Language：English   Publishing type：Research paper (scientific journal)  

We previously demonstrated that thicker periosteal sheets with enhanced cell layering maintain their component cells at relatively immature stages of differentiation but express a high in vivo osteogenic potential. As it has been recently proposed that stiff scaffolds provide a mechanical cue to various cell types that promotes differentiation, we postulated that the maintenance of immature cells in our periosteal sheets is due to the mechanical stiffness of the multilayered-cell architecture. To demonstrate the biomechanical characteristics of our periosteal sheets, we have determined their stiffnesses with atomic force microscopy (AFM) and evaluated the expression of extracellular matrix (ECM) components specifically by both immunocytochemistry and a complementary DNA microarray technology. Compared to osteoblastic Saos2 cells, the cytoskeletal fibers were developed more in the periosteal cells, but the periosteal cells in monolayer culture developed before either the cells in the peripheral or central regions of the periosteal sheets developed. However, the nanoindentation by AFM distinguished the central region from the peripheral region. The peak stiffness values of cells were ordered as follows: tissue culture polystyrene (1.66. GPa). ⋙. dispersed (9.99. kPa). &gt
. central region (5.20. kPa). &gt
. peripheral regions (3.67. kPa). Similarly, the degree of development of α-smooth muscle actin (αSMA) filaments within cells was dispersed. &gt
. central region. &gt
. peripheral region. In conjunction with the abundantly deposited ECM in the periosteal sheets, these findings suggest that the order of cell stiffness may depend on the integration of the stiffness of individual ECM components and the extent of cytoskeletal fiber formation. Because recently published data have demonstrated that the optimal stiffness for osteogenic differentiation is 25-40. kPa, it is plausible that the periosteal cells residing in the less-stiff multilayer regions could be maintained at relatively immature stages under the control of the stem-cell medium in vitro but start differentiating when exposed to the proper stiffness upon release from the culture conditions at the implantation site. © 2013 Elsevier Ltd.

DOI： 10.1016/j.micron.2013.02.001

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DOI： 10.3892/br.2022.1504

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Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αβ structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2-S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C. albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.

DOI： 10.1016/j.jbiosc.2020.02.011

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DOI： 10.1016/j.jbiosc.2019.09.009

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In this study, we used the commercial soybean protein hydrolysate Hinute-DC6 as a novel starting material from which to purify and identify multifunctional cationic peptides. After fractionation, Hinute-DC6 was separated into 20 fractions with varying isoelectric points (pI) by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we purified and identified the cationic peptides from fractions 19 and 20, which had pI values greater than 12, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectrometry. Of the 83 cationic peptides identified, 14 had high pI values and net charges greater than +2, and were chemically synthesized and assayed for various bioactivities, including hemolytic, antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. None of the 14 cationic peptides tested exhibited hemolytic activity toward mammalian red blood cells at concentrations up to 1000 μM. Five of the cationic peptides exhibited antimicrobial activities against at least one of four human-pathogenic microorganisms tested. In addition, in chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, the 50% effective concentrations of these 14 peptides were between 0.069 and 5.2 μM. Tube-formation assays in human umbilical vein endothelial cells showed that each of the 14 cationic peptides exhibited significant angiogenic activities at 10 μM, with values similar to those of the positive control LL-37. Our results demonstrate that the 14 identified cationic peptides have multiple functions with negligible hemolytic activity. These data indicate that the cationic peptides isolated from Hinute-DC6 and fractions containing these cationic peptides have the potential to be used as multifunctional ingredients for healthcare applications.

DOI： 10.1016/j.jbiosc.2019.06.016

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In our previous study, we identified multifunctional cationic peptides from enzymatic hydrolysates of rice bran proteins (RBPs) that have antimicrobial and lipopolysaccharide-neutralizing activities. In this study, we investigated the potential of the peptides RBP-LRR, RBP-EKL, and RBP-SSF to promote proliferation, angiogenesis (tube formation), and migration in human umbilical vein endothelial cells (HUVECs). To determine mechanisms of wound healing actions, angiogenic and migration-promoting activities of these peptides were evaluated following pretreatments of HUVECs with specific inhibitors. In these experiments, the cationic peptides RBP-LRR, RBP-EKL, and RBP-SSF induced cell proliferation at low concentrations of 0.1 μM or 1 μM. Moreover, the three cationic peptides had angiogenic activities at concentrations more than 1 μM in tube formation assays, and their effects were similar to those of LL-37. Subsequent scratch migration assays exhibited that RBP-LRR, RBP-EKL, and RBP-SSF promote wound closure at optimum concentrations of 10, 10, and 0.1 μM, respectively. In further studies, we performed tube formation assays using HUVECs pretreated with SU5416, which inhibits vascular endothelial growth factor (VEGF) receptors, and suggested the possibility that the three cationic peptides induce angiogenesis by activating VEGF receptors. In corresponding scratch migration assays using HUVECs, pretreatment with the proliferation inhibitor mitomycin C did not alter the effects of RBP-LRR and RBP-EKL, and significant contribution to wound closure were mediated by cell migration regardless of proliferation rates. In contrast, RBP-SSF contributed to wound closure exclusively by promoting cell proliferation. The present data indicate that RBP-LRR, RBP-EKL, and RBP-SSF are candidates for use as wound healing agents.

DOI： 10.1016/j.jbiosc.2019.02.002

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DOI： 10.3390/biomedicines7020045

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DOI： 10.1016/j.heliyon.2019.e01490

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In this study, we investigated the lipopolysaccharide (LPS)-neutralizing and angiogenic activities of cationic peptides derived from the traditional Japanese fermented product Natto, which is made by fermenting cooked soybeans using Bacillus subtilis. Initially, we prepared 20 fractions of Natto extracts with various isoelectric points (pI's) using ampholyte-free isoelectric focusing (autofocusing). Cationic peptides were then purified from fractions 19 and 20, whose pH values were greater than 12, using reversed-phase high-performance liquid chromatography, and were identified using matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among the 13 identified cationic peptides, seven (KFNKYGR, FPFPRPPHQK, GQSSRPQDRHQK, QRFDQRSPQ, ERQFPFPRPPHQK, GEIPRPRPRPQHPE, and EQPRPIPFPRPQPR) had pI's greater than 9.5, positive net charges, and differing molecular weights. These peptides were then chemically synthesized and applied to chromogenic LPS-neutralizing assays using Limulus amebocyte lysates, and 50% effective (neutralizing) concentrations of 2.6-5.5 μM were demonstrated. In addition, tube formation assays in human umbilical vein endothelial cells revealed angiogenic activities for all but one (GEIPRPRPRPQHPE) of these seven cationic peptides, with increases in relative tube lengths of 23-31% in the presence of peptides at 10 μM. Subsequent experiments showed negligible hemolytic activity of these peptides at concentrations of up to 500 μM in mammalian red blood cells. Collectively, these data demonstrate that six cationic peptides from Natto extracts, with the exception of GEIPRPRPRPQHPE, have LPS-neutralizing and angiogenic activities but do not induce hemolysis.

DOI： 10.1016/j.jbiosc.2018.09.016

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In this study, we prepared fractions containing multifunctional cationic peptides by separating the commercial soybean protein hydrolysate Hinute-AM into 20 fractions. These fractions contained peptides with various isoelectric points (pI), as indicated by ampholyte-free isoelectric focusing (autofocusing). Thus, we purified and identified the cationic peptides from fractions 19 and 20, which had pH values greater than 10, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among 19 identified cationic peptides, NKNAKPPSPR, PGKKNAIV, KSGPGMSPR, NVSKPPRVV, RKVGAGGRKPLG, and LPCVIGGVPKRV had high pI values and were included as chemically synthesized peptides in assays of various functions, including lipopolysaccharide (LPS)-neutralizing and angiogenic activities. Chromogenic LPS-neutralizing assays using Limulus amebocyte lysates showed that 50% effective concentrations of these six peptides were between 1.63 and 2.65 μM, and were higher than that (0.12 μM) of polymyxin B. Moreover, in tube-formation assays in human umbilical vein endothelial cells, all of the six cationic peptides except LPCVIGGVPKRV exhibited angiogenic activities similar to those of the positive control LL-37. In addition, the six identified cationic peptides had no hemolytic activity at concentrations up to 500 μM in mammalian red blood cells. Our results demonstrate that five of the identified cationic peptides, excluding LPCVIGGVPKRV, have multiple functions and little or no hemolytic activity. These data indicate that fractions containing cationic peptides from Hinute-AM have the potential to be used as dietary supplements and functional ingredients in food products.

DOI： 10.1016/j.jbiosc.2018.07.013

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DOI： 10.1186/s40729-018-0140-8

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DOI： 10.1038/s41598-018-29715-w

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In our previous studies, we showed that AmyI-1-18 and its single amino acid-substituted analogs have antimicrobial, anti-inflammatory, and anti-endotoxic activities and cause little or no hemolysis or cytotoxicity. In this study, we investigated the potential of these peptides to promote proliferation, angiogenesis (tube formation), and migration in human umbilical vein endothelial cells (HUVECs). Among five single amino acid-substituted analogs, [N3L]AmyI-1-18 induced cell proliferation in a concentration-dependent manner with similar efficacy to AmyI-1-18. In tube formation assays, AmyI-1-18 and [N3L]AmyI-1-18 had angiogenic activities at 1 μM and their effects were similar to those of LL-37. Moreover, scratch migration assays showed that AmyI-1-18, [N3L]AmyI-1-18, and LL-37 promote cell migration with optimum concentrations of 10, 1, and 0.1 μM, respectively. Subsequently, we performed tube formation assays using HUVECs pretreated with SU5416, which is an inhibitor of vascular endothelial growth factor (VEGF) receptors, and revealed that AmyI-1-18 and [N3L]AmyI-1-18 induce angiogenesis by activating VEGF receptors. Similarly, after pretreating HUVECs with mitomycin C, which inhibits cell proliferation, [N3L]AmyI-1-18 significantly contributed to wound closure in scratch migration assays. Moreover, enhancements of hydrophobicity following substitution of AmyI-1-18 asparagine with leucine led to greater increases in cell migration. The present data indicate that both peptides, particularly [N3L]AmyI-1-18, are candidates for use as wound healing agents.

DOI： 10.1016/j.peptides.2018.04.017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media S.A.  

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

DOI： 10.3389/fbioe.2018.00004

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Language：English   Publisher：ELSEVIER SCIENCE BV  

DOI： 10.1016/j.reth.2017.10.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

In this study, we hydrolyzed rice endosperm protein (REP) with pepsin and generated 20 fractions containing multifunctional cationic peptides with varying isoelectric point (pI) values using ampholyte-free isoelectric focusing (autofocusing). Subsequently, we determined antimicrobial activities of each fraction against the pathogens Prophyromonas gingivalis, Propionibacterium acnes, Streptocossus mutans, and Candida albicans. Fractions 18, 19, and 20 had pI values greater than 12 and exhibited antimicrobial activity against P. gingivalis, P. acnes, and C. albicans, but not against S. mutans. In further experiments, we purified and identified cationic peptides from fractions 18, 19, and 20 using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. We also chemically synthesized five identified peptides (RSVSKSR, RRVIEPR, ERFQPMFRRPG, RVRQNIDNPNRADTYNPRAG, and VVRRVIEPRGLL) with pI values greater than 10.5 and evaluated antimicrobial, lipopolysaccharide (LPS)-neutralizing, and angiogenic activities. Among these synthetic peptides, only VVRRVIEPRGLL exhibited antimicrobial activity against P. gingivalis, with an IC50 value of 87 mu M. However, all five cationic peptides exhibited LPS-neutralizing and angiogenic activities with little or no hemolytic activity against mammalian red blood cells at functional concentrations. These present data show dual or multiple functions of the five identified cationic peptides with little or no hemolytic activity. Therefore, fractions containing cationic peptides from REP hydrolysates have the potential to be used as dietary supplements and functional ingredients in food products.

