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

Currently, injury of foodborne pathogen in agricultural environment is unclear. So, we observed survival of foodborne pathogens on common procedure for growing vegetable by cultivation on two types of agar medium that possess different selectivity. In case of Salmonella enterica serovar Infantis (SI) on the process of manure preparation, injury was estimated by cultivation on selective agar medium and resuscitation on non-selective agar medium. While in Listeria monocytogenes (LM) on cultivation environment of tomato, physiological changes were confirmed by salt content in agar medium. In manure environment, insufficient elevation of temperature in piled feedstock caused injured SI. LM survived for long term and part of these survived as injured LM in soil and on the surface of tomato fruits. It might be possible to estimate injured SI in actual condition of manure preparation by above procedure. On the contrary, there are practical difficulties for discriminating injured LM in actual agricultural environment by only salt sensitivity.

DOI： https://doi.org/10.3136/nskkk.65.205

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

Outbreaks of food-borne illness caused by Listeria monocytogenes in or on fresh produce are sometimes reported. Tomatoes have been considered as one of the most implicated vehicles for produce-associated outbreaks. In the present paper, using tomato plants and three isolates of L. monocytogenes showing different serotypes (1/2a, 1/2b, 4b), viability and injury of L. monocytogenes in soil and in or on tomato plants during cultivation was investigated. Soil was artificially contaminated with L. monocytogenes at levels of 2, 4, 6 or 8 log CFU/g, followed by cultivation of tomato plants in the contaminated soils. The population of L. monocytogenes in the soil decreased to less than the detection limit (< 2 log CFU/g) 6 - 8 weeks after the start of cultivation. L. monocytogenes was not detected in any harvested fruit after a 16-week cultivation, although it was detected qualitatively from soil samples. Artificial injury of the root did not induce contamination of tomato fruit by L. monocytogenes via vessels. Thus, the possibility of internalization or contamination of tomato fruit by L. monocytogenes from contaminated soil is considered quite low during cultivation. However, L. monocytogenes on the fruit surface survived up to 2 - 3 weeks. Regardless of contamination level, hygienically inappropriate handling by workers might lead to contamination of tomato fruit by bacteria such as L. monocytogenes and thus to food poisoning.

DOI： 10.3136/fstr.22.349

Other Link： https://www.jstage.jst.go.jp/article/fstr/22/3/22_349/_article/-char/en

Repository Public URL： https://hdl.handle.net/2324/7183499

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

Approaches of Improvement of Freezing-Stress Tolerance of Yeast

Repository Public URL： https://hdl.handle.net/2324/7183497

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

Foodborne illnesses associated with the consumption of fresh produce such as raw vegetables have become a major health concern worldwide in recent years. In the present study, we investigated the possible routes of Salmonella contamination in leafy lettuce via soil during cultivation. After 10-week cultivation of lettuce plants in soils inoculated with S. Enteritidis expressing green fluorescent protein (SE-EGFP), the bacterium was detected in soil inoculated with >10(4) cfu/g and from most lettuce leaves cultivated in soils inoculated with >4.4 x 10(7) cfu/g. As Salmonella was not detected in intact lettuce leaves or lettuce leaves with root injury cultivated in highly contaminated soils and after surface disinfection, the lettuce plants were not considered to internalize the bacterium. Overhead irrigation led to the contamination of one in 10 lettuce plants; however, all sets of three leaves of the plant were contaminated (>110 MPN/g). In an effort to prevent Salmonella contamination from soils, we investigated the effects of mulch on contamination levels during cultivation. Mulch effectively reduced Salmonella contamination levels of lettuce plants cultivated in highly contaminated soils.

DOI： 10.3136/fstr.20.961

Other Link： https://www.jstage.jst.go.jp/article/fstr/20/5/20_961/_article/-char/en

Repository Public URL： https://hdl.handle.net/2324/7183500

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

Repository Public URL： https://hdl.handle.net/2324/7183501

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

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.

DOI： 10.1371/journal.pone.0045988

Repository Public URL： https://hdl.handle.net/2324/7183502

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

To investigate functions of trehalose 6 phosphate synthase (TPS) and trehalose 6 phosphate phosphatase (TPP) from a tobacco plant, Nicotiana tabacum, the corresponding cDNA clones were isolated. Those genes were designated NtTPS and NtTPP, respectively NtTPS included a N-terminal extension according to comparison of deduced amino acid sequence of NtTPS with those of TPSs from Escherichia coli and yeast. The NtTPS was genetically modified to lack a region for the N-terminal extension and the modified gene was designated Delta NNtTPS. The genes were expressed in yeast tps1 mutant as two separate proteins and as a NtTPS (or Delta NNtTPS) NtTPP fusion protein. Western blot analysis showed that the NtTPS, NtTPP, and NtTPS NtTPP were expressed abundantly in yeast, while the Delta NNtTPS and Delta NNtTPS-NtTPP were not detected. Interestingly, high levels of trehalose were accumulated in yeast expressing Delta NNtTPS and Delta NNtTPS-NtTPP in spite of their low level expressions. Furthermore, stress tolerances of yeast against osmotic, freezing thawing, and heat stresses were significantly improved by the expression of the tobacco gene, and the increased levels in tolerance were proportional to their trehalose levels. Our results showed that NtTPS and NtTPP functioned in trehalose synthesis by the removal of N-terminal extension of NtTPS and several environmental stress tolerances.

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

Taurine is known to function as a protectant against various stresses in animal cells. In order to utilize taurine as a compatible solute for stress tolerance of yeast, isolation of cDNA clones for genes encoding enzymes involved in biosynthesis of taurine was attempted. Two types of cDNA clones corresponding to genes encoding cysteine dioxygenase (CDO1 and CDO2) and a cDNA clone for cysteine sulfinate decarboxylase (CSD) were isolated from Cyprinus carpio. Deduced amino acid sequences of the two CDOs and that of CSD showed high similarity to those of CDOs and those of CSDs from other organisms, respectively. The coding regions of CDO1, CDO2, and CSD were subcloned into an expression vector, pESC-TRP, for Saccharomyces cerevisiae. Furthermore, to enhance the efficiency of synthesis of taurine in S. cerevisiae, a CDO-CSD fusion was designed and expressed. Expression of CDO and CSD proteins, or the CDO-CSD fusion protein was confirmed by Western blot analysis. HPLC analysis showed that the expression of the proteins led to enhancement of the accumulation level of hypotaurine, a precursor of taurine, rather than taurine. The yeast cells expressing corresponding genes showed tolerance to oxidative stress induced by menadione, but not to freezing-thawing stress.

DOI： 10.1007/s00726-009-0328-6

Repository Public URL： https://hdl.handle.net/2324/7183503

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

Enhanced glucose 6-phosphate dehydrogenase (E C 1 1 1 49, G6PDH) activity has been identified as a hardening-induced intracellular change of Chlorella vulgaris, which acquires freezing tolerance during hardening. In the present study, a full-length cDNA clone corresponding to a gene encoding a chloroplastic isoform of G6PDH, designated CvchG6PDH, was isolated from C vulgarus C-27. By comparing the deduced amino acid sequence of CvchG6PDH with the N-terminal aminoacid sequence of mature G6PDH(2) protein isolated previously, a DNA region encoding mature CvchG6PDH was determined and designated mCvchG6PDH. The deduced amino acid sequence of CvchG6PDH showed higher homology to those of plant plastidic G6PDH genes than those of cytosolic ones. A recombinant mCvchG6PDH protein expressed in Escherichia coli showed similar enzymatic properties to previously isolated Chlorella G6PDH(2), suggesting that the gene encoded plastidic G6PDH(2) protein. Expression of CvchG6PDH was induced transcriptionally throughout 24-h hardening, while the translation was induced up to 9-h hardening and then decreased, and the change did not reflect the enhanced G6PDH activity during hardening. Furthermore, the mCvchG6PDH alleviated both freezing and menadione-induced oxidative stresses in yeast. We showed the contribution of CvchG6PDH in menadione stress tolerance as one of its functions in the acquisition of freezing tolerance of Chlorella.

Repository Public URL： https://hdl.handle.net/2324/17797

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

Trehalases (TREs) function in trehalose hydrolysis and commonly found in most organisms. It is said that most fungi have two types of TREs, acid trehalase and neutral trehalase, but other organisms including plants have only one type. To investigate the function of TRE from plants, a full-length cDNA clone encoding TRE was isolated and designated NtTRE. A conserved region of common trehalase can be found in deduced amino acid sequence of NtTRE, thus the gene was recognized to encode NtTRE enzyme. The NITRE was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein to investigate the function of the expressed protein as trehalase. SDS-PAGE profile of the protein extract of E. coli showed that the expressed GST-NtTRE protein appeared to be cleaved into two polypeptides, which were approximately 56 and 28 kDa in size, and to form an inclusion body. Based on the results of N-terminal amino acid sequencing of the 56-kDa protein, it contained almost all parts of NtTRE protein. Thus, the protein expressed in E. coli was used only for production of anti-NtTRE antibodies. Function of NtTRE was investigated using yeast expressing NtTRE. The NtTRE protein was expressed as a soluble protein. Trehalase activity of protein extract of the transformed yeast was significantly higher than that of control yeast carrying an empty vector. In addition, intracellular trehalose content in yeast cells was significantly reduced by expression of NtTRE. Those data provided a possibility to construct a modified tobacco plant that can accumulate trehalose in the cells by suppression of the expression and/or the activity of NtTRE.

Repository Public URL： https://hdl.handle.net/2324/16107

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

A chloroplastic NADPH-dependent thioredoxin reductase gene was identified from Chlorella vulgaris and designated CvNTRC. Mature CvNTRC protein (mCvNTRC) was expressed in Escherichia coli, and it showed both NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx)-like dithiol-disulfide oxidoreductase activities. The transcript of CvNTRC increased throughout 24-h hardening, whereas the encoded protein amount and total NTR activity decreased once and then increased during hardening. By in vitro pull-down assay, a 21.2-kDa protein bound to mCvNTRC was isolated and identified as a 2-Cys peroxiredoxin (2-Cys Prx) based on the N-terminal sequence. These data suggest that CvNTRC is maintained at a constant level during hardening and functions as an antioxidant with 2-Cys Prx in the acquisition of freezing tolerance of Chlorella.

