミ化ピペコリン酸を材料として，D-アミノ酸酸化酵素と化学的還元剤の共役反応を用いることにより，特異的かつ高収率でL-ピペコリン酸が生成することを見いだした．

Bioscience, Biotechnology, and Biochemistry


Chemical Modification of Urokinase with Bis-imidoesters and Properties of the Intramolecularly Cross-linked Enzyme.

二価性架橋試薬を用いる血栓溶解酵素(ウロキナーゼ)の分子内架橋により，耐熱性や血中安定性等の物理的·生物化学的性質を改善した．


Chemical Modification of Urokinase with Human Serum Albumin Fragments.

血液中での半減期が1分間以内という極めて不安定なウロキナーゼに，ヒト血清アルブミンの断片を化学的に結合させることにより，ウロキナーゼの免疫化学的性質をアルブミンに変換させると共に，血液中での安定性が極めて高い修飾酵素を作製した．


Urokinase Quality: Analysis of Different Preparations.

血栓溶解酵素の性質を酵素化学，生物化学，免疫化学的に解明した．特に，不純タンパク質に対する抗体を用いた純度分析は，微量の不純タンパク質の検出に有用であった．


Purification of High Molecular Weight Urokinase by Reverse-immunoadsorption.

人尿中に極めて微量存在する血栓溶解酵素ウロキナーゼを，本酵素以外の多種類のタンパク質に対する抗体を固定化した逆免疫吸着体を用いて，迅速かつ高収率で精製する新しい精製方法を確立した．


Effect of Microwave Radiation on Escherichia coli O157:H7 in foods

食品中の大腸菌に対するマイクロ波の影響

We examined the survival and acid resistance of enterohemorrhagic Escherichia coli O157 (EHEC O157) in foods irradiated by microwave (2.45 GHz, 700 W, 7 s) with a domestic microwave oven. When the microwave irradiated foods inoculated with the cells at the cell density of 3 x 106 cells/g, the living cell number decreased below 65%. The cells in curry showed the lowest survival ratio (2%) among those in foods examined. Acid resistance of the cells in foods irradiated by the microwave was also effectively reduced below 10% irrespective of foods surveyed. Microwave could prove useful for reducing the living cell number and acid resistance of EHEC O157 in foods.

Natural Science Research, Faculty of Integrated Arts and Sciences, The University of Tokushima

徳島大学総合科学部自然科学研究


Determination of Living Cells of Lactic Bacteria in Foods with Petri-film

ペトリフイルムを用いる食品中の乳酸菌の生菌数測定

We examined the living cell number of lactic bacteria in foods by anaerobic culture with Petri-filmTM (PFAC).As the reference medium, the de Man, Rogosa and Sharpe agar medium (MRSA) and an agar mediumsupplemented with bromocresol purple (BCPA) were used. When we determined the living cell number oflactic bacteria in various products of pickled vegetables (29 products), kimuchi (10 products), meats (13products), lactic fermenting beverages (24 products) and cultured milks (6 products), the cell numbersdetermined with PFAC showed high correlations with those determined with MRSA and BCPA.

徳島大学総合科学部自然科学研究


Production of Human Type Glycosylated Tissue Plasminogen Activator and the Role of Its Carbohydrate Moiety

To begin the examination of the role of human type carbohydrate moiety of tissue plasminogen activator(t-PA) on the binding of the enzyme to fibrin，the naturally glycosylated enzyme was produced by microcarrierculture of human cells established from normal uterine muscle. The cells grown on microcarrers in Haunks' MEM supplemented with 10 % FBS (1.6 x 10 6 cells/ml) rapidly detached themselves from microcarrier in aserum-free medium (t-PA production medium) within 5 days, and it was difficult to produce t-PA for long time (t-PA production: an average of 3 IU/ml/day over 5 days). Addition of 0.5% beef extract to the serum-freemedium suppressed their detaching from microcarriers. By regulating the pH (7.4) and dissolved oxygen(4 ppm) of the serum free medium，the cell density of microcarrier culture increased to 1.2 x10 7 cells/ml and t-PAwas produced over 38 days (t-PA production: an average of 836 IU/ml/day over 38 days). Native t-PAs purifiedto homogeneity from the culture broth had the molecular weight of 63,000 and 65,000 containing 5.6 and 8.5 %carbohydrate，respectively (molecular mass of protein moiety was calculated to be 59,500). By enzymaticdigestion of carbohydrate moiety in native t-PA，we obtained partially deglycosylated t-PA with molecularweights of 60,000 and 62,000. Completely deglycosylated t-PA was obtained by t-PA production in thepresence of 10μg/ml tunicamycin (N-glycosylation inhibitor). This suggests that N-glycosylationof t-PAoccurs while O-glycosylation is absent. The binding strength of these enzymes to fibrin increased withdecrease of the carbohydrate content. The carbohydrate moiety of human type glycosylated t-PA probablymodulates the binding strength of the enzyme to fibrin.

Natural Science Research, Faculty of Integrated Arts and Sciences, The University of Tokushima