DOI： 10.1016/j.peptides.2017.09.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In this study, to prepare the fraction containing multifunctional cationic peptides, we first hydrolyzed rice bran protein (RBP) with pepsin. We separated the enzymatic hydrolysate of RBP into 20 fractions containing peptides with different isoelectric point (pI) values by ampholyte-free isoelectric focusing (autofocusing). Subsequently, we examined the antimicrobial activity of each fraction against four pathogens. In addition, we purified the cationic peptides from fractions exhibiting antimicrobial activity by reversed phase high-performance liquid chromatography and identified them by matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Of five cationic peptides identified, we chemically synthesized three peptides with high pI values and evaluated their multiple functions, including antimicrobial, lipopolysaccharide-neutralizing and angiogenic activities. Our results demonstrated that the three identified cationic peptides exhibited multiple functions with little or no haemolytic activity. Fractions containing cationic peptides obtained from RBP hydrolysate have the potential to be used as dietary supplements and functional ingredients in food products. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2017.04.046

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

In this study, we identified and chemically synthesized three cationic and amphipathic peptides (Glycinin-17, BCAS-16, and BCBS-11) from soybean proteins. These peptides had high isoelectric points, high positive net charges, and included multiple hydrophobic amino acids. Subsequently, we identified multiple functions of these peptides, including antimicrobial, lipopolysaccharide-neutralizing, and angiogenic activities, and examined their cytotoxic activities against mammalian red blood cells. Glycinin-17, BCAS-16, and BCBS-11 exhibited antimicrobial activity against Porphyromonas gingivalis and Candida albicans whereas Glycinin-17 did not possess antimicrobial effects on Propionibacterium acnes and Streptococcus mutans. Membrane-depolarization assays and flow cytometric analyses showed that the antimicrobial properties of Glycinin-17, BCAS-16, and BCBS-11 against P. gingivalis, P. acnes, and S. mutans were dependent on membrane-disrupting potential. In contrast, major antimicrobial activities of these peptides against C. albicans were dependent on interactions with targets other than cell membranes. Furthermore, chromogenic Limulus amebocyte lysate assays showed that 50% effective concentrations (EC50, 0.12-0.31 mu M) of these three peptides neutralize LPS with similar potency (EC50: 0.11 mu M) to that of polymyxin B. Moreover, tube-formation assays in human umbilical vein endothelial cells showed similar angiogenic activities of the three peptides as that following treatment with LL-37. Although BCAS-16 exhibited hemolytic activity, the rate of hemolysis for Glycinin-17 and BCBS-11 in the presence of 500-mu M Glycinin-17 and BCBS-11 was less than 2%. These results demonstrate that cationic and amphipathic peptides from soybean proteins, particularly Glycinin-17 and BCBS-11, have potential as multifunctional ingredients for healthcare applications.

DOI： 10.1002/bip.23023

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DOI： 10.1186/s40729-017-0068-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI-1-18 from rice a-amylase (AmyI-1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI1-18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI-1-18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI-1-18. In the present study, anti-inflammatory (anti-endotoxic) activities of five AmyI-1-18 analogs containing arginine or leucine substitutions were investigated. Two single arginine-substituted and two single leucine-substituted AmyI-1-18 analogs inhibited the production of LPS-induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI-1-18. These data indicate that enhanced cationic and hydrophobic properties of AmyI-1-18 are associated with improved anti-endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC50) of the three AmyI-1-18 analogs (G12R, D15R, and E9L) were 0.11-0.13 mu M, indicating higher anti-endotoxic activity than that of AmyI-1-18 (IC50, 0.22 mu M), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI-1-18 analogs. In addition, AmyI-1-18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of antiinflammatory and LPS-neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine-substituted and leucine-substituted AmyI-1-18 analogs with improved antiendotoxic and antimicrobial activities have clinical potential as dual-function host defense agents. Copyright (C) 2017 European Peptide Society and John Wiley & Sons, Ltd.

DOI： 10.1002/psc.2983

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Society for Biotechnology  

DOI： 10.1016/j.jbiosc.2016.05.008

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Other Link： http://search.jamas.or.jp/link/ui/2017254575

Language：English   Publishing type：Research paper (scientific journal)   Publisher：THE MEMBRANE SOCIETY OF JAPAN  

Here, we prepared a poly(L–lactic acid) (PLLA) microporous membrane with open finger–like pores as a scaffold for tissue engineering by a nonsolvent–induced phase separation method with the aid of a surfactant. The increase of surfactant content in the polymer solution caused the mold (glass plate) side of the membrane to adopt an indented structure with open finger–like pores to enhance the internal surface area. Osteoblast–like cells inoculated on the indented side grew on and in the PLLA membrane scaffold to reach 2 ～ 3 times higher cell density per unit apparent area compared to that attained in monolayer cultures. The cells on the membrane deposited calcium compounds by osteoinduction with ascorbic acid 2–phosphate, dexamethasone, and β– glycerophosphate. The PLLA membrane with open finger–like pores will likely be useful as scaffolds to support the implantation of osteogenic cells in bone tissue engineering.

DOI： 10.5360/membrane.41.304

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Other Link： http://search.jamas.or.jp/link/ui/2017289540

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, Amyl-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; Amyl-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that Amyl-1-18 strongly bound to RNA with a ICD value of 1.4 x 10(-8) M but not to DNA and that Amyl-1-18 specifically bound to DnaK with a K-D value of 4.4 x 10(-8) M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2016.03.004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Rice (Oryza sativa) is consumed as a staple food globally, and rice bran, the byproduct, is an unused biomass that is ultimately discarded as waste. Thus, in the present study, a technique for producing tyrosinase inhibitory peptides from rice bran protein (RBP) was developed. Simultaneous treatment of RBP with chymotrypsin and trypsin produced numerous peptides. Subsequently, six tyrosinase inhibitory peptides were isolated from the hydrolysate fractions in a multistep purification protocol, and their amino acid sequences were determined. Three of these peptides had a C-terminal tyrosine residue and exhibited significant inhibitory effects against tyrosinase-mediated monophenolase reactions. Furthermore, peptide CT-2 (Leu-Gln-Pro-Ser-His-Tyr) potently inhibited melanogenesis in mouse B16 melanoma cells without causing cytotoxicity, suggesting the potential of CT-2 as an agent for melanin-related skin disorder treatment. The present data indicate that RBP is a potent source of tyrosinase inhibitory peptides and that simultaneous treatment of RBP with chymotrypsin and trypsin efficiently produces these peptides.

DOI： 10.1021/acs.jnatprod.6b00449

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Tyrosinase, a rate-limiting enzyme in melanin biosynthesis, catalyzes the hydroxylation of L-tyrosine to 3,4-dihydroxy-L-phenylalanine (L-dopa) (monophenolase reaction) and the subsequent oxidation of L-dopa to L-dopaquinone (diphenolase reaction). Thus, tyrosinase inhibitors have been proposed as skin-lightening agents; however, many of the existing inhibitors cannot be widely used in the cosmetic industry due to their high cytotoxicity and instability. On the other hand, some tyrosinase inhibitory peptides have been reported as safe. In this study, we found that the peptide TH10, which has a similar sequence to the characterized inhibitory peptide P4, strongly inhibits the monophenolase reaction with a half-maximal inhibitory concentration of 102 mu M. Seven of the ten amino acid residues in TH10 were identical to P4; however, TH10 possesses one N-terminal tyrosine, whereas P4 contains three tyrosine residues located at its N-terminus, center, and C-terminus. Subsequent analysis using sequence-shuffled variants indicated that the tyrosine residues located at the N-terminus and center of P4 have little to no contribution to its inhibitory activity. Furthermore, docking simulation analysis of these peptides with mushroom tyrosinase demonstrated that the active tyrosine residue was positioned close to copper ions, suggesting that TH10 and P4 bind to tyrosinase as a substrate analogue. (C) 2015 The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2015.10.010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. (C) 2015, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2015.09.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

AmyI-1-18, an antimicrobial peptide, is a cationic -helical octadecapeptide derived from -amylase of rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antifungal activity of AmyI-1-18 against Candida albicans, 11 analogs bearing substitutions with alanine, leucine, and/or arginine, which were designed on the basis of the helical wheel projection of AmyI-1-18, were synthesized, and their antifungal activity was investigated. The antifungal activities of four analogs obtained by replacing arginine or lysine with alanine were significantly reduced. The results suggested that the cationic arginine and lysine residues in AmyI-1-18 are important for its antifungal activity. The antifungal activities of two single leucine-substituted analogs were not improved, but among three single arginine-substituted analogs, AmyI-1-18(D15R) had approximately a twofold higher antifungal activity [50% growth-inhibitory concentration (IC50): 31 M] than AmyI-1-18 (IC50: 64 M) and exhibited low hemolytic activity (4% at 100 M). Flow cytometric analysis using propidium iodide revealed that the antifungal activity of AmyI-1-18(D15R) was dependent on its membrane-disrupting activity in a manner different from that of AmyI-1-18. Further enhancement of the cationicity and hydrophobicity of AmyI-1-18(D15R) resulted in no improvement in antifungal activity and a significant increase in hemolytic activity. In this study, the results demonstrated that the antifungal activity of AmyI-1-18 against C. albicans was enhanced through increasing its membrane-disrupting activity by replacing aspartic acid at position 15 with arginine without a significant increase in hemolytic activity. (c) 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 219-229, 2016.

DOI： 10.1002/bip.22817

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

We have previously reported that AmyI-1-18, an octadecapeptide derived from alpha-amylase (AmyI-1) of rice, is a novel cationic alpha-helical peptide that exhibited antimicrobial activity against human pathogens, including Porphyromonas gingivalis, Pseudomonas aeruginosa, Prop ionibacterium acnes, Streptococcus mutans, and Candida albicans. In this study, to further investigate the potential functions of AmyI-1-18, we examined its inhibitory ability against the endotoxic activities of lipopolysaccharides (LPSs, smooth and Rc types) and lipid A from Escherichia coli. AmyI-1-18 inhibited the production of endotoxin-induced nitric oxide (NO), an inflammatory mediator, in mouse macrophages (RAW264) in a concentration-dependent manner. The results of a chromogenic Limulus amebocyte lysate assay illustrated that the ability [50% effective concentration (EC50): 0.17 mu M] of AmyI-1-18 to neutralize lipid A was similar to its ability (EC50: 0.26 mu M) to neutralize LPS, suggesting that AmyI-1-18 specifically binds to the lipid A moiety of LPS. Surface plasmon resonance analysis of the interaction between AmyI-1-18 and LPS or lipid A also suggested that AmyI-1-18 directly binds to the lipid A moiety of LPS because the dissociation constant (K-D) of AmyI-1-18 with lipid A is 5.6 x 10(-1)0 M, which is similar to that (4.3 x 10(-10) M) of AmyI-1-18 with LPS. In addition, AmyI-1-18 could block the binding of LPS-binding protein to LPS, although its ability was less than that of polymyxin B. These results suggest that AmyI-1-18 expressing antimicrobial and endotoxin-neutralizing activities is useful as a safe and potent host defense peptide against pathogenic Gram-negative bacteria in many fields of healthcare. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2015.11.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Platelet-rich fibrin (PRF) was developed as an advanced form of platelet-rich plasma to eliminate xenofactors, such as bovine thrombin, and it is mainly used as a source of growth factor for tissue regeneration. Furthermore, although a minor application, PRF in a compressed membrane-like form has also been used as a substitute for commercially available barrier membranes in guided-tissue regeneration (GTR) treatment. However, the PRF membrane is resorbed within 2 weeks or less at implantation sites; therefore, it can barely maintain sufficient space for bone regeneration. In this study, we developed and optimized a heat-compression technique and tested the feasibility of the resulting PRF membrane. Freshly prepared human PRF was first compressed with dry gauze and subsequently with a hot iron. Biodegradability was microscopically examined in vitro by treatment with plasmin at 37 degrees C or in vivo by subcutaneous implantation in nude mice. Compared with the control gauze-compressed PRF, the heat-compressed PRF appeared plasmin-resistant and remained stable for longer than 10 days in vitro. Additionally, in animal implantation studies, the heat-compressed PRF was observed at least for 3 weeks postimplantation in vivo whereas the control PRF was completely resorbed within 2 weeks. Therefore, these findings suggest that the heat-compression technique reduces the rate of biodegradation of the PRF membrane without sacrificing its biocompatibility and that the heat-compressed PRF membrane easily could be prepared at chair-side and applied as a barrier membrane in the GTR treatment. (c) 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 103B: 825-831, 2015.