DOI： 10.1271/bbb.80761

Repository Public URL： https://hdl.handle.net/2324/7183505

Language：English   Publishing type：Research paper (international conference proceedings)  

Machida, T., Kato, E., Ishibashi, A., Ohashi, N., Honjoh, K., and Miyamoto, T.,Molecular characterization of low-temperaure-inducible NTR-C in Chlorella vulgaris,Nucleic Acids Symposium Series,No. 51, 463-464,2007.11.

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

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

The effect of taurine on the survival of Saccharomyces cereuisiae after freezing and oxidative stress was investigated. Proline and NaCl were used in comparison. The accumulation of taurine in yeast cells seemed to lead to the enhancement of tolerance to both freezing and oxidative stress in yeast. Although taurine appeared to be less effective than proline in the development of freezing tolerance, when based on intracellular amounts taurine protected cells more effectively than proline. In order to clarify the effect of taurine on stress tolerance, the expression patterns of stress-responsive genes were observed using RT-PCR. In addition, the contents of glycerol and trehalose as well as the redox states of glutathione in the yeast cells were investigated. Our results suggest that taurine, as well as proline, may function as a cryo-protectant and/or an antioxidant in yeast.

DOI： 10.3136/fstr.13.145

Repository Public URL： https://hdl.handle.net/2324/7183517

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

A cDNA clone corresponding to the gene for glucose 6-phosphate dehydrogenase (E.C.1.1.1.49) was isolated from a cDNA library constructed from poly(A)+RNA from Chlorella vulgaris C-27, a frost hardy strain. The cDNA clone was designated as Cvcg6pdh. The length of Cvcg6pdh was 1845 bp and the clone coded for 521 amino acids. The deduced amino acid sequence of Cvcg6pdh showed sequence homology to cytosolic G6PDHs from other higher plants rather than chloroplastic ones. Northern blot analysis of a transcript of Cvcg6pdh gene showed that the gene was down-regulated once and then induced after 12-h hardening. Activity staining also showed that the expression pattern of one G6PDH isoform was similar to that of the transcript of the gene. Southern blot analysis of genomic DNA showed that Cvcg6pdh seems to hybridize with, at least, one copy of g6pdh genes. The coding region of the clone was amplified by PCR and the product was introduced into Saccharomyces cerevisiae by using a pTG887 expression vector. The activity of G6PDH in transformed yeast was enhanced up to 8.7 times that of the control strain. Furthermore, after freezing-thawing, the viability of the yeast transformed with pTG887/Cvcg6pdh was significantly higher than that of the control yeast cells carrying pTG887. © 2006 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.plantsci.2006.12.004

Repository Public URL： https://hdl.handle.net/2324/7183533

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

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

DOI： 10.1271/bbb.67.1888

Repository Public URL： https://hdl.handle.net/2324/7183545

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

Protoplasts from Chlorella vulgaris K-73122 were obtained by enzymatic digestion with a mixture of Acromopeptidase, Cellulase ONOZUKA R-10, Chitosanase KI, Gluczyme, and Uskizyme. The formation of naked protoplasts was confirmed by fluorescence microscopy using fluorescent brightner 28, which stains cell walls. About 88&#37; of C. vulgaris K-73122 cells were converted into osmotically-labile cells. Furthermore, a method for regeneration of intact cells from the protoplasts was developed. Utilization of 0.5 M sucrose as an osmoticum, Fe-EDTA as an iron source, and bacto-agar as a supporting was shown to help regeneration of the cell walls of two strains, C. vulgaris K-73122 and C-27.

Repository Public URL： https://hdl.handle.net/2324/4494

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

Two low-temperature-inducible Chlorella genes for D12 and w-3 fatty acid desaturase (FAD): Isolation of D12 and w-3 fad cDNA clones, expression of D12 fad in Saccharomyces cerevisiae , and expression of w-3 fad in Nicotiana tabacum, Suga, K., Honjoh, K., Furuya, N., Shimizu, H., Nishi, K., Shinohara, F., Hirabaru, Y., Maruyama, I., Miyamoto, T., Hatano, S., and Iio, M., Biosci. Biotechnol. Biochem., 66(6): 1314-1327 (2002)

Repository Public URL： https://hdl.handle.net/2324/7183546

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

Two low-temperature-inducible chlorella genes for Δ12 and ω-3 fatty acid desaturase (FAD): Isolation of Δ12 and ω-3 fad cDNA clones,… expression of Δ12 fad in saccharomyces cerevisiae, and Expression of ω-3 fad in nicotiana tabacum
In an attempt to clarify the involvement of fatty acid desaturases (FADs) in the freezing tolerance of Chlorella vulgaris IAM C-27, developed by hardening, we have isolated cDNA clones for two types of FADs from the Chlorella strain, based on the sequence information of genes for Δ12 and ω-3 FADs, respectively desaturating oleic acid (18:1) to linoleic acid (18:2) and linoleic acid (18:2) to linolenic acid (18:3). The deduced amino acid sequence of the first clone, designated CvFad2, showed about 66% similarity to the microsomal Δ12 FADs from several higher plants and this gene had Δ12 FAD activity when expressed in Saccharomyces cerevisiae. The predicted protein encoded by a second gene, designated CvFad3, showed about 60% similarity to the microsomal and plastidial ω-3 FADs from several higher plants. The features of the amino acid sequences of the C- and N-terminal regions of CvFAD3 and fatty acid analysis of polar lipids in transgenic tobacco plant expressing the CvFad3 gene suggested that this gene encodes the microsomal ω-3 FAD. Southern blot analysis showed that both genes were single-copy genes in the genome of the Chlorella strain. Different transcriptional patterns were observed with the two genes during hardening in Northern blot analysis. © 2002 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

DOI： 10.1271/bbb.66.1314

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

A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at -1, -3, and -4°C, with the exception that their freezing temperature was -2°C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance tests at -1-4°C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at -3°C.

DOI： 10.1271/bbb.65.1796

Repository Public URL： https://hdl.handle.net/2324/7183548

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

Cryoprotective activities of group3 late embryogenesis abundant proteins from Chlorella vulgaris C-27, Honjoh, K., Matsumoto, H., Shimizu, H., Ooyama, K., Tanaka, K., Oda, Y., Takata, R., Joh, T., Suga, K., Miyamoto, T., Iio, M. and Hatano, S. , Biosci. Biotechnol. Biochem., 64(8): 1656-1663 (2000)

DOI： 10.1271/bbb.64.1656

Repository Public URL： https://hdl.handle.net/2324/7183550

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

The hiC6 gene of Chlorella vulgaris, sharing sequence similarity with a late embryogenesis abundant (lea) gene, was introduced into Saccharomyces cerevisiae. It was inserted on a multicopy plasmid under the transcriptional control of the yeast GAL1 promoter. Expression of HIC6 protein was confirmed by immunochemical methods. Expression level of the protein was increased gradually with the induction-time by galactose. With maximum induction time, the freeze-tolerance of yeast transformed with hiC6 gene was approximately 3.3 times (from 20&#37; to 66&#37; survival rate) higher than that of the control yeast.

DOI： 10.1016/S0176-1617(99)80046-7

Repository Public URL： https://hdl.handle.net/2324/7183555

Language：Japanese  

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

Hardening-induced soluble proteins of Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27) were isolated and purified by two-dimensional high-performance liquid chromatography (2D-HPLC) on an anion-exchange column, with subsequent reversed-phase chromatography. Some of the proteins were resolved by SDS-PAGE, characterized by amino-terminal sequencing and identified by searching for homologies in databases. Separation of the soluble proteins during the hardening of Chlorella by a combination of 2D-HPLC and SDS-PAGE revealed that at least 31 proteins were induced or increased in abundance. Of particular interest was the induction after 12 h of a 10-kDa protein with the amino-terminal amino acid sequence AGNKPITEQISDAVGAAGQKVG and the induction after 6 h of a 14-kDa protein with the amino-terminal sequence ALGEESLGDKAKNAFEDAKDAVKDAAGNVKEAV. The amino-terminal sequences of these proteins indicated that they were homologous to late embryogenesis abundant (LEA) proteins. Furthermore, the level of a 22-kDa protein also increased after 12 h. The amino-terminal sequence of this protein, AAPLVGGPAPDFTAAAVFD, indicated that it was homologous to thioredoxin peroxidase. Copyright © 1995. The Japanese Society of Plant Physiologists.

Repository Public URL： https://hdl.handle.net/2324/7183556

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

To investigate the effects of hardening on gene expression in Chlorella vulgaris Beijerink IAM C-27 (formerly Chlorella ellipsoidea Gerneck IAM C-27), a frost-hardy strain, 17 cDNA clones corresponding to hardening-induced Chlorella (hiC) genes were isolated by differential screening of a cDNA library from 6-h hardened cells. Northern blot analysis of transcripts of hiC genes showed that these genes are specifically induced by hardening and that their patterns of induction vary. Southern blots of genomic DNAs from two strains (Chlorella ellipsoidea Gerneck IAM C-102, chilling-sensitive; and C. vulgaris C-27, frost-hardy) of Chlorella indicated that ten hiC clones out of 17 hybridized only with DNA of strain C-27 and the other seven clones hybridized with DNA of both strains. However, of these seven clones, transcripts corresponding to six clones did not accumulate in strain C-102 at low temperatures. The sequence of a deduced protein encoded by the most abundant clone, hiC6, exhibited homology to sequences of Group III LEA (late embryogenesis abundant) proteins and had an amino-terminal amino acid sequence that was similar to the sequences of chloroplast transit peptides. © 1995 The Japanese Society of Plant Physiologists (JSPP).