DOI： 10.1002/jbm.b.33262

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Platelet-rich plasma (PRP) has been widely applied in regenerative therapy due to its high concentration of growth factors. Previous in vitro and in vivo studies have provided evidence supporting the angiogenic activity of PRP. To more directly demonstrate how PRP acts on endothelial cells, we examined the PRP-induced changes in the motility of human umbilical vein endothelial cells by examining the involvement of VEGF. Time-lapse quantitative imaging demonstrated that in the initial phase (approximate to 2 h) of treatment, PRP substantially stimulated cell migration in a wound-healing assay. However, this effect of PRP was not sustained at significant levels beyond the initial phase. The average net distance of cell migration at 10 h was 0.45 +/- 0.16 mm and 0.82 +/- 0.23 mm in control and PRP-stimulated cells, respectively. This effect was also demonstrated with recombinant human VEGF and was significantly attenuated by a neutralizing anti-VEGF antibody. Immunofluorescent examination of paxillin and actin fibers demonstrated that PRP concomitantly up-regulated focal adhesion and cytoskeletal formation. Western blotting analysis of phosphorylated VEGFR2 demonstrated that PRP mainly stimulated the phosphorylation of immature VEGFR2 in a dose- and time-dependent manner, an action that was completely blocked by the neutralizing antibody. Taken together, these data suggest that PRP acts directly on endothelial cells via the activation of VEGFR2 to transiently up-regulate their motility. Thus, the possibility that PRP desensitizes target endothelial cells for a relatively long period of time after short-term activation should be considered when the controlled release system of PRP components is designed. (c) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/cm.21221

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Microfiltration membranes of poly(L-lactic acid) (PLEA) have been prepared by a nonsolvent-induced phase separation method with the aid of surfactants. Surfactants with hydrophilic-lipophilic balance (HLB) values of 14.9-15.6 were found to be useful in reducing the shrinkage in thickness of the PLEA membrane. Among the surfactants examined, Tween 80 was the best for preparing microfiltration membranes. The surfactant allowed instantaneous phase separation and seemed to enhance the diffusion of water in the PLEA solution during structure formation. The membrane had asymmetric finger-like structures and showed low membrane resistance and high bacterial cell retention when the membrane was prepared from a 10 wt% PLEA solution in 1,4-dioxane containing 10 wt.% Tween 80. Bovine serum albumin molecules passed through the membrane suggesting that the membrane functions as a microfiltration membrane. The membrane was stable at 25 degrees C but degradable at 60 degrees C in wet conditions. The membrane can be applied as a compostable microfiltration membrane in food and biochemical industries. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.memsci.2015.01.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Amyl-1-18, an octadecapeptide derived from a-amylase (Amyl-1) of rice (Oryza sativa L. japonica), is a novel cationic a-helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of Amyl-1-18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, Amyl-1-18 exhibited antimicrobial activity against nine. Its 50% growth-inhibitory concentrations (ICs50) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 mu M, respectively. Amyl-1-18 had little or no hemolytic activity even at 500 mu M, and showed negligible cytotoxicity up to 1200 mu M. The degree of 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Amyl-1-18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA-intercalating dye, were 89%, 43%, and 3%, respectively, when Amyl-1-18 was added at a concentration equal to its 43IC(50). Therefore, the antimicrobial activity of Amyl-1-18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane-disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that Amyl-1-18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare. (C) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/bip.22605

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CL(14-25), a dodecapeptide, exhibits antimicrobial activity against Porphyromonas gingivalis with the 50% growth-inhibitory concentration (IC50) value of 145 mu M, and arginine-specific gingipain (Rgp)-inhibitory activity. Kinetic analysis revealed that CL(14-25) is a mixed-type inhibitor, with inhibition constants (K-i and K-i' values) of 1.4 x 10(-6) M and 4.3 x 10(-6) M, respectively. To elucidate the contributions of four cationic amino acid residues at the N- and C-termini of CL(14-25) toward Rgp-inhibitory activity, we investigated the Rgp-inhibitory activities of truncated and alanine-substituted analogs of CL(14-25). Rgp-inhibitory activities significantly decreased by truncated analogs, CL(15-25) and CL(1625), whereas those of CL(14-24) and CL(14-23) were almost as high as that of CL(14-25). Rgp-inhibitory activities of alanine-substituted analogs, CL(R14A) and CL(R14A, R15A) also significantly decreased, whereas those of CL(K25A) and CL(R24A, K25A) were higher than that of CL(14-25). These results suggest that the arginine residue at position 15 substantially contributes to the Rgp-inhibitory activity and that the arginine residue at position 14 plays important roles in exerting Rgp-inhibitory activity. In this study, we demonstrated that CL(K25A) was a potent, dual function, peptide inhibitor candidate, exhibiting Rgp-inhibitory activity with Ki and K-i(l) of 9.6 x 10(-7) M and 1.9 x 10(-6) M, respectively, and antimicrobial activity against P. gingivalis with an IC50 value of 51 mM. (C) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/bip.22525

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An atmospheric-pressure plasma (APP) treatment was recently reported to render titanium (Ti) surfaces more suitable for osteoblastic cell proliferation and osteogenesis. However, the mechanism of action remains to be clearly demonstrated. In this study, we focused on cell adhesion and examined the effects of the APP treatment on the initial responses of human prenatal-derived osteoblastic cells incubated on chemically polished commercially pure Ti (CP-cpTi) plates. In the medium containing 1% fetal bovine serum, the initial cell adhesion and the actin polymerization were evaluated by scanning electron microscopy and fluorescence microscopy. The expression of cell adhesion-related molecules and osteoblast markers at the messenger RNA level was assessed by real-time quantitative polymerase chain reaction. Although the cells on the APP-treated CP-cpTi surface developed fewer cytoskeletal actin fibers, they attached with higher affinity and consequently proliferated more actively (1.46-fold over control at 72 h). However, most of the cell adhesion molecule genes were significantly downregulated (from 40 to 85% of control) in the cells incubated on the APP-treated CP-cpTi surface at 24 h. Similarly, the osteoblast marker genes were significantly downregulated (from 49 to 63% of control) at 72 h. However, the osteoblast marker genes were drastically upregulated (from 197 to 296% of control) in these cells by dexamethasone and beta-glycerophosphate treatment. These findings suggest that the APP treatment improves the ability of the CP-cpTi surface to support osteoblastic proliferation by enhancing the initial cell adhesion and supports osteoblastic differentiation when immature osteoblasts begin the differentiation process. (C) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/jbm.b.33114

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

AmyI-1 is an alpha-amylase from Oryza sativa (rice) and plays a crucial role in degrading starch in various tissues and at various growth stages. This enzyme is a glycoprotein with an N-glycosylated carbohydrate chain, a unique characteristic among plant alpha-amylases. In this study, we report the first crystal structure of AmyI-1 at 2.2-angstrom resolution. The structure consists of a typical (beta/alpha)(8)-barrel, which is well-conserved among most alpha-amylases in the glycoside hydrolase family-13. Structural superimposition indicated small variations in the catalytic domain and carbohydrate-binding sites between AmyI-1 and barley alpha-amylases. By contrast, regions around the N-linked glycosylation sites displayed lower conservation of amino acid residues, including Asn-263, Asn-265, Thr-307, Asn-342, Pro-373, and Ala-374 in AmyI-1, which are not conserved in barley alpha-amylases, suggesting that these residues may contribute to the construction of the structure of glycosylated AmyI-1. These results increase the depths of our understanding of the biological functions of AmyI-1.

DOI： 10.1080/09168451.2014.917261

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Background and Objective: Porphyromonas gingivalis is a major etiological agent in the development and progression of periodontal diseases. In this study, we isolated a cell growth inhibitor against P. gingivalis species from rice protein extract.
Material and Methods: The cell growth inhibitor active against P. gingivalis was purified from polished rice extract using a six-step column chromatography process. Its antimicrobial properties were investigated through microscope analysis, spectrum of activity and general structure.
Results: The inhibitor was identified as AmyI-1, an alpha-amylase, and showed significant cell growth inhibitory activity against P. gingivalis species. Scanning electron microscopy micrograph analysis and bactericidal assay indicated an intriguing possibility that the inhibitor compromises the cell membrane structure of the bacterial cells and leads to cell death. Moreover, alpha-amylases from human saliva and porcine pancreas showed inhibitory activity similar to that of AmyI-1.
Conclusions: This is the first study to report that alpha-amylases cause cell death of periodontal pathogenic bacteria. This finding highlights the potential importance and therapeutic potential of alpha-amylases in treating periodontal diseases.

DOI： 10.1111/jre.12079

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The antimicrobial activity of analogs obtained by substituting arginine and lysine in CL(14-25), a cationic alpha-helical dodecapeptide, with alanine against Porphyromonas gingivalis, a periodontal pathogen, varied significantly depending on the number and position of cationic amino acids. The alanine-substituted analogs had no hemolytic activity, even at a concentration of 1 mM. The antimicrobial activities of CL(K20A) and CL(K20A, K25A) were 3.8-fold and 9.1-fold higher, respectively, than that of CL(14-25). The antimicrobial activity of CL(R15A) was slightly lower than that of CL(14-25), suggesting that arginine at position 15 is not essential but is important for the antimicrobial activity. The experiments in which the alanine-substituted analogs bearing the replacement of arginine at position 24 and/or lysine at position 25 were used showed that arginine at position 24 was crucial for the antimicrobial activity whenever lysine at position 25 was substituted with alanine. Helical wheel projections of the alanine-substituted analogs indicate that the hydrophobicity in the vicinity of leucine at position 16 and alanines at positions 18 and/or 21 increased by substituting lysine at positions 20 and 25 with alanine, respectively. The degrees of diSC(3)-5 release from P. gingivalis cells and disruption of GUVs induced by the alanine-substituted analogs with different positive charges were not closely related to their antimicrobial activities. The enhanced antimicrobial activities of the alanine-substituted analogs appear to be mainly attributable to the changes in properties such as hydrophobicity and amphipathic propensity due to alanine substitution and not to their extents of positive charge (cationicity). (C) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/bip.22399

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

CL(14-25), a dodecapeptide, that is a partial region near N-terminus of cyanate lyase (CL, EC 4.3.99.1) from rice (Oryza sativa L. japonica), contains three arginine and two lysine residues. It was a novel cationic alpha-helical antimicrobial peptide. The antimicrobial activity of CL(14-25) against Porphyromonas gingivalis, a periodontal pathogen, was quantitatively evaluated by a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentration of CL(14-25) against P. gingivalis cells was 145 mu M. CL(14-25), even at a concentration of 800 mu M, had no hemolytic activity. When giant unilamellar vesicles (GUVs) that mimic the membrane composition of Gram-negative bacteria were used, microscopy image analysis suggested that CL(14-25) disrupted GUVs in a detergent-like manner. Therefore, CL(14-25) appears to exhibit antimicrobial activity through membrane disruption. To investigate the contribution of cationic amino acid residues in CL(14-25) to its antimicrobial activity, we synthesized four truncated CL analogs, in which one or two cationic amino acid residues were deleted from the N- and C- termini of CL(14-25). The degrees of calcein leakage from large unilamellar vesicles (LUVs) and 3,3'-dipropylthiadicarbocyanine iodide (diSC(3)-5) release from P. gingivalis cells induced by truncated CL analogs were closely related to their antimicrobial activities. CL analogs, which were truncated by removing an arginine residue from the N-terminus and a lysine residue from the C-terminus maintained their antimicrobial activity. However, CL analogs, which were further truncated by removing two arginine residues from the N-terminus, and an arginine and a lysine residue from the C-terminus, rarely exhibited antimicrobial activity. (c) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2012.12.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Flexible microporous scaffold membranes of poly(L-lactic acid) (PLLA)/poly(epsilon-caprolactone) (PCL) blend were developed for bone regeneration. The new membranes overcome the fragility problems of PLLA membranes. When the PCL content in the blend was increased to 50%, the mechanical property for elongation was significantly improved without sacrificing the pores on the membrane surface that help tissue segments adhere the membrane. However, further increases in PCL content reduced the surface pores. In addition, spontaneous cell invasion and formation of cell-multilayers were observed in the membrane of PLLA/PCL blend (50:50). These results indicate that the porous membrane of PLLA/PCL blend (50:50) is useful for the preparation of periosteal sheets in tissue engineering. (C) 2012 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.matlet.2012.12.076

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Using a co-cultivation system developed previously, positive interaction for cell growth between Bifidobacterium adolescent-is and Propionibacterium freudenreichii was evaluated. The total dry cell weight (DCW) of these two strains obtained in the co-cultivation system was 1.5-1.7-fold of the sum of the DCWs obtained in two single cultivations of each bacterium. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2012.09.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Inc.  

Hsp70(241-258), an octadecapeptide derived from the heat shock protein 70 (Hsp70) of rice (Oryza sativa L. japonica), is a novel cationic β-helical antimicrobial peptide (AMP) that contains four lysine, two arginine, and two histidine residues. The antimicrobial activity of Hsp70(241-258) against Porphyromonas gingivalis, a periodontal pathogen, and Candida albicans, an opportunistic fungal pathogen, was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. The 50% growth-inhibitory concentrations of Hsp70(241-258) against P. gingivalis and C. albicans cells were 63μM and 70μM, respectively. Hsp70(241-258) had little or no hemolytic activity even at 1 mM, and showed negligible cytotoxicity up to 300M. The degrees of calcein leakage from large unilamellar vesicles, which mimic the membranes of Gram-negative bacteria, and 3,3 β- dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of Hsp70(241-258) increased in a concentrationdependent manner. When Hsp70(241-258) was added to calcein-acetoxymethyl ester-loaded C. albicans cells, calcein release from the cells increased in a concentration-dependent manner. Flow cytometric analysis also showed that the percentages of C. albicans cells stained with propidium iodide, a DNAintercalating dye, increased as the concentration of Hsp70(241-258) added was increased. Therefore, Hsp70(241-258) appears to exhibit antimicrobial activity against P. gingivalis and C. albicans through membrane disruption. These results suggest that Hsp70(241-258) could be useful as a safe and potent AMP against P. gingivalis and C. albicans in many fields of health care, especially in the control of oral infections. © 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2013.08.011

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Positive interactions between Bifidobacterium longum or Bifidobacterium breve and Propionibacterium freudenreichii that enhance cell growth were evaluated using a co-cultivation system. The total dry cell weight of B. longum and P. freudenreichii from co-cultivation was 10.6 g. This value is 1.2-fold higher than the sum (9.1 g) of the maximum dry cell weights obtained from the cultivation of each bacterium. Currently, the total dry cell weight of co-cultivated B. breve and P. freudenreichii was 21.3 g. The total dry cell weight of B. breve and P. freudenreichii is 1.6-fold higher than the sum (13.2 g) of the maximum dry cell weights from two single cultivations of each bacterium. These data are direct evidence for a cooperative interaction between the two microorganisms. Copyright © 2013 The Society of Chemical Engineers, Japan.

DOI： 10.1252/jcej.12we231

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Depth filter microfiltration membranes produced from biodegradable poly(l-lactic acid) (PLLA) are reported in this paper. The asymmetric porous membranes reported here were formed via a process that combined nonsolvent-induced and thermally induced phase separation. The membranes served as efficient and rapid depth filters to retain bacterial cells when cell suspensions were filtered from the porous or rough side of the membrane, while serving as screen filters when the suspension was fed from the smooth skin side. After use, the depth filters were disposed of by non-enzymatic degradation - a process that was significantly accelerated in wet conditions at 60 degrees C, conditions under which composting is usually operated. The fact that the PLLA depth filters reported here can be disposed of by composting will help reduce industrial wastes in biochemical and food industries when they are used as a pre-filter. (C) 2012 Elsevier B. V. All rights reserved.

DOI： 10.1016/j.memsci.2012.01.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SAGE PUBLICATIONS LTD  

In this in vitro study, novel porous poly(L-lactic acid) membranes were developed to improve periosteal sheets by promoting initial adhesion of periosteal tissue segments and stimulating the formation of a viable multilayered cellular sheet. The biocompatibility, biodegradability, and osteogenicity were evaluated using human periosteal tissue segments cultured on porous poly(L-lactic acid) membranes; the periosteal sheets were osteogen induced and were then implanted in the dorsal subcutaneous tissue of nude mice. In vivo, the membrane degraded into clusters of membrane particles separated by wide cracks; fibroblastic cells invaded along with small blood vessels from the surrounding mouse connective tissue. In osteoinduced periosteal sheets, the membrane clusters were surrounded by numerous capillaries and a number of tartrate-resistant acid phosphatase-positive, multinucleated cells. Neither severe inflammation nor fibrous encapsulation was observed throughout the implantation (similar to 12 weeks). These porous poly(L-lactic acid) membranes were highly biocompatible and functioned well as biodegradable scaffolds that could enhance the use of osteogenic periosteal sheets in therapy.

DOI： 10.1177/0883911512438306

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC CHEMICAL ENG JAPAN  

Filtration is an important industrial production processes to treat particle dispersion systems. In this paper we show the filtration characteristics of suspensions of agglomerated streptobacteria (chain bacteria), Bacillus amyloliquefaciens, by asymmetric depth filter membranes of poly(L-lactic acid) (PLLA). The permeation flux was higher in the filtration with the depth filter membrane than in that with a screen filter membrane at a transmembrane pressure of 50-150 kPa. The bacterial cell layer had a high compressibility index of the specific resistance (0.60) in the filtration with the screen filter membrane. However, the cell layer formed in the internal structure of the depth filter membrane of PLLA showed a low apparent compressibility index (0.11). Scanning electron micrographs revealed that the agglomerated cells were captured on the depth filter membrane at 10 kPa while those cells were captured within the membrane at 150 kPa. Thus the filtration performance with the depth filter membrane increased at higher transmembrane pressures. The biodegradable PLLA membrane showed high performance in the filtration of bacterial cell suspensions and has an advantage in disposal after use because it can be compostable with bacterial cells clogged in the membrane.

DOI： 10.1252/jcej.12we044

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Human cultured periosteal sheets, which are developed from small excised periosteum tissue segments (PTSs) in culture dishes by simple expansion culture, have been applied as a promising autologous osteogenic grafting material for periodontal regenerative therapy. However, the weak initial adhesion of PTSs to dish surfaces often hampers cellular outgrowth and limits the number of preparations. To correct this weakness and still avoid the use of animal-derived adhesion biomolecules, we have developed a novel, biodegradable, porous poly(L-lactic acid) (pPLLA) membrane. Freshly excised PTSs bound well to the highly porous pPLLA membrane, possibly due to the presence of semihemispheric 20-30 mu m diameter openings on the upper surface. Global gene expression analysis demonstrated that periosteal sheets cultured on pPLLA membranes upregulated expression of many adhesion molecules. Osteogenic induction stimulated the production of proteoglycans by these cells and concomitantly enhanced their expansion and penetration into the deep pore regions of the membrane in parallel with the progression of in vitro mineralization. These findings suggest that our pPLLA membranes not only facilitate initial adhesion, primarily mediated by adsorbed proteins, but also enhance biological adhesion by inducing endogenous adhesion molecules in periosteal sheet cultures. Therefore, the efficacy of periosteal sheets in therapy should be greatly enhanced by using this new pPLLA membrane. (C) 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 98A: 100-113, 2011.

DOI： 10.1002/jbm.a.33074

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC CHEMICAL ENG JAPAN  

Microfiltration membranes of poly(L-lactic acid) (PLLA), which is a major biodegradable plastic, were prepared from PLLA-1,4-dioxane-water solutions via the thermally induced phase separation. The resulting membranes are used to retain bacterial cells and permeate protein molecules. Only 20% of the bacterial cells in cell suspensions were retained by the membranes prepared from 10-12.5% polymer solution. Increasing the polymer concentration from 12.5 to 15% markedly elevated the bacterial cell retention to 60%. The retention further increased to 90% by elevating the casting temperature from 40 to 50 degrees C. By drying the polymer solution for 2 min before quenching, the cell retention reached 99%. Casting at temperatures of 60 degrees C or higher and drying for periods of 5 min or longer made the membrane uneven and/or increased the membrane resistance. By drying the polymer solution before quenching, the porous structure near the membrane surface was altered so that the membrane retains the bacterial cells. The membrane retained bacterial cells in the manner of a screen filter. The membranes developed in this research will be useful as a pre-filter in biochemical processes to enhance the life time of the final filters and to reduce industrial wastes due to being made of a compostable polymer.

DOI： 10.1252/jcej.11we030

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of sake spoiling hiochi bacteria (Lactobacillus hilgardii, Lactobacillus fructivorans, and Lactobacillus paracasei) in namazake, which is raw (unpasteurized) sake. The bacteriocin concentrations required for decreasing the viable cell concentrations of L hilgardii and L fructivorans below the detection limit (1.0 x 10(2) cells/ml) in 24 h from the initial concentration of 4.0-9.5 x 10(5) cells/ml in the namazake at pH 4.5 and at 4 degrees C, were 18-35 U/ml and 5.6 U/ml for the bacteriocin from C101910 and NBRC 12007, respectively. To decrease the viable cell concentration of L paracasei from the initial concentration of 7.5 x 10(5) cells/ml to below the detection limit (1.0 x 10(2) cells/ml) in 24 h, 350 U/ml bacteriocin from C101910 and 140 U/ml bacteriocin from NBRC 12007 were required. In experiments using McIlvaine buffer (pH 4.5) with 15% ethanol instead of namazake as the medium, the viable cell concentrations of L. hilgardii and L paracasei decreased to less than 1.0 x 10(2) cells/ml, whereas those of L fructivorans decreased to less than 1.0 x 10(3) cells/ml, when bacteriocins were added at the concentrations that had proven effective in namazake. The membrane depolarization assay using a fluorescent probe showed that the presence of ethanol stimulated the collapse of the membrane potential induced by bacteriocins. The ethanol induced collapse of the membrane potential suggests that the application of bacteriocins at the storage stage of namazake is more beneficial than when used in other stages of the sake brewing process. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.11.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：DESALINATION PUBL  

Permeable microporous foams and membranes of biodegradable polyesters are currently used in the area of tissue engineering and drug delivery systems. Their mechanical properties are useful in the medical applications. The foams should mechanically fit to the tissues where the foams were implanted. In this study, we investigated the mechanical properties of the foams of biodegradable polyesters by compression tests. Microporous foams of poly(L-lactic acid), poly (epsilon-caprolactone), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) were prepared by thermally induced phase separation method. The compression tests were performed at 23-24 degrees C with a universal testing machine. The structure of the microporous foams depended on the kinds of polymers, polymer concentrations, and quenching temperatures. The cell size of the foams was smaller when the polymer concentration was higher or the quenching temperature was lower. We analyzed the stress-strain diagrams of the foams in the compression test. The lower the relative density of the foams to the solid materials the lower the elastic limit stress was. The relative Young's modulus and relative elastic limit stress of the foams were approximately proportional to the square of their relative density and less dependent on their cell size. The dependences were similar to those of open-cell foams of polyurethane.

DOI： 10.5004/dwt.2010.1696

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：DESALINATION PUBL  

Membrane filtration is often used in purification of carbon nanotubes and in preparation of their thin films (buckypaper). Filtration characteristics of multiwall carbon nanotubes (MWCNT) suspended in aqueous solution of detergents was investigated in this study. Although the diameter of the MWCNT was estimated at around 50 nm from the scanning electron micrograph, a microfiltration membrane whose nominal pore size was 0.2 mu m retained the carbon nanotubes. The specific resistance of the filter cake of the carbon nanotubes was 7 x 10(13) m kg(-1) at 98 kPa and the compressibility index was 0.12. The carbon nanotubes were entangled in the buckypaper formed on the microfiltration membrane and the internal structure of the buckypaper was similar under the applied pressure in the preparation of 10-98 kPa.

DOI： 10.5004/dwt.2010.1717

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Microporous membranes of poly(1,4-butylene succinate) (PBS), which is a biodegradable biomass plastic, were prepared from PBS-chloroform solutions via nonsolvent and thermally induced phase separation with a coagulation bath of methanol. The permeation resistance of a membrane prepared from a 10% polymer solution at 25 degrees C was more than 10(14) m(-1). The membrane resistance decreased with increasing pre-incubation temperature. Retention of bacterial cells (0.7 phi x 2.5 mu m) decreased with increasing pre-incubation temperature. Increasing polymer concentration increased the permeation resistance and the retention of the cells. Membranes prepared from the 10% PBS solution pre-incubated at 47.5 degrees C show a permeation resistance of 10(11) m(-1) and retention of the bacterial cells of 99%. The PBS membranes will be useful in biosepartion processes as a prefilter that can be disposed by composting after use.

DOI： 10.5004/dwt.2010.1715

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Enzymatic oxidative polymerization of methoxyphenols by a fungal laccase was investigated to synthesize polymethoxyphenols, which would be environmentally benign materials because they are degraded by lignin degrading fungi. An industrially produced thermostable laccase from Trametes sp. Ha1 polymerized o-methoxyphenol and 2,6-dimethoxyphenol in McIlvaine buffer (pH 4.5) both with and without organic solvents while the enzyme needed organic solvents to polymerize phenol, alkylphenols, and m- and p-methoxyphenols. The reaction rates correlated with the energies of the highest occupied molecular orbitals (HOMO) of the phenols. The poly(o-methoxyphenol) synthesized was soluble in N,N-dimethylformamide (DMF) while 80% of the poly(2,6-dimethoxyphenol) was insoluble in DMF. The weight-average molecular weight of the poly(o-methoxyphenol) was 7000-11,000. The reaction rate of polymerization in McIlvaine buffer without organic solvents increased until an o-methoxyphenol concentration of 500 mmol dm(-3) although the Michaelis constant calculated from the oxygen consumption rate was 0.4 mmol dm(-3). The o-methoxyphenol and 2,6-dimethoxyphenol would be suitable for production of environmentally benign phenolic resins by enzymatic oxidative polymerization using laccase without formaldehyde, hydrogen peroxide, or organic solvent. (C) 2009 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.ces.2009.05.041

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Language：English   Publisher：The Society for Biotechnology, Japan  

The effects of steam explosion (1.5MPa, 1min) on the treatment of rice straw with Pleurotus ostreatus were evaluated in terms of the change in composition of the components and the susceptibility to enzymatic hydrolysis. When rice straw was pretreated with a steam explosion prior to biological treatment, the treatment time required for obtaining a 33% net glucose yield was reduced to 36 days from 60 days. The reduction is probably due to loosening of networks of Klason lignin with sugar moieties and partial collapse of the structure during the biological treatment.