Repository Public URL： https://hdl.handle.net/2324/7183564

Language：English  

DOI： 10.1080/87559129.2025.2503474

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

Salmonella Schwarzengrund and Escherichia coli O157:H7 are ones of foodborne pathogens that can produce biofilms and cause serious food poisoning. Bacteriophages are an emerging antibacterial strategy used to prevent foodborne pathogen contamination in the food industry. In this study, the combined antibacterial effects of the polyvalent phage PS5 and sodium hexametaphosphate (SHMP) against both pathogens were investigated to evaluate their effectiveness in food applications. The combined treatment with phage PS5 (multiplicity of infection, MOI = 10) and 1.0% SHMP inhibited the growth of S. Schwarzengrund and E. coli O157:H7, and the viable counts of both decreased by more than 2.45 log CFU/mL. In KAGOME vegetable and fruit mixed juice, the combined treatment with PS5 (MOI = 100) and 1.0% SHMP also resulted in significant pathogen inactivation at 4 °C after 24 h. PS5 (1010 PFU/mL) and 1.0% SHMP showed stronger synergistic effects on biofilm formation and the removal of established biofilms on polystyrene plates. Additionally, we evaluated their combined effects on reducing the biofilms of S. Schwarzengrund and E. coli O157:H7 on glass tubes and cabbage leaves at 4 °C. These findings indicate the utility of this approach in the biocontrol of the planktonic and biofilm cells of S. Schwarzengrund and E. coli O157:H7 on foods and food contact surfaces.

DOI： 10.1016/j.fm.2024.104680

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Archives of Virology  

Campylobacter jejuni is a leading cause of foodborne illness worldwide. The application of bacteriophages offers a promising approach to specifically target and reduce C. jejuni contamination in food products. In this study, two C. jejuni phages were characterized, and their ability to inhibit bacterial growth in combination with ethylenediaminetetraacetic acid (EDTA) was investigated. Both phages exhibited tolerance to a wide range of temperature (4-60 °C) and pH (3-9). Phage vB_CjeM-PC10 and vB_CjeM-PC22 were found to have a latent period of 30 min and 20 min and a burst size of 7 and 35 PFU/cell, respectively. Phage vB_CjeM-PC10 has a linear double-stranded DNA (dsDNA) genome of 51,148 bp with 77 ORFs and 29% GC content. Phage vB_CjeM-PC22 has a circular dsDNA genome of 32,543 bp with 56 ORFs and 28% GC content. At 42 °C, the combination of these phages (MOI = 10) and EDTA decreased the count of viable C. jejuni by 5.2 log10 and inhibited the regrowth of resistant cells for 48 h. At 4 °C, phage vB_CjeM-PC10 alone (MOI = 1000) reduced the count of viable C. jejuni by 3 log10 in brain heart infusion (BHI) broth and 2 log10 on chicken skin after incubation for 48 h. Although these phages were effective against C. jejuni, they cannot be utilized directly for food safety applications because they are lysogenic. Nevertheless, these findings expand the genome library of C. jejuni phages and enrich data resources by highlighting potential strategies for controlling C. jejuni infections.

DOI： 10.1007/s00705-024-06169-2

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Language：English   Publisher：Journal of Food Science  

Theaflavin 3,3′-digallate (TF3), a major polyphenolic component of black tea, exhibits antibacterial effects against many foodborne pathogens. However, the antibacterial mechanisms of TF3 against Listeria monocytogenes remain unclear. In this study, we investigated the effects of TF3 on viability, biofilm, and membrane function of L. monocytogenes by the conventional plating method, crystal violet staining, and microscopy using fluorescent dyes JC-1 and Laurdan, respectively. It was found that TF3 showed excellent antibacterial activity against L. monocytogenes with the minimum inhibitory concentration of 62.5 mg/L. The viable count determined on TSA decreased by 3 log after the treatment for 2 h with TF3 at 62.5 mg/L. The viable count determined on TSA containing 4% NaCl decreased by more than 4 log after the treatment for 30 min with TF3 at the same concentration, suggesting that TF3 gave damage on the cells, enhancing the antibacterial action of 4% NaCl, but the damage was recoverable in the absence of 4% NaCl. To explore the antibacterial mechanisms of TF3, the effects of TF3 on membrane potential and membrane fluidity were investigated. TF3 reduced both membrane potential and fluidity of L. monocytogenes at 62.5 mg/L, suggesting that TF3 damaged the structural integrity of the cell membrane. TF3 reduced biofilm mass of mature biofilm of L. monocytogenes. Moreover, THEAFLAVIN TF40, a commercially available Camellia sinensis leaf extract containing TF3, reduced viable count of L. monocytogenes by 2 log on apple skin. These results suggest the potential of theaflavins as a natural anti-Listeria disinfectant.

DOI： 10.1111/1750-3841.17321

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DOI： 10.3390/antibiotics13090884

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

This study focused on the isolation and characterization of bacteriophages with specific activity against toxin-producing and multidrug-resistant strains of Bacillus cereus sensu stricto (B. cereus s. s.). Ten different samples yielded six bacteriophages by utilizing the double-layer agar technique. The most promising phage, vB_BceS-M2, was selected based on its broad host range and robust lytic activity against various B. cereus s. s. strains. The phage vB_BceS-M2 had a circular double-stranded DNA genome of 56,482 bp. This phage exhibited stability over a wide range of temperatures and pH values, which is crucial for its potential application in food matrices. The combined effect of phage vB_BceS-M2 and nisin, a widely used antimicrobial peptide, was investigated to enhance antimicrobial efficacy against B. cereus in food. The results suggested that nisin showed synergy and combined effect with the phage, potentially overcoming the growth of phage-resistant bacteria in the broth. Furthermore, practical applications were conducted in various liquid and solid food matrices, including whole and skimmed milk, boiled rice, cheese, and frozen meatballs, both at 4 and 25 °C. Phage vB_BceS-M2, either alone or in combination with nisin, reduced the growth rate of B. cereus in foods other than whole milk. The combination of bacteriophage and nisin showed promise for the development of effective antimicrobial interventions to counteract toxigenic and antibiotic-resistant B. cereus in food.

DOI： 10.1016/j.foodres.2024.114685

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Food Microbiology  

Nowadays, the discovery of alternative natural antimicrobial substances such as bacteriophages, essential oils, and other physical and chemical agents is developing in the food industry. In this study, nine bacteriophages were isolated from various parts of raw chickens and exhibited lytic activities against L. monocytogenes and various Listeria spp. The characterization of phage vB_LmoS-PLM9 was stable at 4 to 50 °C and pH range from 4 to 10. Phage vB_LmoS-PLM9 had a circular, double-stranded genomic DNA with 38,345 bp having endolysin but no antibiotic resistance or virulence genes. Among the eight essential oils tested at 10 %, cinnamon bark, and cassia oils showed the strongest antilisterial activities. The combined use of phage vB_LmoS-PLM9 and cinnamon oils indicated higher efficiency than single treatments. The combination of phage (MOI of 10) and both cinnamon oils (0.03 %) reduced the viable counts of L. monocytogenes and inhibited the regrowth of resistant cell populations in broth at 30 °C. Furthermore, treatment with the combination of phage (MOI of 100) and cinnamon oil (0.125 %) was effective in milk, especially at 4 °C by reducing the viable count to less than lower limit of detection. These results suggest combining phage and cinnamon oil is a potential approach for controlling L. monocytogenes in milk.

DOI： 10.1016/j.ijfoodmicro.2024.110797

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Publisher：European Food Research and Technology  

Bacillus cereus is a spore-forming foodborne pathogen that causes emetic and diarrheal food poisoning. An emetic strain of B. cereus produces spores and an emetic toxin called cereulide, which exhibits high heat resistance. In this study, we investigated the effects of epigallocatechin gallate (EGCg), theaflavin-3’-gallate (TF2b), and theaflavin-3,3’-gallate (TF3), polyphenolic components of tea extracts, on the survivability, spores, and toxin of B. cereus. Among them, EGCg at 62.5 μg/mL and 250 μg/mL showed bacteriostatic and bactericidal effects on B. cereus by damaging the cellular membrane, causing leakage of cellular nucleic acid related substances, and thus inhibiting the growth of B. cereus. However, no significant effects were shown on spore germination by EGCg, TF2b, or TF3 at a concentration of 250 μg/mL in this investigation. Their inhibitory effects were primarily confined to the outgrowth of daughter cells after spore germination. Furthermore, quantitative polymerase chain reaction (qPCR) showed that they suppressed the activity of cesA, a gene associated with cereulide production. At concentrations of 125 µg/mL for TF2b and 62.5 µg/mL for TF3, a discernible attenuation of cytotoxic effects induced by cereulide on HEp-2 cells was evident.

DOI： 10.1007/s00217-024-04593-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Food Microbiology  

The continuous detection of multi-drug-resistant enterococci in food source environments has aroused widespread concern. In this study, 198 samples from chicken products, animal feces, raw milk, and vegetables were collected in Japan and Egypt to investigate the prevalence of enterococci and virulence characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed for species identification and taxonomic analysis of the isolates. The results showed that the rates of most virulence genes (efaA, gelE, asa1, ace, and hyl) in the Japanese isolates were slightly higher than those in the Egyptian isolates. The rate of efaA was the highest (94.9 %) among seven virulence genes detected, but the cylA gene was not detected in all isolates, which was in accordance with γ-type hemolysis phenotype. In Enterococcus faecalis, the rate of kanamycin-resistant strains was the highest (84.75 %) among the antibiotics tested. Moreover, 78 % of E. faecalis strains exhibited multi-drug resistance. Four moderately vancomycin-resistant strains were found in Egyptian isolates, but none were found in Japanese isolates. MALDI-TOF MS analysis correctly identified 98.5 % (68/69) of the Enterococcus isolates. In the principal component analysis dendrogram, strains isolated from the same region with the same virulence characteristics and similar biofilm-forming abilities were characterized by clustered distribution in different clusters. This finding highlights the potential of MALDI-TOF MS for classifying E. faecalis strains from food sources.

DOI： 10.1016/j.ijfoodmicro.2024.110768

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

Pseudomonas spp., such as P. fluorescens group, P. fragi, and P. putida, are the major psychrophilic spoilage bacteria in the food industry. Bacteriophages (phages) are a promising tool for controlling food-spoilage and food-poisoning bacteria; however, there are few reports on phages effective on food-spoilage bacteria such as Pseudomonas spp. In this study, 12 Pseudomonas phages were isolated from chicken and soil samples. Based on the host range and lytic activity at 30 °C and 4 °C and various combinations of phages, phages vB_PflP-PCS4 and vB_PflP-PCW2 were selected to prepare phage cocktails to control Pseudomonas spp. The phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 showed the strongest lytic activity and retarded regrowth of P. fluorescens and P. putida at 30 °C, 8 °C, and 4 °C at a multiplicity of infection of 100. Nucleotide sequence analysis of the genomic DNA indicated that vB_PflP-PCS4 and vB_PflP-PCW2 phages were lytic phages of the Podoviridae family and lacked tRNA, toxin, or virulence genes. A novel endolysin gene was found in the genomic DNA of phage vB_PflP-PCS4. The results of this study suggest that the phage cocktail consisting of vB_PflP-PCS4 and vB_PflP-PCW2 is a promising tool for the biocontrol of psychrophilic food-spoilage pseudomonads during cold storage and distribution.