DOI： 10.1016/j.jbiosc.2010.04.014

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：SOC CHEMICAL ENG JAPAN  

Filtration characteristics of an asymmetric depth filter (nominal pore size: 0.2 mu m) were investigated by using suspensions of two bacteria, Lactobacillus plantarum and Bacillus amyloliquefaciens. In the filtration of L. plantarum suspensions, the permeation flux was 3-8 times higher with the depth filter than with a screen filter at 10-150 kPa. Apparent specific resistance of the cell layer in the depth filter reduced to about 1/10-1/100 of that of the filter cake formed on the screen filter. Scanning electron microscopy revealed that the trapped microbial cells were dispersed to a depth of 2/3 from the inlet side of the depth filter. In the filtration of B. amyloliquefaciens suspensions, the permeation flux of depth and screen filters was comparable at 10-50 kPa, and filter cakes formed on both filters. However, the permeation flux increased remarkably at 100-150 kPa with the depth filter but scarcely increased with the screen filter. The smallness of increase with the screen filter was due to the high compressibility of the microbial filter cake, while the remarkable increase with the depth filter was due to the penetration of the chain bacterium into the filter. The increase in transmembrane pressure was highly effective in the filtration of agglomerated chain bacteria such as B. amyloliquefaciens in trapping cells in the asymmetric depth filter.

DOI： 10.1252/kakoronbunshu.36.494

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To enzymatically hydrolyze cellulose of rice straw to glucose, the effect of pretreatment of rice straw with 15 strains of basidiomycetes is evaluated in terms of the quantitative changes in the components of pretreated rice straw and their susceptibility to enzymatic hydrolysis. Pleurotus ostreatus was found to be one of the most suitable white-rot fungi for biological pretreatment of rice straw. Of 11 strains of P. ostreatus tested, ATCC 66376 was found to have the properties superior to the other strains. Thus, in the pretreatment for 48 d, P. ostreatus ATCC 66376 degraded 39% Klason lignin and retained 79% cellulose in the pretreated rice straw. When the rice straw pretreated with this fungus was hydrolyzed with a commercial cellulase, the net yield of glucose determined on the basis of the weight of cellulose fraction of the untreated rice straw was the highest.

DOI： 10.1252/jcej.09We193

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Language：Japanese   Publisher：公益社団法人 化学工学会  

DOI： 10.11491/scej.2010f.0.1072.0

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DOI： 10.11491/scej.2010f.0.716.0

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DOI： 10.11491/scej.2010f.0.998.0

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Depth filter membranes of conventional synthetic polymers have problems at disposal after their uses although they can avoid the formation of filter cakes which cause a large increase in membrane resistance. In this study we developed microfiltration membranes of poly(L-lactic acid), one of the biodegradable biomass plastics. The asymmetric porous structures of the membranes were formed via thermally and nonsolvent-induced phase separation process. The membranes retained bacterial cells in a manner of depth filters. Depth filter membranes of biodegradable biomass plastics can be degraded in composting machines to reduce industrial wastes in biochemical and food industries.

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DOI： 10.1016/j.jbiosc.2009.09.024

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DOI： 10.11301/jsfe.10.45

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Depth filters trap particles internally, and thus the compaction of the particle layer is partially repressed by the internal structure of the filter to yield a high permeation flux at a low transmembrane pressure. We filtered suspensions of three kinds of polystyrene latex particles (incompressible, spherical, 0.30, 0.54, and 0.70 mu m in diameter) with an asymmetric depth filter, SE20 (nominal pore size=0.20 mu m), at different transmembrane pressures and concentrations. The filtration resistance per unit weight of trapped particles with the depth filter was half of that with a screen filter in the filtration of latex particles. The particles were trapped first near the outlet side of the filter and then near the inlet side, unlike the standard blocking model for the membranes with cylindrical pores and the deep bed filtration model for sand filtration. A filtration model was proposed for the filtration of latex suspension with the depth filter. The model suggested that the asymmetric depth filter showed high performance because the filaments in the filter increased the porosity of the particle layer in contact with them.

DOI： 10.1252/kakoronbunshu.35.81

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Natural and synthetic estrogens from sewage treatment systems are suspected to influence the reproductive health of the animals in the rivers. In this article, we investigated the enzymatic treatment of three estrogens (estrone, 17 beta-estradiol, and 17 alpha-ethynyletstradiol) by a fungal laccase which oxidize phenolic compounds with dissolved oxygen. The elimination of the estrogenic activities by enzymatic oxidation was demonstrated by medaka vitellogenin assay. In addition, we developed an enzymatic treatment system comprised of beta-D-glucuronidase and the laccase for 17 beta-estradiol 3-(beta-D-glucuronide) degradation. The two enzymes eliminated 17 beta-estradiol 3-(beta-D-glucuronide) and the intermediate, 17 beta-estradiol, efficiently.

DOI： 10.1016/S1001-0742(08)62332-3

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Microporous foams of polymer blends of two biodegradable polyesters, poly(L-lactic acid) (PLLA) and poly(E-caprolactone) (PCL), were prepared via thermally induced phase separation method. The phase behaviors of the solutions of the polymer blends in 1,4-dioxane containing water were similar, which would be due to the similar Solubility parameters of the two polymers (20.5-21.1 and 21.2 MPa(0.5)),). However, the porous structure of the polymer blends dependent oil the blend ratio under the same conditions other than the ratio. Hepatic cells (HepG2 cells) were Cultured on the porous polymer blends. The cells invaded into the scaffold of PCL and PLLA-PCL-blend (1:4) by 1.0 and 1.5 mm, respectively, while they grew near the Surface of the PLLA foams. Blending of biodegradable polyesters has the potential of controlling the porous structure of biodegradable foams.

DOI： 10.1016/j.desal.2007.09.084

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Enhanced production of bacteriocin by Staphylococcus sp. NPSI 38 (NPSI 38) using koji extract medium was investigated. The bacteriocin produced by NPSI 38 in MRS medium was found to be effective for inhibiting the growth of Lactobacillus hilgardii NBRC 15886T, one of the representative hiochi-bacteria. In cultivations using test tubes without pH control, meat extract and Polypepton were effective for bacteriocin production by NPSI 38 with 10%(v/v) koji extract medium. When the koji extract medium supplemented with hydrolyzate of a rice protein preparation was used instead of meat extract and Polypepton, NPSI 38 produced 160 U/ml of bacteriocin in the cultivation with pH control, which was almost as high as that (156 U/ml) observed in cultivation using MRS medium with pH control. When the cells of L. hilgardii NBRC 15886 T collected at logarithmic growth and stationary phases were inoculated into fresh modified MRS medium with 11 U/ml of the bacteriocin, negligible cell growth was observed, irrespective of different growth phases of cells inoculated. Little or no increase in cell concentrations in the media containing bacteriocin showed that the action of the bacteriocin produced by NPSI 38 was bacteriostatic against the hiochi-bacterium.

DOI： 10.3136/fstr.14.239

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Enhanced production of bacteriocin by Staphylococcus sp. NPSI 38 (NPSI 38) using koji extract medium was investigated. The bacteriocin produced by NPSI 38 in MRS medium was found to be effective for inhibiting the growth of Lactobacillus hilgardii NBRC 15886(T), one of the representative hiochi-bacteria. In cultivations using test tubes without pH control, meat extract and Polypepton were effective for bacteriocin production by NPSI 38 with 10%(v/v) koji extract medium. When the koji extract medium supplemented with hydrolyzate of a rice protein preparation was used instead of meat extract and Polypepton, NPSI 38 produced 160 U/ml of bacteriocin in the cultivation with pH control, which was almost as high as that (156 U/ml) observed in cultivation using MRS medium with pH control. When the cells of L. hilgardii NBRC 15886 T collected at logarithmic growth and stationary phases were inoculated into fresh modified MRS medium with 11 U/ml of the bacteriocin, negligible cell growth was observed, irrespective of different growth phases of cells inoculated. Little or no increase in cell concentrations in the media containing bacteriocin showed that the action of the bacteriocin produced by NPSI 38 was bacteriostatic against the hiochi-bacterium.

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Production of a bifidogenic growth stimulator (BGS) by Propionibacterium freudenreichii subsp. shermanii (Propionibaeterium shermanii) using lactic acid as a carbon source was investigated using different cultivation methods. When a continuous bioreactor system with a filtration device was used at a dilution rate of 0.075 h(-1), the average BGS concentration was 2.4 mg/l, which corresponds to a BGS productivity per cultivation time of 1.8x10(-1)mg(.)l(-1.)h(-1). The BGS productivity per cultivation time in continuous cultivation with filtration was 1.9-fold that (9.4x10(-2) mg(.)l(-1.)h(-1)) in a conventional batch cultivation. In fed-batch cultivation with feed-back control using an online lactic acid controller with a lactic acid biosensor, it was possible to prevent substrate inhibition by maintaining the lactic acid concentration in culture broth low at 3.3 g/l, and an enhanced BGS production (31 mg/l) was successfully attained. The BGS productivity per cultivation time (2.1x 10(-1) mg (.)l(-1.)h(-1)) in the fed-batch cultivation with feed-back control was 2.2-fold that in the conventional batch cultivation. A new bioreactor system was developed by coupling a continuous bioreactor system with a filtration device to an on-line lactic acid controller. Using the new bioreactor system, we produced BGS continuously at a high level of 47 mg/l. The BGS productivities per cultivation time (3.5 mg(.)l(-1.)h(-1)) and the total volume of medium used (1.7x 10(-1) mg(.)l(-1.)h(-1)) obtained in the new bioreactor system were 37-fold and 2.1-fold those in the conventional batch cultivation, respectively. These results described above clearly demonstrate the positive effects of both the continuous filtration for removal of metabolites (propionic and acetic acids) inhibitory to cell growth and feed-back control of lactic acid concentration in the culture broth on BGS production by P shermanii. This paper is the first report on BGS production by the propionic acid bacterium using lactic acid as a carbon source.

DOI： 10.1263/jbb.105.184

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Production of a bifidogenic growth stimulator (BGS) by propionibacterial strains was investigated using lactic acid as a carbon source. The maximum concentration (1.9 mg/L) of BGS was obtained in the mono-cultivation of Propionibacterium shermanii using 20 g/L of lactic acid. The co-cultivation, in which lactose was gradually converted only to lactic acid by a homo-fermentative lactic acid bacterium and subsequently the lactic acid was used as a carbon source for BGS production by P. shermanii, resulted in the successful BGS production. The BGS concentration obtained in the co-cultivations P. shermanii and Lactobacillus casei or P. shermanii and Lactobacillus bulgaricus seas 1.8 or 6.4 mg/L, respectively.

DOI： 10.1252/jcej.07we309

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Production of a bifidogenic growth stimulator (BGS) by propionic acid bacteria was investigated under anaerobic and aerobic culture conditions. To measure the concentration of extracellular BGS produced by propionic acid bacteria, we evaluated the effects of bioassay conditions using Bifidobacterium longum as a test microorganism on the formation of a growth-stimulation zone. The diameter of the growth-stimulation zone was significantly affected by both the component concentrations and the pH of a bioassay medium. The optimum component concentrations and pH of a bioassay medium were one-half of the normal values and 8.5, respectively. Using the bioassay method, we can measure the concentration of BGS produced by propionic acid bacteria ranging in concentrations from 0.1 mu g/l to 1 mg/l using 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as standards. Of six dairy propionic acid bacterial strains tested, the four strains (Propionibacterium freudenreichii ET-3, P. shermanii PZ-3, P. acidipropionici JCM 6432, and P. jensenii JCM 6433) produced BGS at a concentration range of 4-23 mg/l under the anaerobic culture conditions. Analysis of high performance liquid chromatography (HPLC) showed that more than 70% of total BGS produced in supernatant samples was DHNA and no ACNQ was produced by the strains. The effect of oxygen supply on BGS production was investigated for the four BGS-producing strains. The aerobic conditions exerted in positive effects on BGS production by only P. acidipropionici JCM 6432. The concentration of BGS obtained in the aerobic cultivation of P. acidipropionici JCM 6432 was 1.3-fold than that in anaerobic cultivation. Different properties (BGS production as well as cell growth and glucose metabolism) occurring in response to the aerobic conditions were observed, depending on the propionic acid bacterial strain used. This paper is the first report on BGS production by propionibacterial strains except for P. freudenreichii.

DOI： 10.1263/jbb.103.464

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Membrane-surface liquid culture (MSLC) is a promising method for the bioproduction of highly aerobic filamentous fungi [A. Ogawa, A. Yasuhara, T. Tanaka, T. Sakiyama, K. Nakanishi, Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704, J. Ferment. Bioeng. 80 (1995) 35-40]. This paper reports on the production of laccase by Trametes versicolor on a microporous membrane of poly(L-lactic acid) (PLLA), which can be biodegraded via composting after use. The membrane was stable as a support for 24 days at 30 degrees C. During the first 9 days in MSLC, the fungus produced half as much laccase as it did in liquid-surface culture (LSC); however, the mycelium on the membrane was able to be re-used five times for laccase production. The laccase production was accelerated in the repeated use of the culture while the mycelium in LSC ceased to produce the enzyme. This study shows that compostable PLLA microporous membranes can be used for enzyme production by MSLC of filamentous fungi. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2006.10.002

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DOI： 10.11491/scej.2007f.0.535.0

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DOI： 10.11491/scej.2007f.0.812.0

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Biodegradable filtration membranes can be applied in food and biochemical processes to clarify products or to yield particles from suspensions, and then degrade in composting processes. In this study we developed microfiltration membranes from a polymer blend of poly(F-caprolactone) (PCL) and poly(L-lactic acid) (PLLA). The membranes were formed via the thermally induced phase separation process and were used to separate yeast cells from their suspension. The blend ratio (PCL:PLLA) of 4:1 was important to prepare the biodegradable polymer-blend membrane with the ability of the retention of yeast cells and without exfoliation of the membrane. The permeation resistance of PCL-PLLA blend (4: 1) membranes was as high as that of PCL membranes and was three-order lower than that of a PLLA membrane. In the filtration of yeast cell suspensions the increase of the permeation resistance during the filtration was much lower with a PCL-PLLA (4: 1) membrane than with a PLLA membrane. The PCL-PLLA (4: 1) membrane captured the cells as a depth filter while the PLLA membrane did as a screen filter.

DOI： 10.1016/j.desal.2005.06.068

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Microfiltration membranes that can be composted after service were developed from biodegradable polyesters, poly(L-lactic acid) (PLLA) and poly(epsilon-caprolactone) (PCL). The membranes were formed via the thermally induced phase separation method. A 10 wt% PLLA solution in a mixed diluent of 1,4-dioxane and water (87:13 by weight) was prepared in a flat mold and quenched from 52 degrees C (4 degrees C above cloud point temperature) to 0 degrees C. After diluent extraction, the membrane separated yeast cells (6 mu m) from their suspensions. A PCL membrane formed by the same method did not reject yeast cells. PCL membranes formed by quenching a 16 wt% PCL solution to 0 degrees C and quenching a 10 wt% PCL solution to -196 degrees C did separate yeast cells from their suspensions. The permeation flux was much higher in the filtration of 1 kg.m(-3) yeast cell suspension with the PLLA and PCL membranes formed by quenching a 10 wt% PLLA or PCL solution to -196 degrees C than in the filtration with the PLLA membrane formed by quenching a 10 wt% PLLA solution to 0 degrees C. The higher flux would be due to the lower resistance of the membranes formed by liquid nitrogen quenching (-196 degrees C) and the mode of depth filtration. Porous biodegradable microfiltration membranes prepared from these polymers have the potential to serve as disposable filters in food and biochemical industries.

DOI： 10.1252/jcej.39.144

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Production of D-lactic acid from rice bran, one of the most abundant agricultural by-products in Japan, is studied. Lactobacillus delbrueckii subsp. delbrueckii IFO 3202 and defatted rice bran powder after squeezing rice oil were used for the production. Since the rice bran contains polysaccharides as starch and cellulose, we coupled saccharification with amylase and cellulase to lactic acid fermentation. The indigenous bacteria in the rice bran produced racemic lactic acid in the saccharification at pH 6.0-6.8. Thus the pH was controlled at 5.0 to suppress the growth of the indigenous bacteria. L. delbrueekii IFO 3202 produced 28 kg m(-3) lactic acid from 100 kg m(-3) rice bran after 36 h at 37 degrees C. The yield based on the amount of sugars soluble after 36-h hydrolysis of the bran by amylase and cellulase (36 kg m(-3) from 100 kg m(-3) of the bran) was 78%. The optical purity of produced D-lactic acid was 95% e.e. (c) 2005 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.biortech.2005.02.025

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DOI： 10.11491/scej.2006f.0.402.0

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DOI： 10.11491/scej.2006f.0.401.0

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On the basis of growth rate at low PH, yield of lactic acid from glucose, and optical purity of lactic acid produced, we selected lactic acid bacteria favorable for production of optically pure L-lactic acid from defatted rice bran without sterilization. Of 21 strains tested, strains Nos.13 and 16 produced 27-29 kg m(-3) of lactic acid with high optical purity from 100 kg m(-3) Of unsterilized defatted rice bran in simultaneous saccharification and fermentation (SSF) with MRS medium at PH 4.5, a level at which the growth of indigenous lactic acid bacteria in defatted rice bran was suppressed. In a SSF process using strain No.16 in which McIlvaine buffer (PH 4.5) was used instead of MRS medium, no growth of indigenous lactic acid bacteria was observed in defatted rice bran, and 28kg m(-3) of lactic acid with 92% L-type content was produced from 100 kg m(-3) of unsterilized defatted rice bran. In SSF using McIlvaine buffer (PH 4.5), the protein fraction of defatted rice bran was found to play a significant role as a nitrogen source for the growth of lactic acid bacteria. By increasing the initial cell concentration to OD660=1.0 for SSF using strain No. 16 and McIlvaine buffer (PH 4.5), the proportion of L-lactic acid produced was enhanced to 95%.

DOI： 10.3136/fstr.11.400

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The effects of biological pretreatment of rice straw using four white-rot fungi (Phanerochaete chrysosporium, Trametes versicolor, Ceriporiopsis subvermispora, and Pleurotus ostreatus) were evaluated on the basis of quantitative and structural changes in the components of the pretreated rice straw as well as susceptibility to enzymatic hydrolysis. Of these white-rot fungi, P ostreatus selectively degraded the lignin fraction of rice straw rather than the holocellulose component. When rice straw (water content of 60%) was pretreated with P ostreatus for 60 d, the total weight loss and the degree of Klason lignin degraded were 25% and 41%, respectively. After the pretreatment, the residual amounts of cellulose and hemicellulose were 83% and 52% of those in untreated rice straw, respectively. By enzymatic hydrolysis with a commercial cellulase preparation for 48 111, 52% holocellulose and 44% cellulose in the pretreated rice straw were solubilized. The net sugar yields based on the amounts of holocellulose and cellulose of untreated rice straw were 33% for total soluble sugar from holocellulose and 32% for glucose from cellulose. The SEM observations showed that the increase in susceptibility of rice straw to enzymatic hydrolysis by pretreatment with P ostreatus is caused by partial degradation of the lignin seal. When the content of Klason lignin was less than 15% of the total weight of the pretreated straw, enhanced degrees of enzymatic solubilization of holocellulose and cellulose fractions were observed as the content of Klason lignin decreased.

DOI： 10.1263/jbb.100.637

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DOI： 10.11491/scej.2005.0.408.0

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The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation ( in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.

DOI： 10.1007/s00253-004-1671-x

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Microfiltration membranes of poly(L-lactic acid) (PLLA) were prepared from PLLA-1,4-dioxane-water solutions via the thermally induced phase separation process. Ternary phase diagrams for this system, determined at 10, 48, and 80 C, indicate that essentially polymer-free droplets form in a matrix phase of PLLA-1,4-dioxane-water solution. Rapid solidification of the PLLA after the initial liquid-liquid phase separation was necessary to obtain membranes with open pores at the membrane surface and low permeation resistance. Using filtration of cell suspensions, the effective pore size of the best membrane formed in this study was found to be between 0.6 and 4.4 mum. (C) 2004 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.memsci.2004.03.020

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Co-culture systems containing two microorganisms for the production of useful substances are described. We developed a novel co-culture system composed of two fermentors and two microfiltration modules. The proposed co-culture system allowed regulation of the dissolved oxygen concentration at a level suitable for an individual microorganism in each fermentor, as well as the successful exchange of culture medium between two fermentors. By co-culture, using a combination of Pichia stipitis and Saccharomyces cerevisiae, ethanol was produced efficiently from a mixture of glucose and xylose. Moreover, the useful probiotic cells were simultaneously produced with a high productivity by our co-culture using a combination of Bifidobacterium and Propionibacterium. Kefiran production by Lactobacillus kefiranofaciens alone under the culture conditions, established by mimicking the presence and activities of yeast cells in kefir grains, was also investigated. The results obtained showed that under the culture conditions established by mimicking the actions of yeast cells on L. kefiranofaciens in kefir grains, the amount of kefiran produced was enhanced, even when the lactic acid bacterium alone was used.

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The effects of operation conditions on reaction rate in the enzymatic oxidative treatment of nonylphenol with laccase in a rotating reactor were studied. Sea sand that adsorbed nonylphenol was used as a polluted soil model. Nonylphenol concentration decreased with laccase treatment five times faster at 40degreesC than at 10degreesC and the apparent activation energy of the enzyme reaction was 39 kJ mol(-1), which was in the range of the values reported for similar laccase reactions. Reaction rate increased when the angle of the axis of the rotating reactor from the vertical line increased and when the speed of revolution was increased to 10 rpm at different volumes of the enzyme solution. Thus, mixing is important for the oxidation of nonylphenol with laccase. The nonylphenol released from the sand into the enzyme solution in the initial stage of the treatment was easily oxidized. However, the nonylphenol adsorbed on the sand reacted slowly. Reaction rate increased nearly proportional to the square root of enzyme concentration, which suggests that the nonylphenol radical reacted with unoxidized nonylphenol (nonenzymatic propagation) during the enzymatic oxidative treatment. The dependence of reaction rate on the nonylphenol concentration was similar to the Michaelis-Menten type. The residual estrogenic activity of the treated sand was measured by medaka vitellogenin assay. The estrogenic activity decreased to 1/6-1/90 after 24 h of the treatment.

DOI： 10.1016/S1389-1723(04)70147-4

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The kinetics of enzymatic oxidative polymerization of 4-chloroguaiacol by laccase was studied. 4-Chloroguaiacol, a model compound of the chloroguaiacols formed in the bleaching process in the pulp industry, was oxidized by laccase from Trametes sp. Ha1 to eliminate chloroguaiacols from pulp wastewater by polymerization and precipitation. On the basis of the reaction rate obtained from the decrease in the 4-chloroguaiacol concentration, the optimum pH of the reaction was 4-5 and the apparent Michaelis constant K. for the reaction was more than 5 mmol dm(-3). However, the value of K. obtained from the dependence of oxygen consumption rate on the concentration of 4-chloroguaiacol was 0.092 mmol dm(-3). The reaction rate was expressed as the sum of the enzymatic oxidation rate of 4-chloroguaiacol and the polymerization rate of the radical formed in the enzymatic oxidation. The kinetics of the enzymatic oxidative polymerization shown in this study will be useful to understand those of the enzymatic oxidation of other phenolic compounds.

DOI： 10.1252/jcej.36.1101

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The structural change of a filter cake and its effect on permeation flux in crossflow filtration of a suspension containing particles different in size were studied. A suspension containing yeast cells (5 mum in average diameter) and polystyrene latex particles (0.5 mum) with different compositions was used as a model. The change in the structure of the filter cake formed on the membrane surface was observed by using a scanning electron microscope. When the pore size was smaller than the latex particle size, a yeast cell layer containing latex particles was formed on the membrane at the initial stage of filtration. Then, the yeast cell layer was replaced by the latex particle layer. The increase of the circulation flow rate decreased the amount of the filter cake to increase the permeation flux in this case. On the other hand, when the membrane pore size was larger than the latex particle size, a yeast cell layer was first formed on the membrane surface followed by deposition of latex particles on it. The permeation flux was relatively higher than the former case. However, the permeation flux slightly increased with the increase of the circulation flow rate although the weight of the filter cake decreased in the latter case. Thus, a small amount of the latex particles in the cell suspension considerably decreased the permeation flux in crossflow filtration of the suspension. In addition the dependence of the permeation flux on the circulation flow rate was proved to be different whether the small particles were smaller than the membrane pores. These findings including the structure of the filter cakes will be helpful to understand the permeation behaviors in the crossflow filtration of microbial broths which usually contain small particles.