DOI： 10.1007/s10123-023-00479-2

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

Bacillus cereus sensu stricto is a foodborne pathogen that causes food poisoning. Their spore and biofilm-forming abilities persist in various environments and foods. This study investigated the prevalence, virulence, antibiotic resistance, and genetic diversity of B. cereus s. s. strains isolated from various food samples. Of 179 samples, 22.34% were positive for B. cereus s. s., with significantly high detection rates in milk products and raw chicken meat. Forty strains were isolated from positive samples. Matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis revealed nine distinct clusters and multi-locus sequence typing revealed 34 sequence types including 23 novel sequences, demonstrating high genetic diversity among the isolates. PCR analysis revealed that all the strains contained at least one toxin gene, but none contained the cytK gene. Antibiotic resistance tests revealed that all isolates were classified as multidrug-resistant, with high resistance levels, particularly to β-lactam antibiotics and vancomycin, but were susceptible to gentamicin. All isolates showed variations in biofilm formation. This study highlights the significant public health risk due to B. cereus s. s. and underscores the need for stringent monitoring and control measures in food production to manage antimicrobial resistance and ensure food safety.

DOI： 10.3390/antibiotics13080774

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DOI： doi: 10.3136/fstr.FSTR-D-23-00186

Publisher：Food Control  

Phage-encoded peptidoglycanases (phage endolysins) are hydrolyzing enzymes that break peptidoglycan bonds within infected bacterial cell walls at the end of the lytic cycle. They are promising antibacterial agents capable of controlling major foodborne pathogens. Here, we cloned, overexpressed, and purified the phage-encoded protein LysCPQ7, a putative endolysin from the Clostridium perfringens phage CPQ7. The predicted amino acid sequence indicated that LysCPQ7 exhibited amidase activity to digest the peptidoglycan bond between muramic acid and peptide in bacterial cell walls. LysCPQ7 was characterized as an alkalophilic and thermostable endolysin. It exhibited the highest lytic activity against C. perfringens cells at pH values ranging from 9.0 to 11.0, and was stable at temperatures from −20 to 60 °C. The lytic activity of LysCPQ7 was not significantly (p < 0.05) influenced in the absence/presence of 100, 200, and 300 mM of NaCl. The antibacterial test confirmed that LysCPQ7 reduced the viability of C. perfringens cells by 4.1 log after 6 h of incubation in broth. LysCPQ7 significantly (p < 0.05) decreased the viability of C. perfringens in artificially-contaminated milk and sliced cheese under refrigeration and room temperatures. Our results showed that LysCPQ7 has great potential as a newly emerging biocontrol agent against C. perfringens in dairy and fermented food products.

DOI： 10.1016/j.foodcont.2023.110157

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Applied Microbiology  

AIMS: The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. METHODS AND RESULTS: A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. CONCLUSION: PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells.

DOI： 10.1093/jambio/lxad318

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Language：English   Publisher：Japanese Society for Food Science and Technology  

<p>In this study, the <i>dnaK</i> gene-deletion mutant strain of <i>Escherichia coli</i> BW25113 showed a higher susceptibility than the wild-type strain of <i>E. coli</i> BW25113 to phage S127BCL3. Flavonoids, myricetin and quercetin which had been reported to suppress the role of DnaK were tested to examine their effects on the phage susceptibility of <i>E. coli</i> BW25113. A 6-h pretreatment with 500 µmol/L myricetin or quercetin increased the phage susceptibility of <i>E. coli</i> BW25113. A similar result was observed in <i>E. coli</i> O157:H7. Real-time quantitative polymerase chain reaction (qPCR) was conducted to investigate the effects of flavonoids on the transcription of chaperone genes (<i>dnaK, dnaJ, groEL</i>, and <i>grpE</i>) in <i>E. coli.</i> Pretreatment of wild-type <i>E. coli</i> BW25113 with flavonoids decreased the transcription of chaperone genes. This is the first report demonstrating the enhancement of the phage susceptibility of both <i>E. coli</i> BW25113 and <i>E. coli</i> O157:H7 by flavonoids. The results of this study on the combined effects of flavonoids involved in foods and phages on <i>E. coli</i> provide scientific bases for development of a novel biocontrol method of foodborne bacteria.</p>

DOI： 10.3136/fstr.fstr-d-23-00186

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DOI： 10.1128/MRA.00504-23

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Language：English   Publisher：AMB Express  

Contaminated food with antibiotic-resistant Enterococcus spp. could be the vehicle for transmitting Enterococcus to humans and accordingly cause a public health problem. The accumulation of biogenic amines produced by Enterococcus faecalis (E. faecalis) in food may have cytological effects. Bacteriophages (phage in short) are natural antimicrobial agents and can be used alone or in combination with other food preservatives to reduce food microbial contaminants. The aim of this study was to isolate a novel phage against E. faecalis and determine its host range to evaluate its potential application. Bacteriophage, vB_EfKS5, with a broad host range, was isolated to control the growth of E. faecalis. The vB_EfKS5 genome is 59,246 bp in length and has a GC content of 39.7%. The computational analysis of phage vB_EfKS5 genome confirmed that it does not contain any lysogenic, toxic, or virulent genes. Phage vB_EfKS5 exhibited lytic activity against most E. faecalis isolates with different multiplicities of infections and it infected 75.5% (22/29) of E. faecalis isolates and 42.3% (3/7) of E. faecium isolates. It was also able to destroy the biofilm formed by E. faecalis with different MOIs. Phage vB_EfKS5 alone or in combination with nisin could control the growth of E. faecalis in broth and milk. Based on its high productivity, stability, short latent period, and large burst size, phage vB_EfKS5 has a high potential for applications both in food and medical applications.

DOI： 10.1186/s13568-023-01628-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Journal of Applied Microbiology  

AIMS: Isolation and characterization of Enterococcus phages and application of phage cocktail to control E. faecalis in milk. METHODS AND RESULTS: For phage isolations, double layer agar method was used. Host range of the phages were determined by the spot test. Twelve phages with varying host ranges were isolated. Phages PEF1, PEF7b, and PEF9 with different host ranges and lytic activities were selected for phage cocktails. Compared to two-phages cocktails tested, the cocktail containing all the three phages displayed stronger antibacterial and biofilm removal activities. The cocktail treatment reduced viable E. faecalis in biofilm by 6 log within 6 h at both 30°C and 4°C. In milk, the cocktail gradually reduced the viable count of E. faecalis and the count reached below the lower limit of detection at 48 h at 4°C. CONCLUSION: The strong bactericidal and biofilm removal activities of the phage cocktail suggest the potential of this cocktail as a natural biocontrol agent for combating E. faecalis in milk.

DOI： 10.1093/jambio/lxad250

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DOI： https://doi.org/10.1093/jambio/lxad250

Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Food Microbiology  

Salmonella spp., one of the most frequently reported bacteria, causes foodborne illness and economic losses. Due to the threat of increasing antibiotic resistant foodborne pathogens, application of bacteriophages as novel antibacterial agents in food matrices has become an emerging strategy. In this study, a novel Salmonella phage PS3-1 with high lytic activity against Salmonella Typhimurium was identified from previously isolated phages. PS3-1 belonged to the class Caudoviricetes with a broad host range, and had relatively short latent period (15 min), large burst size (92 PFU/cell), high pH stability (pH 3.0-11.0) and thermal tolerance (4-60 °C). Genome sequencing analysis showed that PS3-1 genome consisted of 107,110 bp DNA, without antibiotic resistance and virulence related genes. The results of growth curve and time-kill assay showed that PS3-1 not only inhibited the growth of S. Typhimurium, but also effectively decreased the viable cell counts (0.30-4.72 log) after 24-h incubation at 7, 25 and 37 °C (P < 0.05). Moreover, >1.28 log of established biofilm cells were effectively removed after 24-h treatment with PS3-1. Besides, PS3-1 significantly reduced the viability of S. Typhimurium in milk, lettuce, raw pork meat and ready-to-eat steamed-chicken breast at different temperatures (P < 0.05). These results demonstrated that PS3-1 may be an excellent antibacterial agent for controlling S. Typhimurium in food industry.

DOI： 10.1016/j.ijfoodmicro.2023.110295

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DOI： https://doi.org/10.1016/j.heliyon.2023.e20727

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DOI： https://doi.org/10.1016/j.foodcont.2023.110157

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

This study investigated the prevalence, serotype, antimicrobial resistance (AMR), virulence potential, and biofilm formation of Listeria monocytogenes isolated in 2022 in Japan and compared their profiles with those of isolates in 2012 and 2017. A total of 85 chicken samples were randomly collected from different supermarkets in Fukuoka in 2022. L. monocytogenes were isolated by conventional method and characterized by MALDI-TOF MS. Among 85 samples tested in 2022, 9 (10.6%) were positive for L. monocytogenes and 17 strains were isolated from the positive samples. The isolates were serotyped as 1/2b (41.2%), 3a (29.4%), 3b (23.5%) and 1/2a (5.9%). Antimicrobial susceptibility tests of the 2022 isolates showed susceptibility to majority of the antibiotics, except cefoxitin, oxacillin, and fosfomycin. Compared to the previous surveillance results, the prevalence of L. monocytogenes in 2022 (10.6%) was significantly lower (p < 0.05) than those of the isolates in 2017 (24%) and 2012 (52.9%). The distribution of serotypes 1/2a and 1/2b decreased over time, and serotype 4b was not detected in the 2022 isolates. The proportion of multidrug resistant strains in 2022 (16.7%) was significantly lower than those in 2012 (46.7%) and 2017 (82.6%). Moreover, a total of 36 isolates (12 isolates/ year) were used to detect the virulence genes (hlyA, plcA, clpC, and inlA) and biofilm-forming capacity. Most of the isolates from different years harboured four virulence genes. The biofilm formation of the 2022 isolates was significantly weaker (p < 0.05) than those of the 2012 and 2017 isolates. Thus, despite the low rates of contamination in chicken meat and AMR of the isolates, virulent L. monocytogenes contamination in food should still be acknowledged.