DOI： 10.1252/jcej.34.1524

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DOI： 10.1263/jbb.92.312

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Kefiran production by lactobacillus kefiranofaciens alone under the culture conditions established by mimicking the presence and activities of yeast cells in kefir grains was investigated. When the pH of the culture broth was controlled at pH 5.5 by adding a 4 N-NaOH solution during cultivation, cell growth and kefiran production were stimulated as compared with those in the cultivation without pH control. The addition of 5 g/l of yeast extract to the medium was essential for kefiran production. By sparging a mixed gas of N2 and CO2 at a volume ratio of 9:1 into the culture broth at 0.3 vvm throughout the cultivation, a slight increase in the amount (670 mg/l) of kefiran produced was observed as compared with that (650 mg/l) in the cultivation without aeration. When 10 g/l or 20 g/l of ethanol was added to the medium containing 50 g/l of lactose, the kefiran concentrations were about 930 mg/l or 840 mg/l at 8 days, respectively. The maximum concentration of kefiran obtained was about 1040 mg/l at 10 days, when 10 g/l of ethanol was added to the medium containing 75 g/l of lactose. These results showed that under the culture conditions established by mimicking the actions of yeast cells on L. kefiranofaciens in kefir grains the amount of kefiran produced was enhanced even when only the lactic acid bacterium was used.

DOI： 10.3136/fstr.7.333

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Five endocrine disrupting chemicals were degraded enzymatically in model bottom sediments. Since alkylphenols, bisphenol A, synthetic estrogen and phthalic esters are hydrophobic and found to be in bottom sediments in the water environment, we used a modal polluted sediment in which those chemicals were adsorbed on sea sand. The concentration of alkylphenols (nonylphenol and octylphenol) and synthetic estrogen (ethynylestradiol) were decreased by fungal laccases which oxidize phenols with the aid of molecular oxygen. Bisphenol A was degraded by laccase from basidiomycetes with the aid of extracellular polymers. Dibutyl phthalate was hydrolyzed by lipase from Candida cylindracea. These results will be useful in the remediation of the bottom sediments polluted by endocrine disrupting chemicals.

DOI： 10.2166/wst.2000.0556

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The antimicrobial activities of standard solutions of three organic acids (lactic, acetic, and propionic acids) were compared using Micrococcus luteus, Pseudomonas sp. and Staphylococcus aureus as test microorganisms. At the same concentrations of the undissociated form, the antimicrobial activities of acetic and propionic acids were higher than that of lactic acid, irrespective of test microorganisms. In a single cultivation of Bifidobacterium longum, a mixture of lactic (17 g/l) and acetic (20 g/l) acids was produced from 50 g/l lactose and its antimicrobial activities against M. luteus, Pseudomonas sp., and S. aureus correspond to that of 32, 19, and 25 g/l of acetic acid, respectively. To increase the total antimicrobial activity, a co-culture of B. longum and Propionibacterium freudenreichii, in which lactic acid produced once from lactose by B. longum was converted to acetic and propionic acids by P. freudenreichii, was done using TPY medium containing commercially available peptones as a nitrogen source. By the sequential conversion of lactose using the two microorganisms, the culture supernatant containing a mixture of acetic (27 g/l) and propionic (13 g/l) acids without lactic acid was produced. The antimicrobial activities of the mixture against nl. luteus, Pseudomonas sp., and S. aureus were 35, 30, and 26 g/l as a concentration of acetic acid, respectively, higher than that obtained in the cultivation of B. longum alone. When the medium containing an enzymatic hydrolyzate of whey proteins with a protease was used in the co-culture of B. longum and P. freudenreichii, the culture supernatant containing the mixture of organic acids was also obtained in the same manner as the co-culture using TPY medium and the activities were 43, 29, and 29 g/l as a concentration of acetic acid for M. luteus, Pseudomonas sp. and S. aureus, respectively.

DOI： 10.1271/bbb.62.1522

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The behavior of the permeation flux in crossflow filtration of Corynebacterium glutamicum broth was studied. In the beginning of filtration the permeation flux changed in accordance with the cake filtration model. The permeation flux agreed with the value calculated from the weight of the cell layer formed on the membrane per unit filtration area and the specific resistance of the cell layer measured in dead-end filtration. After cell deposition on the membrane, permeation resistance rapidly increased. The increase was due to the formation of the gel layer of the proteins and polysaccharides in the supernatant of the broth. When the circulation flow rate was raised, the weight of the cell layer reduced. However, the beginning of the rapid increase of permeation resistance came earlier and the apparent specific resistance of the cell layer increased. The higher the circulation flow rate, the higher the permeation flux at the beginning of the rapid increase of resistance. When the transmembrane pressure was lowered or the cell concentration was raised, the beginning of the rapid increase of the permeation resistance came earlier. The permeation flux at which the formation of the gel layer began was almost constant, even if the transmembrane pressure and the cell concentration were changed.

DOI： 10.1080/01496399808544784

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Characteristics of crossflow filtration of cell types were compared. Although the cell sizes were similar for the six yeast cells, the specific resistance of the cake measured in dead-end filtration and permeation flux were different. The flux decreased with increasing specific resistance. However, the dependencies of the steady-state flux on the shear stress were similar for all the yeast cells. The steady state permeation flux of the yeast cell suspensions was correlated with the shear stress to the 0.6-0.9th power and with the cell concentration to the -0.3rd power regardless of the transmembrane pressure. The permeation flux in the unsteady state was well simulated by the correlation for the lift velocity of the cells obtained from the steady-state flux.

DOI： 10.1080/01496399708000743

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Filtration behaviors in the unsteady-state phase of crossflow filtration of broths of Bacillus subtilis, Escherichia coli, and Lactobacillus delbrueckii, which are rod-shaped, were studied from the viewpoint of the changes in the specific resistance and in the structure of the microbial cake formed on the membrane surface. The permeation flux followed the cake filtration law at the initial stage of the crossflow filtration of the broths of B. subtilis and E. coli, where the cells deposited randomly on the membrane. filtration of at. delbrueckii broth, the period of random deposition was shorter. cake formed at the initial stage agreed with that measured in dead-end filtration. started to increase in comparison with that measured in dead-end filtration due to shear-induced arrangement of the cells. The extent of the increase in specific resistance became higher and the time taken to start the cell arrangement became shorter with increasing circulation flow rate. The increase in specific resistance due to the shear-induced arrangement was more appreciable in the crossflow filtration of the broth oft. delbrueckii than that of B. subtilis and E. coli. The average permeation flux was increased considerably by applying periodical backwashing with appropriate time intervals. The permeation flux was well predicted by the cake filtration law, since the cells deposited in a way similar to that for dead-end filtration during a sufficiently short period of crossflow filtration in a backwashing mode.

DOI： 10.1252/jcej.29.973

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The periodical stopping of permeation flow was applied to increase the permeation flux in crossflow filtration of commercially available baker's yeast cell suspension. The permeation flux after 3 h filtration in the crossflow filtration increased to 8 x 10(-5) m(3)/m(2) s (290 L/m(2) h) from 2 x 10(-5) m(3)/m(2) s (72 L/m(2) h) by applying the periodical stopping of permeation. Introduction of air bubbles during the stopping period of permeation further increased the flux. (C) 1995 John Wiley and Sons, Inc.

DOI： 10.1002/bit.260470313

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N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester, a precursor of the synthetic sweetener, aspartame, was synthesized from N-(benzyloxycarbonyl])-L-aspartic acid and L-phenylalanine methyl ester with an immobilized thermolysin (EC 3.4.24.4) in the mixed organic solvent system of tert-amyl alcohol and ethyl acetate. A mixed solvent consisting of tert-amyl alcohol and ethyl acetate at a ratio of 33:67 (v/v) was found to be the most suitable with respect to synthetic rate and stability of the immobilized enzyme. The reaction continued to proceed quite successfully in a column reactor at 40 degrees C and at a space velocity of 3.6 h(-1) with a yield of 99%, using 40 mM Z-Asp and 200 mM PheOMe dissolved in the mixed solvent as the substrate. (C) 1995 John Wiley & Sons, Inc.

DOI： 10.1002/bit.260460617

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Membrane-surface liquid culture (MSLC) developed by the authors (Yasuhara er (al., 1994) was applied to the production of kojic acid, using Aspergillus oryzae var. oryzae IFO 30113. By the fed-batch MSLC with intermittent glucose addition, the amount of kojic acid increased to over 50 times that obtained by means of the culture in shake flasks.

DOI： 10.1007/BF00224417

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Membrane-surface liquid culture (MSLC) was applied to the production of kojic acid using Aspergillus oryzae NRRL484. The characteristics of kojic acid fermentation by MSLC were compared particularly with those by shaking culture. The maximum concentration of kojic acid produced and the production rate of kojic acid by the MSLC were usually higher than those by the shaking culture. In the shaking culture, the kojic acid production was the highest with 0.05-0.25% yeast extract and the maximum concentration was around 20 mg/ml. In the MSLC, the highest kojic acid concentration of about 30 mg/ml was obtained with 0.25-0.5 % yeast extract. By addition of powdered glucose at a final concentration of 10%, 2-3 times at appropriate intervals during the batch MSLC, the concentration of kojic acid increased to over 100 mg/ml and kojic acid crystals formed in the medium. Repeated fed-batch production of kojic acid by MSLC was quite successful. The concentration of kojic acid produced in each batch was maintained at 75 mg/ml or more with a yield of around 50% for 10 batches and 75 d when the medium contained 0.25% yeast extract. The kojic acid productivity by the repeated fed-batch MSLC was 14.2 g/l/d, about 6 times higher than that by the shaking culture with the medium containing 0.25% yeast extract. After the 11th batch, the production rate decreased, probably due to an increased amount of cells formed on the membrane.

DOI： 10.1016/0922-338X(95)98174-J

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A membrane-surface liquid culture (MSLC), in which microorganisms were grown on a microporous membrane surface exposed to the air with the other side of the membrane in contact with liquid medium, was applied to the production of neutral protease from Aspergillus oryzae LAM2704. The amount of protease produced by batch MSLC was much higher than that produced by shaking culture, liquid-surface culture, or agar plate culture. In MSLC the amount of protease produced per milligram of dry cells as well as the amount of protease produced were dependent particularly on the glucose concentration. By decreasing the glucose concentration from 1.5% to 0.2%, the amount of protease produced and its specific amount were increased by 60% and around 7 times, respectively. Using a medium containing 0.2% glucose and 0.4% casein, long-term repeated-batch production of the enzyme was conducted by exchanging the medium every 12 to 48 h with a fresh one. Protease production continued in more than 15 successive batches, although the amount of enzyme produced in each batch gradually decreased. When the medium was exchanged every 24 h, the degree of the decrease was the smallest, and the cumulative amount of enzyme produced was more than 20 and 400 times those obtained in the batch MSLC and shaking culture, respectively.

DOI： 10.1016/0922-338X(95)98173-I

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N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the synthetic sweetner asparatame, was synthesized from N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp) and L-phenylalanine methyl ester (PheOMe) with an immobilized thermolysin in various organic solvents. We found that in tert-amyl alcohol containing a small amount of water the immobilized enzyme showed a high activity comparable to that in ethyl acetate with quite a high stability. The immobilized enzyme was fully stable up to 70 degrees C in tert-amyl alcohol in the absence of the substrate, and up to 50 degrees C in the presence of the substrate. The high stability in the presence of the substrate was found due to the fact that the release of calcium ions, the stabilizing factor of thermolysin, is suppressed.
The substrate concentration dependence of th initial synthetic rate with the immobilized enzyme was quite different from that with the free enzyme in the biphasic system, in contrast to that in ethyl acetate. Finally, Z-AspPheOMe was continuously synthesized in a column reactor using 200 mM PheOMe and 120 mM Z-Asp as the substrate for over 300 h at 45 degrees C and a space velocity of 1 h(-1) without any loss of activity. (C) 1994 John Wiley & Sons, Inc.

DOI： 10.1002/bit.260431116

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A broth of yeast cells cultivated in molasses was cross-filtered with a thin-channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 mu m). The steady-state flux was one-twentieth that calculated for a cake filtration model from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead-end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 mu m in diameter) in the molasses. The mechanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form a cake in a similar way to dead-end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.8 mu m, but using larger pores of 3 to 5 mu m it returned almost to the base line. (C) 1994 John Wiley & Sons, Inc.

DOI： 10.1002/bit.260431113

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N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalamine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPheOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AspPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (C) 1994 John Wiley & Sons, Inc.

DOI： 10.1002/bit.260431115

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A novel cultivation method using a microporous membrane (membrane-surface liquid culture) was developed, in which moulds are grown on the membrane surface with its opposite side being in contact with a liquid medium. The amount of neutral protease from Aspergillus oryzae produced was more than 10 times higher than that produced by the conventional liquid culture.

DOI： 10.1007/BF00155416

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The specific resistance of the cake formed in the crossflow filtration of Escherichia coli was higher than that formed in the dead-end filtration. The scanning electronmicrographs revealed that the cells in the cake formed in the crossflow filtration were oriented in the direction of the circulation flow, while the cells deposited at random in the dead-end filtration. The shear-induced arrangement of cells might increase the specific resistance of the cake in crossflow filtration.