DOI： 10.1016/j.micpath.2023.106333

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DOI： https://doi.org/10.1016/j.micpath.2023.106333

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DOI： https://doi.org/10.1016/j.ijfoodmicro.2023.110295

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DOI： https://doi.org/10.3390/antibiotics12061077

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

Bacterial food poisoning cases due to Salmonella and E. coli O157:H7 have been linked with the consumption of a variety of food products, threatening public health around the world. This study describes the combined effects of a phage cocktail (STG2, SEG5, and PS5), EDTA, nisin, and polylysine against the bacterial cocktail consisting of S. typhimurium, S. enteritidis, and E. coli O157:H7. Overall, phage cocktail (alone or in combination with nisin or/and polylysine) not only showed great antibacterial effects against bacterial cocktail at different temperatures (4 °C, 24 °C, and 37 °C), but also totally inhibited the emergence of phage resistance during the incubation period. These results suggest that the combination of phages with nisin or/and polylysine has great potential to simultaneously control S. typhimurium, S. enteritidis, and E. coli O157:H7.

DOI： 10.3390/antibiotics12061077

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DOI： 10.1016/j.jfp.2023.100044

Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Food Microbiology  

As one major foodborne pathogen, Salmonella can cause serious food poisoning outbreaks worldwide. Bacteriophage therapy is increasingly considered as one of the promising antibacterial agents for the biocontrol of foodborne pathogens. In the current study, a lytic phage STG2 capable of infecting S. enteritidis and S. typhimurium was characterized, and its efficacy in reducing these foodborne pathogens in both planktonic and biofilm forms was evaluated on cabbage and various surfaces. Genomic characterization revealed that phage STG2 was Siphoviridae phage (Epseptimavirus genus) with a dsDNA genome comprising of 114,275 bp and its genome does not contain any genes associated to antibiotic resistance, toxins, lysogeny, or virulence factors. Additionally, phage STG2 exhibited great efficacy in reducing (>2 Log) planktonic cells on cabbage as well as the biofilms formed on cabbage, polystyrene, and stainless steel, suggesting that phage STG2 is capable of simultaneously controlling both S. enteritidis and S. typhimurium contaminations on food and food-related surfaces.

DOI： 10.1016/j.ijfoodmicro.2022.109999

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Toxicology in Vitro  

Enterohemorrhagic or Shiga toxin-producing Escherichia coli is a food-poisoning bacterium that grows in the intestine to produce Shiga toxin (Stx). In this study, the effects of 20 polyphenols on the cytotoxicity of Stx1 and Stx2 in Vero cells were investigated. Among these, epigallocatechin gallate, butein, isorhapontigenin, hesperetin, morin, luteolin, resveratrol, and rhapontigenin showed inhibitory effects on the cytotoxicity of Stxs at 0.4 mmol/L. Furthermore, Vero cells pre-treated with these polyphenols were resistant to Stx at 0.4 mmol/L. However, luteolin showed the most potent inhibitory and cytoprotective effect against Stxs at 0.08 mmol/L or more. This inhibitory mechanism of luteolin was determined using a cell-free protein synthesis system and quantitative reverse transcription PCR assay to detect depurination of 28S rRNA in Vero cells. Luteolin did not inhibit the cell-free protein synthesis by Stxs, suggesting that the enzymatic activity of the Stx A subunit was not inhibited by luteolin. The depurination of 28S rRNA by Stxs was also investigated in Vero cells. The 28S rRNA depurination by Stxs was suppressed in Vero cells treated with Stxs which had been pretreated with luteolin. These results suggest that luteolin inhibits the incorporation of Stxs into Vero cells. This is the first report to show that luteolin inhibits the cytotoxicity of both Stx1 and Stx2 by inhibiting the incorporation of Stxs into Vero cells.

DOI： 10.1016/j.tiv.2022.105537

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DOI： 10.3136/fstr.FSTR-D-22-00074

Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Food Microbiology  

Clostridium perfringens is a major cause of foodborne disease in developed countries. The aim of this study was to isolate and characterize phages specific to C. perfringens to evaluate the most efficient phage cocktail for the biocontrol of C. perfringens, both in vitro and in curry roux. In this study, four phages were isolated from chicken meat and were morphologically and genetically characterized along with two phages previously isolated in our laboratory that display different host lysis spectra. Phage cocktail CP11, consisting of phages CPQ3, 7, 8, and 10, showed the broadest host range. Electron micrograph images suggested that all four phages belong to the Podoviridae family, and none of them carry any antibiotic resistance or toxin genes. Notably, the phages were stable at various pH values and in curry roux. Cocktails consisting of six, five, and four phages at the same concentrations were examined to determine the most effective phage cocktail. Phage cocktail PC11 significantly decreased the viable count of C. perfringens to a value less than the lower detection limit up to 48 h at both 8 and 37 °C in broth and at 24 °C in the curry roux. These results suggest that phage cocktail PC11 is a promising natural biocontrol agent against C. perfringens in vitro and in curry roux.

DOI： 10.1016/j.ijfoodmicro.2022.109886

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

Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persist in leafy green vegetables and subsequently produce biofilms. Biofilm formation is an ongoing concern in the food industry as biofilms are relatively resistant to a variety of antimicrobial treatments. In the present study, we evaluated the combined effects of phage FP43 and mild-heated slightly acidic hypochlorous water (SAHW) in reducing established biofilms on lettuce. Prior to the sequential treatments involving phage-SAHW and SAHW-phage for long-term storage, equal inoculum densities of E. coli O157:H7 and E. coli O91:H- were added on iceberg lettuce surfaces and the lettuce samples were stored at 10 °C for 48 h to allow biofilm formation. The sequential treatment with phage FP43 and SAHW significantly decreased the number of adhered cells, especially the combination of phage FP43 at 25 °C for 2 h and mild-heated SAHW, which considerably eliminated E. coli viable biofilm cells to undetectable levels (>3 log CFU/piece). However, the biofilms were not completely removed, as evidenced via SEM observation. Additionally, sequential treatment with SAHW and phage caused continuous reductions in viable counts, decreasing the viability of E. coli O157:H7 and total E. coli to the lower limit of detection after incubation for 5 d. Meanwhile, bacterial regrowth was observed after treatment with SAHW alone. These results indicated that the combination of phage and SAHW could be considered as a promising strategy to control the formation of E. coli O157:H7 biofilms on lettuce.

DOI： 10.1016/j.fm.2022.104010

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：International Journal of Food Microbiology  

Salmonella enterica subsp. enterica serotype Typhimurium (S. Typhimurium) is one of the most prevalent foodborne pathogens responsible for food poisoning and is spread through the consumption of contaminated poultry products. In this study, four antimicrobial peptides (AMPs) with varying hydrophobicity and helical structure-forming tendencies were designed and synthesized based on the amino acid sequences of peptides from egg white hydrolysate. Two of these AMPs, P1R3 (KSWKKHVVSGFFLR) and P1C (KSWKKHVVSGFFLRLWVHKK), exhibited inhibitory activity against S. Typhimurium and compromised its biofilm-forming ability. Investigation of their modes of action revealed that P1R3 and P1C interact with and permeabilize the cytoplasmic membrane of bacteria, leading to membrane potential dissipation, damage to membrane integrity, and consequent bacterial death. P1R3 also bound to S. Typhimurium DNA, resulting in DNA aggregation or precipitation. Moreover, both peptides showed negligible cytotoxicity to Vero cells, and P1C displayed significant antimicrobial activity in chicken meat. Peptides P1R3 and P1C, therefore, have the potential to be developed as promising food preservatives, especially against pathogenic S. Typhimurium.

DOI： 10.1016/j.ijfoodmicro.2022.109802

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Clostridium perfringens is one of the most important foodborne pathogens in developed countries. It causes severe food poisoning outbreaks worldwide, along with mortality and economic losses. Recently, bacteriophages have been investigated as an alternative tool to control pathogenic bacteria in the food industry. In this study, 19 Clostridium perfringens and 6 Clostridium perfringens bacteriophages were isolated from chicken meat. According to host range and stability tests, bacteriophage CPQ1 showed high thermostability and the broadest host range. The electron micrograph image of this bacteriophage suggested that it belongs to the Picovirinae subfamily of the Podoviridae family. Nucleotide sequence analysis of the genomic DNA indicated the absence of any antibiotic resistance, toxin, or virulence genes. In broth, CPQ1 showed strong lytic activity with a low MOI of 1, decreasing the OD600 of Clostridium perfringens cell suspension from 0.2 to 0.02 at 37 °C in 2 h. In pasteurized milk and chicken meat, CPQ1 with an MOI of 10 also caused a significant decrease in viable counts of Clostridium perfringens compared to the bacteriophageless control at both 24 °C and 37 °C. This is the first report on the application of bacteriophage to control Clostridium perfringens in foods.

DOI： 10.1016/j.ijfoodmicro.2021.109446

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The gene encoding LysSTG2, a novel endolysin from Salmonella-lytic bacteriophage STG2, has been cloned, overexpressed, and characterized previously. In this study, LysSTG2 was used to control Pseudomonas aeruginosa and P. putida, which showed high sensitivity to LysSTG2, in bottled water, milk, chicken breast, salmon, and the biofilms formed on the surface of polystyrene resin and stainless steel. In bottled water, LysSTG2 combined with EDTA reduced the viable counts of P. aeruginosa and P. putida by 2.2 log and to lower than the limit of detection, respectively. However, there was no significant decrease in viable counts in pasteurized milk artificially contaminated with these bacteria. Meanwhile, the addition of 1 mg/mL LysSTG2 alone reduced the viable counts of P. aeruginosa and P. putida by 0.6 log in chicken and 1.1 log in salmon samples contaminated with these bacteria, respectively. Further reduction in viable counts in combination with EDTA was not observed. Additionally, a strong effect of LysSTG2 on the removal of P. aeruginosa and P. putida biofilms was observed on the polystyrene resin and stainless steel surfaces, displaying more than 99.9% decrease in viable biofilm cells after 2 h of treatment at 37 °C. The results of this study suggest that LysSTG2 has the potential to control both planktonic and biofilm cells of Pseudomonas. Further investigations are required to optimize and improve the use of the endolysin for application in food and food processing facilities.