DOI： 10.1007/BF00207634

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A suspension of various microorganisms was cross-filtered and the filtration characteristics were studied. In crossflow filtration of Corynebacterium glutamicum, an ellipsoidal-shaped bacterium, the experimental permeation flux was in agreement with the calculated value based on the filtration theory, using the specific resistance measured in dead-end filtration and the amount of cake per unit filtration area. The cells deposited on the membrane in the same manner as in dead;end filtration. On the other hand, in crossflow filtration of Bacillus species, all of which are rod-shaped cells, the cells in the cake formed on the membrane were oriented toward the direction of the circulation flow. This arrangement of the cells increased the specific resistance of the cake, which made the flux lower than the calculated value, using the specific resistance measured in dead-end filtration. Furthermore, we clarified that the degree of the cell arrangement was dependent on the operational conditions in the crossflow filtration of rod-shaped cells.

DOI： 10.1016/0922-338X(94)90046-9

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To improve the performance of cross-flow membrane filtration of pullulan broth from Aureobasidium pullulans, the effect of the cultivation conditions was examined. In particular, the sucrose concentration in the medium was changed over a wide range. By decreasing the sucrose concentration the distribution of morphology of the microbial cells in the broth changed; the yeast-like form became predominant and, as a result, the specific resistance of the microbial cake was lowered. When the broth was fermented with a sucrose concentration of 2.5% or lower, the filtration characteristics were greatly improved by periodic closure of permeation during cross-flow filtration.

DOI： 10.1007/bf00166842

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Cross-flow filtration of culture broth from Aureobasidium pullulans, which elaborates pullulan, was done with a thin channel-type module and microfiltration membranes made of different materials and with different pore sizes. Various factors affecting the results of the filtration were studied. The specific resistance of the microbial cake was found to be higher than that of bakers' yeast, the cells of which are about the same size as an A. pullulans cell, and resistance increased with cultivation time. The flux and transmission of pullulan through the membrane decreased with cultivation time as the specific resistance increased. The flux and transmission of pullulan depended on the structure and pore size of the membrane and also on the pH of the broth. With a polysulphone membrane with a nominal pore size of 2.0 mum, transmission was nearly 100% with negligible leakage of cells and the flux was high when the PH of the broth was adjusted to 2.0.

DOI： 10.1007/bf00166843

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Factors affecting the performance of crossflow filtration were investigated with a thin-channel module and yeast cells. In crossflow filtration of Saccharomyces cerevisiae cells cultivated with YPD medium (yeast extract, polypeptone, and dextrose) and suspended in saline, a steady state was attained within several minutes when the cell concentration was low and the circulation flow rate was high. The steady-state flux and the change in flux during the initial unsteady state were explained well by conventional filtration theory, with the amount of cake deposited and the mean specific resistance to the cake measured in a dead-end filtration apparatus used in calculation. When the circulation flow rate was lower than a critical value, a part of the channel of the cross-flow filtration module was plugged with cell cake, and thus the steady-state flux was low. In crossflow filtration of suspensions of commercially available baker's yeast, the flux gradually decreased, and the flux after 8 h of filtration was lower than the value calculated by filtration theory. Fine particles contaminating the baker's yeast was responsible for the decrease. A similar phenomenon was observed in crossflow filtration of a broth of S. cerevisiae cells cultivated in molasses medium, which also contains fine particles. The YPD medium, which does not contain such particles, had no effect of the permeation flux during crossflow filtration.

DOI： 10.1002/bit.260410604

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DOI： 10.1016/0922-338X(90)90185-Y

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Culture of autologous cell sheets is one of the promising methods in regenerative medicine. Cultured epidermis is used for the coverage of massive burn wounds and cultured periosteum is for the treatment of infrabony periodontal defects. In cell sheet engineering cardiomyocyte sheets prepared with temperature–responsive culture dishes are layered to construct cardiac grafts. Those cultured cell sheets, however, need high techniques in culture and/or surgery. We have been developing several microporous membranes of biodegradable polyesters to handle cultured cell sheets easily. Microporous surfaces of poly(<sub>L</sub>–lactic acid) (PLLA) membrane, which was prepared by thermally induced phase separation method, anchored periosteal tissue segments on the membranes. The cultured periosteal cell sheets on the membranes were successfully osteoinduced in an induction medium. The PLLA membrane implanted with the cell sheet was gradually degraded in a nude mouse without severe inflammation or fibrous encapsulation. Ductility of the membrane with a microporous surface was conferred by blending brittle PLLA with ductile poly(ε–caprolactone) (PCL). Established osteoblast–like (Saos–2) cells grew on a flexible microporous membrane of PCL and hydroxyapatite, the latter of which is a major mineral component of bone and used in bone defect treatment, although the cells grew in a single layer. A finger–like structure of PLLA microporous membrane prepared by nonsolvent–induced phase separation method with a surfactant enhances the cell growth in the membrane possibly by higher diffusion of oxygen, nutrients, and wastes. The biodegradable microporous sheets including those described in the review will complement the existing cultured cell sheet technology.

DOI： 10.5360/membrane.40.124

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DOI： 10.5360/membrane.39.21

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)   Publisher：食品資材研究会  

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生分解性プラスチックの代表であるポリ乳酸は,食品容器やゴミ袋などの日用品から,自動車部品や電子機器の筺体などの耐久消費財の部品にまで幅広く使用されるようになりつつある.ポリ乳酸は,そのモノマーである乳酸が農産副産物などの低利用バイオマスから発酵生産が可能なため,化石資源に依存しないバイオマスプラスチックとしても注目されている.本稿では,ポリ乳酸の幅広い用途開発のための材料の高性能化と我々の取り組んでいる多孔質化・複合化を用いた機能性材料の開発について解説する.

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Language：Japanese   Publisher：THE MEMBRANE SOCIETY OF JAPAN  

Biodegradable plastics, such as poly(L-lactic acid)(PLLA), poly(ε-caprolactone)(PCL)and poly(1,4-butylene suc-cinate)(PBS), degrade in natural environments and composting facilities. We have been developing porous mem-branes of biodegradable plastics to reduce the wastes formed in filtration processes in food and biochemical indus-tries. Microporous membranes of PLLA, PCL and PCL-PLLA-blends were preparedvia thermally induced phase sep-aration. The hydraulic resistance and pore size of the membranes were evaluated by filtration of water and a suspen-sion of yeast cells (6μm), respectively. The PLLA membrane had high permeation resistance (low permeability)although it retained yeast cells on the membrane. The PCL membrane passed the yeast cells although it had highpermeability. Thus we prepared microporous membranes of polymer blend of the two biodegradable plastics to yieldhigh permeability and high cell retention. The resistance of PCL-PLLA-blend (4:1)membrane was as low as that ofthe PCL membrane. The polymer blend membrane retained yeast cells in a manner of depth filtration, which allowedfor fast filtration. Microporous membranes of PBS were prepared from PBS-chloroform solutions via immersion pre-cipitation method with a coagulation bath of methanol. Membranes prepared from 10% PBS solutions pre-incubatedat 47.5℃show a permeation resistance of 1011m–1and a retention of the bacterial cells (0.7φ×2.5μm)of 99%. Themicroporous membranes of biodegradable plastics will be helpful as prefilters to eliminate the waste of filter and filteraids in food and biochemical industries.

DOI： 10.5360/membrane.33.165

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Publisher：Japan Society for Food Engineering  

We investigated the antimicrobial activity of several bacteriocins, which were produced by newly isolated lactic acid bacteria in koji extract medium supplemented with rice protein hydrolyzate (RPH), against strains of NBRC type culture and spoilage bacteria isolated from deteriorated sake. The partial sequences of the 16S rRNA gene of the spoilage bacteria isolated from sake showed a high similarity to those of <I>Lactobacillus fructivorans, Lactobacillus hilgardii</I>, and <I>Lactobacillus paracasei</I>, with 99.6-100% identity. <I>Enterococcus durans</I> C102901 (C102901), <I>Lactococcus lactis</I> subsp. lactis C101910 (C101910), and <I>Lactococcus lactis</I> subsp. <I>lactis</I> NBRC 12007 (NBRC 12007) grew well and produced bacteriocins with high activity in koji extract medium supplemented with RPH. When culture supernatants containing bacteriocins from C101910 and NBRC 12007 were added to the medium at a volume ratio of 10% (v/v), the growth of <I>L. fructivorans</I> NBRC 13954<SUP>T</SUP> was significantly inhibited and the viable cell concentration decreased below the detection limit (1.0&times;10<SUP>2</SUP> cells/ml) at 4 h. Further, by the addition of bacteriocin solutions from C102901, C101910, and NBRC 12007 to the medium at a volume ratio of 1% (v/v), the growth of <I>L. hilgardii</I> NBRC 15886<SUP>T</SUP> and H130 (closely related to <I>L. hilgardii</I>) isolated from putrid sake was bactericidally inhibited and the colony-forming units fell by more than three orders of magnitude within 4-12 h as compared with the initial cell concentration.

DOI： 10.11301/jsfe2000.9.277

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In this review the problems and solutions on the membrane separation of microbial cell suspensions in food and biochemical industries were discussed. The deposition of filter cakes on membranes decreases the permeation flux in the filtration. Currently filter aids such as diatomaceous earth are widely used to decrease the permeation resistance of the filter cakes although the cakes containing filter aids are discharged as industrial wastes. Crossflow filtration is a method to obtain high permeation fluxes by suppressing the formation of filter cakes on the membranes. High permeation fluxes could be obtained in the crossflow filtration of suspensions of spherical cells such as baker&#039;s yeast. However, the authors showed that the permeation flux decreases in the suspensions of rod-shaped cells or microbial suspensions containing fine particles from culture media and biopolymers. It was shown that the permeation fluxes could be recovered by the use of a modified backflushing method by the authors. The development of biodegradable polyester membranes for dead-end filtration to decrease the industrial waste was also discussed.

DOI： 10.11301/jsfe2000.8.221

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Language：English   Publisher：日本食品工学会  

In this review the problems and solutions on the membrane separation of microbial cell suspensions in food and biochemical industries were discussed. The deposition of filter cakes on membranes decreases the permeation flux in the filtration. Currently filter aids such as diatomaceous earth are widely used to decrease the permeation resistance of the filter cakes although the cakes containing filter aids are discharged as industrial wastes. Crossflow filtration is a method to obtain high permeation fluxes by suppressing the formation of filter cakes on the membranes. High permeation fluxes could be obtained in the crossflow filtration of suspensions of spherical cells such as baker's yeast. However, the authors showed that the permeation flux decreases in the suspensions of rodshaped cells or microbial suspensions containing fine particles from culture media and biopolymers. It was shown that the permeation fluxes could be recovered by the use of a modified backflushing method by the authors. The development of biodegradable polyester membranes for dead-end filtration to decrease the industrial waste was also discussed. © 2007, Japan Society for Food Engineering. All rights reserved.

DOI： 10.11301/jsfe2000.8.221

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Cross-linked poly-gamma-glutamic acid (C-L gamma-PGA) at 5 mu g/ml flocculated bentonite suspension pretreated with polyaluminum chloride (PAC) at 2 mu g/ml AI(3+)-PAC to a transparency of approximately 30% after 30 min and more than 90% after 4 h, while Al3+ concentration in the upper phase of the suspension decreased with incubation time. When pretreated with FeCl3 at 16 mu g/ml Fe3+-FeCl3, similar results were obtained. In the case of Escherichia coli suspension, the combination of C-L gamma-PGA and FeCI3 demonstrated a more marked flocculating activity with a satisfactory transparency occurring after 30 min of treatment, accompanied by a decrease in residual Fe3+ concentration. In the above two suspensions pretreated with FeCI3, small visible floats appeared in the early stage of incubation. These floats were found to be due to the direct interaction between FeCl3 and C-L gamma-PGA, indicating the formation of a water-insoluble complex. After allowing the suspension to stand for a long time, elemental analysis and inductively coupled plasma spectroscopy of the precipitates produced suggested that not only the complex was formed due to the interaction between Fe3+ in FeCl3 and COO- in the C-L gamma-PGA molecule, but also Fe2O3 and Fe(OH)(3) might be entrapped in this complex. This could be applied to scavenge metal ions including Fe3+ from polluted water.

DOI： 10.1263/jbb.100.207

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Language：English   Publisher：ELSEVIER SCIENCE BV  

The permeation behavior in crossflow filtration of a model suspension containing yeast cells and polystyrene latex particles was studied. When the membrane pore size was larger than the latex particle size, a yeast cell layer was first formed on the membrane surface and then latex particles were deposited on it. When the pore size was smaller than the latex particle size, a yeast cell layer containing latex particles was formed on the membrane surface at the start of filtration. However, it was then replaced by the latex particle layer.

DOI： 10.1016/S0921-0423(00)80008-8

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