DOI： 10.1016/j.foodcont.2021.108460

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DOI： 10.3136/fstr.FSTR-D-21-00195

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

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

DOI： 10.1016/j.fm.2021.103853

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

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

DOI： 10.1016/j.fm.2021.103791

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

DOI： 10.1128/Spectrum.00141-21

Language：English   Publishing type：Research paper (bulletin of university, research institution)  

DOI： 10.5109/4363550

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

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

© 2020 Elsevier Ltd This study describes the characterization of bacteriophage FP43, active against pathogenic and non-pathogenic strains of Escherichia coli, and its ability to inhibit and remove a mixed biofilm of enterohemorrhagic E. coli O157:H7 and O91:H-. Phage FP43 is a member of the Myoviridae family, having a dsDNA genome consisting of 169,248 bp with good stability. It has a short latent period and large burst size. Phage FP43 decreased the formation of biofilm, comprising E. coli O157:H7 and O91:H-, by 82.4&#37; at 30 °C. After incubation for 6 h with FP43, viable counts of E. coli O157:H7 and total cells in the biofilm were reduced by 2.76 and 2.85 log, respectively. In planktonic cells, viable E. coli O157:H7 and total counts decreased by 3.44 and 3.62 log, respectively, after incubation with the phage for 4 h. Moreover, more than 60&#37; of an established, mixed biofilm was removed after a 6 h exposure to the phage, in which E. coli O157:H7 and total viable counts decreased by 2.07 and 1.93 log, respectively. These results suggest that bacteriophage FP43 has broad host range against both pathogenic and non-pathogenic E. coli, and potential to reduce viable counts of both enterohemorrhagic E. coli O157:H7 and E. coli O91 in mixed-strain biofilm.

DOI： 10.1016/j.lwt.2020.109945

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

© 2020 The Authors Objectives: Staphylococcus aureus is an important nosocomial pathogen that produces various extracellular toxins. Epigallocatechin gallate (EGCg) is a polyphenol that is abundant in green tea. EGCg displays strong antibacterial activity against Gram-positive bacteria. The effect of EGCg on gene expression by S. aureus was investigated to clarify the mechanism underlying its antibacterial action. Methods: Microarray analysis was performed on S. aureus treated with or without 500 mg/L EGCg. Differentially expressed genes were identified and their changes at the transcription level were confirmed using real-time quantitative polymerase chain reaction (qPCR). The membrane potential of cells treated with or without EGCg were observed under fluorescence microscopy. Results: Microarray analysis revealed that EGCg treatment of S. aureus resulted in increased and decreased transcription of 75 and 72 genes, respectively. Increased transcription exceeding 1-log2-fold change of genes related to membrane transport included gntP, gntK, rumA, SAOUHSC_02723, SAOUHSC_01311, and vraS. Decreased transcription was observed in genes involved in toxin production and stress response (hlgA, SAOUHSC_01110, hly, hlgB, efb, and hlgC). All changes in transcription were confirmed using real-time qPCR. The membrane potential of S. aureus treated with 500 mg/L EGCg markedly decreased, indicating that EGCg damaged the cell membrane. Conclusions: S. aureus increases the transcription of genes involved in membrane transport to recover membrane function. EGCg can potentially serve as a natural antibacterial agent to control the growth and toxin production of S. aureus.

DOI： 10.1016/j.jgar.2020.06.006

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

© 2020 American Society for Microbiology. YafQ is an endoribonuclease toxin that degrades target gene transcripts such as that of tnaA, a gene encoding tryptophanase to synthesize indole from tryptophan. DinJ is the cognate antitoxin of YafQ, and the YafQ-DinJ system was reported to regulate persister formation by controlling indole production in Escherichia coli. In this study, we investigated the role of YafQ-DinJ, indole production, and persister population in bacterial heat tolerance. yafQ (ΔyafQ), dinJ (ΔdinJ), and tnaA (ΔtnaA) single-gene knockout mutants showed approximately 10-fold higher heat tolerance than wild-type (WT) E. coli BW25113. Persister fractions of all mutants were slightly larger than that of the WT. Interestingly, these persister cells showed an approximately 100-fold higher heat tolerance than normal cells, but there was no difference among the persister cells of all mutants and the WT in terms of heat tolerance. Indole and its derivatives promoted a drastic reduction of bacterial heat tolerance by just 10 min of pretreatment, which is not sufficient to affect persister formation before heat treatment. Surprisingly, indole and its derivatives also reduced the heat tolerance of persister cells. Among the tested derivatives, 5-iodoindole exhibited the strongest effect on both normal and persister cells.

DOI： 10.1128/AEM.00935-20

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

The pathogenicity of Salmonella Typhimurium, a foodborne pathogen, is mainly attributed to its ability to form biofilm on food contact surfaces. epsilon-polylysine, a polymer of positively charged lysine, is reported to inhibit biofilm formation of both gram-positive and gram-negative bacteria. To elucidate the mechanism underlying epsilon-polylysine-mediated inhibition of biofilm formation, the transcriptional profiles of epsilon-polylysine-treated and untreated Salmonella Typhimurium cells were comparatively analysed. The genome-wide DNA microarray analysis was performed using Salmonella Typhimurium incubated with 0.001&#37; epsilon-polylysine in 0.1&#37; Bacto Soytone at 30 degrees C for 2 h. The expression levels of genes involved in curli amyloid fibres and cellulose production, quorum sensing, and flagellar motility were downregulated, whereas those of genes associated with colanic acid synthesis were upregulated after treatment with epsilon-polylysine. The microarray results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, treatment with epsilon-polylysine decreased the production of colanic acid in Salmonella Typhimurium. The findings of this study improved our understanding of the mechanisms underlying epsilon-polylysine-mediated biofilm inhibition and may contribute to the development of new disinfectants to control biofilm during food manufacturing and storage.

DOI： 10.1007/s00253-020-10575-2

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

Staphylococcus aureus is a notorious foodborne pathogen since it has ability to produce variety of toxins including heat-stable enterotoxin, form biofilm, and acquire resistance to antibiotics. Biocontrol of foodborne pathogens by lytic bacteriophages garners increasing interest from both researchers and food industry. In the present study, 29 phages against S. aureus were successfully isolated from chicken, pork, and fish. Characterization of the isolates revealed that phage SA46-CTH2 belonging to Podoviridae family had a number of features suitable for food industry applications such as wide host range, short latent period, large burst size, high stress tolerance, and a genome free of virulence genes. Furthermore, phage SA46-CTH2 alone or in combination with nisin exhibited great efficacy in reducing planktonic and biofilm cells of S. aureus at various conditions tested. The combination of phage SA46-CTH2 and nisin was also found to be able to inhibit the regrowth of S. aureus at both 37 and 24 degrees C. Key points center dot A total of 29 S. aureus phages were successfully isolated from fish, pork, and chicken products. center dot Phage SA46-CTH2 was characterized by host range, morphology, and genome sequencing. center dot SA46-CTH2 significantly reduced both planktonic and biofilm cells of S. aureus. center dot Combination of SA46-CTH2 and nisin inhibited the regrowth of S. aureus.

DOI： 10.1007/s00253-020-10581-4

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

Salmonella Enteritidis, Salmonella Typhimurium, and Escherichia coli O157:H7 are the most important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages are increasingly considered as novel antibacterial agents to control foodborne pathogens. In this study, 8 Salmonella phages and 10 E. coli O157:H7 phages were isolated from chicken products. A polyvalent phage PS5 capable of infecting S. Enteritidis, S. Typhimurium, and E. coli O157:H7 was further characterized and its efficacy in reducing these foodborne pathogens was evaluated in in vitro and in foods. Morphology, one-step growth, and stability assay showed that phage PS5 was a myovirus, with relatively short latent periods, large burst sizes, and high stability. Genome sequencing analysis revealed that the genome of PS5 does not contain any genes associated to antibiotic resistance, toxins, lysogeny, and virulence factors. In broth, phage PS5 significantly decreased the viable counts of all the three bacterial hosts by more than 1.3 log CFU/mL compared to controls after 2 h of incubation at 4 degrees C and 24 degrees C. In foods, treatment with PS5 also resulted in significant reductions of viable counts of all the three bacterial hosts compared to controls at temperatures tested. This is the first report on single phage capable of simultaneously controlling S. Enteritidis, S. Typhimurium and E. coli O157:H7 in both in vitro and in foods.

DOI： 10.1016/j.foodres.2020.108977

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

We investigated the characteristics of Staphylococcus aureus isolates causing bovine subclinical mastitis (SCM), and their genetic relatedness with the isolates obtained from Egyptian cheese. Twenty-five S. aureus isolates were identified from 150 SCM milk and 75 cheese samples. The antibiogram revealed multidrug-resistant (MDR) isolates. Fifteen isolates were categorised as methicillin-resistant. Antimicrobial resistance and virulence genes were detected. Spa typing and SCCmec classification were performed. More than 50&#37; of isolates were found carrying human-specific virulence determinants, while other isolates characterised were presumed to be of bovine-origin. Spa-types t304, t688, t084 corresponded isolates from SCM milk and cheese samples; the similar genotypes from SCM and cheese displayed divergence in virulence traits. Moreover, our results revealed the novel spa-type t18546. Animals and dairy food could be a reservoir for transformative changes in S. aureus virulence, leading to the emergence of virulent MDR strains that may become potential public-health threats. (C) 2020 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.idairyj.2020.104646

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

© 2018 The Society for Applied Microbiology Aim: The purpose of this study was to clarify the mechanism of the antibacterial action of two high potential and natural food additives, epigallocatechin gallate (EGCg) and theaflavin-3,3′-digallate (TF3), on Clostridium perfringens. Methods and Results: Minimal inhibitory concentrations were determined by the serial dilution method. Afterwards, the cells were treated with 250 or 1000 mg l −1 of EGCg and 125 or 500 mg l −1 of TF3 and morphological changes were observed and cell sizes were also measured under fluorescence microscopy. Our results showed that TF3 had a twice stronger antibacterial activity than EGCg against C. perfringens. Phase-contrast and fluorescence microscopy confirmed that the bacterial cells elongated without DNA segregation and septum formation in the presence of 250 mg l −1 EGCg. While in the higher concentration of EGCg and TF3, cell growth was suppressed. Bacterial cells reached to around 12 μm after the 24 h incubation with 250 mg l −1 EGCg, but the cells were shorter than the control at 1000 mg l −1 of EGCg. After washing and incubating the elongated cells in fresh medium, DNA segregated at 2 h of incubation. The average cell length decreased gradually and reached the normal size at 8 h. Conclusion: It seems that EGCg at a low concentration affected the proteins involved in the septum formation, DNA segregation and cell division. Furthermore, the high concentration of EGCg and TF3 seemed to cause stronger cellular damage to C. perfringens. Significance and Impact of the Study: These polyphenols are widely distributed in all higher plants especially in tea plants, and people tend to use natural food additives rather than synthetic ones. EGCg and TF3, as natural food additives, can prevent C. perfringens food poisoning along with other potential health benefits.

DOI： 10.1111/jam.14134

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

Increased foodborne outbreaks associated with low-moisture foods contaminated with Salmonella have raised the need for further insights into their possible causes and control measures. This study investigated the effects of sucrose-induced low water activity (a(w)) on heat resistance and global gene expression in Salmonella Typhimurium. Following heat treatment at 60 degrees C for 5 min, viable cell counts on TSA of the cells grown in TSB supplemented with 35 &#37; (w/v) sucrose for 24 h and resuspended in the same medium were 3-Log higher than those grown and resuspended in TSB without sucrose, and 1-Log higher than the cells grown in TSB and resuspended in TSB with 35 &#37; sucrose. Viability of the cells directly transferred from TSB to preheated TSB with sucrose was positively correlated with sucrose concentration. DNA microarray analysis identified sixteen up-regulated genes involved in cobalamin biosynthesis in the cells grown in the presence of 35 &#37; sucrose. Deletion of the pocR gene, which positively regulates cobalamin biosynthesis, resulted in suppression of the improvement in heat resistance of S. Typhimurium under sucrose-induced low a(w), suggesting potential contribution of this gene in increasing heat resistance of S. Typhimurium.

DOI： 10.3136/fstr.25.903

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

In this study, the antimicrobial resistance profiles of L. monocytogenes isolated from chicken meat in Fukuoka in 2017 were compared with the isolates of 2012. A total of 85 and 50 chicken meat samples, including different body parts, were collected from different supermarkets in Fukuoka in 2012 and 2017, respectively. Detection, isolation, identification, and characterization of L. monocytogenes were performed according to the conventional methods. Forty-five among 85 samples (53&#37;) were positive for L. monocytogenes in 2012, while 12 among 50 samples in 2017 (24&#37;) tested positive. One hundred fifty-three and 29 L. monocytogenes strains were isolated in 2012 and 2017, respectively. The serotypes of isolates in 2012 were 1/2a (21.5&#37;), 1/2b (73.9&#37;), 1/2c (1.5&#37;), and 4b/4e (3.1&#37;). In contrast, the 2017 isolates showed 1/2a (48.3&#37;) and 1/2b (51.7&#37;) serotypes. While all isolates in 2012 were positive for hlyA (listeriolysin O) in the PCR assay with hlyA primer set 7, only 17 hlyA positive isolates were seen in 2017. Moreover, 75 isolates with different ribotypes in 2012 and 29 isolates in 2017, respectively, were tested for antimicrobial susceptibility by broth microdilution for 18 different antimicrobial agents. Most of the 2012 and 2017 isolates displayed antimicrobial susceptibility. However, among the 2012 and 2017 isolates, 98.7&#37; and 100&#37; of the isolates were resistant to cefoxitin, 57.3&#37; and 95.7&#37; to fosfomycin, 72.0&#37; and 82.6&#37; to oxacillin, 8.0&#37; and 17.4&#37; to clindamycin, respectively. In addition, 2.7&#37; of the isolates in 2012 were resistant to flomoxef and 4.3&#37; of the isolates in 2017 to linezolid. Multidrug resistance (MDR) to 3 or more antimicrobials was observed in 35/75 (46.7&#37;) isolates of 2012 and 19/23 (82.6&#37;) in 2017. Detection of antimicrobial resistance (AMR) genes by PCR showed that the resistant isolates of 2012 were positive for mecA (96.3&#37;) and ermC (83.3&#37;), whereas the resistant isolates in 2017 screened positive for mecA (94.7&#37;) and mefA (25.0&#37;). Other cfxA, ermA, ermB, fosA, fosB, and fosC genes were absent in the PCR assay for any of the isolates. This study investigated for the first time the change in the L. monocytogenes contamination of chicken meat and antibiotic resistance of the isolated L. monocytogenes strains in Fukuoka, Japan, in the course of 5 years. Although the contamination rate of L. monocytogenes in 2017 was found to be lower than that in 2012, AMR of the isolates in 2017 was higher.

DOI： 10.1016/j.ijfoodmicro.2019.05.016

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

Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important foodborne pathogen. Baicalein (5,6,7-trihydroxylflavone), a flavone isolated from the roots of Scutellaria baicalensis, is considered as a potential antibacterial agent to control foodborne pathogens. Among seven compounds selected by in silico screening of the natural compound database, baicalein inhibited the cytotoxicity of both Shiga toxins 1 and 2 (Stx1 and Stx2) against Vero cells after pretreatment at 0.13 mmol/L. In addition, baicalein reduced the susceptibility of Vero cells to both Stx1 and Stx2. Real-time qPCR showed that baicalein increased transcription of stx1 but not of stx2. However, baicalein had no effects on production or secretion of Stx1 or Stx2. Docking models suggested that baicalein formed a stable structure with StxB pentamer with low intramolecular energy. The results demonstrate that inhibitory activity of baicalein against the cytotoxicity of both Stx1 and Stx2 might be due to of the formation of a binding structure inside the pocket of the Stx1B and Stx2B pentamers.

DOI： 10.3390/toxins11090505

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

Sodium chloride maintains osmotic pressure of living cells including bacteria. Heat treatment is common for decontamination of bacteria in food. In this study, effects of NaCl on heat resistance of Salmonella Typhimurium were investigated. After cultivation in TSB containing 0.5&#37; (TSB), 4&#37; (4SC) and 8&#37; (8SC) NaCl, S. Typhimurium cells were heated at 60 degrees C for 20 min. Total viable counts including intact cells and injured but recoverable cells determined by the plating method using TSA of S. Typhimurium cultured in 4SC and 8SC were significantly higher than those of the cells cultured in TSB. Meanwhile, changes of gene transcription were analyzed by DNA microarray. Transcription of genes involved in the colanic acid synthesis largely increased after cultivation in 4SC and 8SC. The amount of colanic acid significantly increased in the cells cultured in 4SC compared to that in M9-glucose medium. After recovery culture for 3 h, the genes involved in the phage shock response strongly up-regulated, suggesting contribution of these gene products in recovery of heat injured cells. The outcome of this study contributes to understand the mechanism of cross protection in Salmonella.

DOI： 10.1016/j.lwt.2019.02.056

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

This study investigated the effect of epigallocatechin gallate (EGCg) on Staphylococcus aureus to determine its mechanism of antibacterial action. Adsorption of EGCg on the cell envelope of S. aureus after EGCg treatment was demonstrated using a FITC-labeled antibody specific to EGCg. After EGCg treatment of S. aureus for 4 h, abnormalities in septum formation and cell segregation were observed at concentrations greater than 250 mg/L, and debris presumed to arise from cell destruction or leakage of cytoplasmic materials was observed around the cells at 500 mg/L. Two-dimensional electrophoresis of proteins prepared from EGCg-treated S. aureus cells revealed the presence of 18 protein spots that disappeared or showed markedly decreased intensity compared to those from control cells. These proteins included DnaK, elongation factor G, DNA-directed RNA polymerase, L-lactate dehydrogenase, pyruvate dehydrogenase, and acetate kinase. Furthermore, S. aureus showed decreased glucose uptake after EGCg treatment. These results suggest that EGCg inhibits the functions of cell-envelope proteins, and it causes cellular damage and disruption of the cells in S. aureus.

DOI： 10.3136/fstr.25.277

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

To develop a method for the identification of shiikuwasha (Citrus depressa Hayata) and calamondin (Citrus madurensis Lour.), trnL-trnF and trnT-trnL intergenic spacer regions of their chloroplast DNA were amplified using PCR and the nucleotide sequences were determined. In each region, a single nucleotide polymorphism (SNP) site specific to the respective citrus species (shiikuwasha and calamondin) was found. For species discrimination using PCR, two forward primers containing the allele-specific SNP site at the 3'-end and a mismatched nucleotide at the 3rd base from the 3'-end were designed. The allele-specific forward primers specific to shiikuwasha and calamondin were respectively designated CiDeLF-F and CiMaTL-F. To confirm the specificity of the designed primers, PCR was carried out with DNA prepared from citrus peel or hand-squeezed juice as the template. Results showed that shiikuwasha and calamondin fruits and juices were identifiable by PCR using the allele-specific primers. Furthermore, this allele-specific PCR method can be applied to industrially processed and concentrated juice by amplifying DNA in advance.

DOI： 10.3136/fstr.25.19

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

Shiga toxin-producing Escherichia coli (STEC) O157:H7 and extended-spectrum beta-lactamase (ESBL) producing E. coli (ESBLEC) are important bacteria of public health concern and frequently isolated from raw beef products. Bacteriophage-based methods have been increasingly exploited to control bacterial contamination in meats. Here, we describe the isolation, characterization, and application of a lytic phage PE37 for the simultaneous bio-control of STEC O157:H7 and ESBLEC. Phage PE37, isolated from the bovine intestine, was morphologically characterized as a member of the Myoviridae family, with a broad host range and great stability under various stress conditions. Sequencing analysis revealed that the genomic DNA of phage PE37 contains genes that contribute to virion structure, replication, assembly, and host lysis. PE37 significantly reduced the viable counts of STEC O157:H7 by 4.9 and 2.6logCFU/mL in broth after 6h of incubation at 25 and 8 degrees C, respectively. Application of phage PE37 to raw beef artificially contaminated with STEC O157:H7 resulted in significant reductions in the viable counts by 2.3 and 0.9logCFU/piece after 24h of storage at 25 and 8 degrees C, respectively. Treatment of raw beef contaminated with a bacterial cocktail of STEC O157:H7 and ESBLEC with PE37 also significantly decreased the viable counts of the bacterial mixture by 1.4 and 1.0logCFU/piece after 24h of incubation at 25 and 8 degrees C, respectively. These findings suggest that bacteriophage PE37 may be a potential bio-agent for controlling STEC O157:H7 and ESBLEC contamination in raw beef.

DOI： 10.1007/s00253-018-9399-1

Language：English   Publishing type：Research paper (bulletin of university, research institution)  

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

Chicken meats are considered as main sources associated with Salmonella infections in humans. In this study, lytic phages against Salmonella were isolated and examined for their efficacy to control Salmonella. Eighteen lytic phages were isolated from raw chicken skin and gizzard. Five phages belonging to Myoviridae and Siphoviridae families were characterized and selected for bacterial challenge tests. The treatment of raw chicken breast samples contaminated with S. Enteritidis and S. Typhimurium at 8 degrees C by the cocktail of five phages significantly reduced (P < 0.05) viable counts by 1.41 and 1.86 log CFU/piece, respectively. When incubated at 25 degrees C, the highest reductions of viable counts of S. Enteritidis and S. Typhimurium in the phage-treated samples were 3.06 and 2.21 log CFU/piece, respectively (P < 0.05). These data suggested that the phages isolated from raw chicken meats are potential agents for controlling Salmonella in raw meats.

DOI： 10.1016/j.lwt.2018.01.072

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

Sublethally heat-injured cells of Salmonella in food can recover under favorable conditions, leading to foodborne illness. To elucidate the molecular mechanism of recovery from heat injury, the global changes in gene transcription of Salmonella Typhimurium were investigated in previous study. In this study, the functions of genes involved in phage shock response (viz., phage shock protein (psp) genes), the transcription levels of which were found in previous study to be increased during recovery from heat injury, were investigated in recovering cells. The increase in pspABCDEFG transcription levels during the recovery process was confirmed by qRT-PCR. To understand the role of psp genes in heat injury recovery, a pspA deletion mutant (Delta pspA) and a pspA-overexpressing strain (S. Typhimurium pBAD30/pspA(+)) were constructed. Delta pspA showed slightly lower viable counts and membrane potential than those of the wild-type strain during recovery. On the other hand, there was no significant difference in the viable counts between S. Typhimurium pBAD30/pspA(+) and the control strains S. Typhimurium pBAD30/pspA (-) and S. Typhimurium pBAD30(+) during recovery. It would seem that a lack of PspA protein alone somewhat affects the recovery of S. Typhimurium from heat injury, but overexpression of PspA alone is not sufficient to overcome this effect.

DOI： 10.4265/bio.23.17

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

The aim was to isolate Campylobacter jejuni-specific lytic phages from meats on the market in Japan. These phages were effectively isolated from 13 of 15 (86.7&#37;) retail chicken meat samples (skin and liver) by the enrichment method using Preston Campylobacter Selective Enrichment Broth and 10 host Campylobacter strains. Among the 26 phage isolates, 14 were extracted by means of C. jejuni L26 as a host strain. Phage PHC10 showed the broadest lytic spectrum: active against 67.4&#37; of the 46 C. jejuni strains tested. The other phage isolates showed different lytic spectra. Because phages PHC5, PHC10, PHC19, PHC22, and PHC25 possess an icosahedral head and a contracted tail, they seem to be members of the Myoviridae family. Effects of 19 phage isolates on viability of C. jejuni were investigated. These phages reduced viable counts of C. jejuni by 1-3 log after 6-12 h of incubation at 42 degrees C as compared to the initial counts. The C. jejuni L26 was found to be suitable as a host because of the wide hosting range. The phages isolated in this study seem to be promising biocontrol agents against C. jejuni in food.

DOI： 10.4265/bio.22.213

Other Link： https://www.jstage.jst.go.jp/article/bio/22/4/22_213/_article/-char/en

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

Detection of foodborne pathogens at very low levels is still a challenge. A custom-built multichannel surface plasmon resonance (SPR) biosensor and simultaneous enrichment broth (SEB) were used to develop a simultaneous detection method for 3 important foodborne pathogens, Escherichia coli O157:H7 (O157:H7), Salmonella enteritidis, and Listeria monocytogenes, at a very low level. These 3 foodborne pathogens at a very low level (14, 6, and 28 CFU/25 g (mL) for O157:H7, S. enteritidis, and L. monocytogenes, respectively) were inoculated in SEB and incubated at 37 C for 24 h. Sample prepared from the simultaneous enrichment culture was analyzed using the multichannel SPR biosensor and sensor chip immobilized with polyclonal antibodies specific to each of the target pathogens. O157:H7, S. enteritidis, and L. monocytogenes in chicken were detected simultaneously at an inoculum dose of 14, 6, and 28 CFU/25 g, respectively. Our method using a custom-built multichannel SPR biosensor and enrichment in SEB is expected as a rapid and simultaneous detection method for low levels of O157:H7, S. enteritidis, and L. monocytogenes in food.

DOI： 10.1111/1750-3841.13843

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

Catechins are major polyphenolic compounds of green tea. To investigate mechanism for antibacterial action of catechins, 11 monoclonal antibodies (MAbs) were raised against a 3-succinyl-epicatechin (EC) -keyhole limpet hemocyanin (KLH) conjugate. Amino acid sequences of variable regions determined for MAbs b-1058, b-1565, and b-2106 confirmed their innovative character. MAb b-1058 strongly interacted with its target substances in the following order of magnitude: theaflavin-3,3'-di-O-gallate (TFDG) > theaflavin-3-O-gallate (TF3G) >= theaflavin-3'-O-gallate (TF3'G) > gallocatechin gallate (GCg) > penta-O-galloyl-beta-D-glucose (PGG) > epigallocatechin gallate (EGCg), as determined using surface plasmon resonance (SPR) on MAb-immobilized sensor chips. The affinity profiles of MAbs b-1058 and b-2106 to the various polyphenols tested suggested that flavan skeletons with both carbonyl oxygen and hydroxyl groups are important for this interaction to take place. S. aureus cells treated with EGCg showed green fluorescence around the cells after incubation with FITC-labeled MAb b-1058. The fluorescence intensity increased with increasing concentrations of EGCg. These MAbs are effective to investigate antibacterial mechanism of catechins and theaflavins.

DOI： 10.1016/j.mimet.2017.03.014

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

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

To understand the effects of pretreatment of lettuce during cultivation with 0.001&#37; epsilon-polylysine (PL) in combination with 0.25&#37; milk serum protein (MSP) on the attachment of Salmonella Enteritidis, lettuce leaves were contaminated with S. Enteritidis after a 1-day treatment with food additives, and harvested after cultivation for 1 day. Viable S. Enteritidis counts on lettuce leaves pretreated with the PL and MSP mixture were significantly reduced from 5.7 log CFU/g to 1 log CFU/g after decontamination by washing with water and a subsequent treatment with NaCIO. The viable S. Enteritidis counts on radish sprouts grown for 7 d in the presence of 0.01&#37; PL from seeds inoculated with S. Enteritidis were reduced to 3.1 log CFU/50 sprouts after NaCIO decontamination. These counts were significantly lower (P < 0.05) than those of plants without additive(s). The treatment with additive(s) did not affect the ascorbic acid and chlorophyll contents of both plants.

DOI： 10.3136/fstr.22.703

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

Extended Spectrum Beta-Lactamase-producing Escherichia coli (ESBLEC) were isolated from 12/13 (92.3&#37;) raw chicken meat samples by using selective culture and PCR. Of the 27 ESBLEC analyzed, 33.3&#37; (9/27) of isolates were positive for ESBL of CTX-M group 1 followed by TEM (22.2&#37;), SHV (22.2&#37;), CTX-M group 2 (11.1&#37;), and CTX-M group 9 (11.1&#37;). None of ESBLEC tested were positive for stx(1), stx(2), eae, ehxA, saa, and subAB genes of Shiga toxin-producing E. coli. Among 22 isolated phages, phages PBL66-CL1 and PBL116-CS6 infected 21 (77.7&#37;) and 20 (74&#37;) of 27 ESBLEC isolates examined, respectively. The remaining phages lysed less than 50&#37; of the hosts tested. Compared to non-treatment, the treatment of isolate EBL116 in the broth medium with phage cocktail of PBL66-CL1 and PBL116-CS6 at 25 degrees C and 5 degrees C after 6 h significantly reduced bacterial viable counts by 5.06 and 133 log CFU/mL, respectively. When the treatment was performed on raw chicken meat samples, viable counts of EBL116 were also decreased by 2.02 and 1.67 log CFU/4 cm(2) meat piece after 6 hat 25 degrees C and 5 degrees C, respectively. This study demonstrates a high prevalence of ESBLEC in raw chicken meat and possible use of lytic phages as bactericide for controlling ESBLEC. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.lwt.2016.04.013

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

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

Subtilase cytotoxin (SubAB) is an important virulence factor of eae-negative Shiga toxin-producing Escherichia coli (STEC). Three variants of SubAB-encoding genes have been reported in the literature; however, the newly described subAB variant (subAB2-2) was found only in STEC strains from deer meat, sheep, and some wild animals. In this study, subAB variants were detected by PCR and DNA sequencing in 5 out of 12 (41.6&#37;) eae-negative STEC strains isolated from patients. Most subAB-positive STEC strains (80&#37;) harbored the subAB1 gene. The subAB2-2 gene was detected for the first time in the clinical STEC O128:H2 strain. Other virulence genes including stx1a, stx1c, stx2b, ehxA, and tia were also detected in this strain. The DNA sequence analyses of the subAB1 and subAB2-2 genes of the clinical STEC strains showed 99&#37; and 100&#37; identity to those of the reference strains 98NK2 and LM27558stx2, respectively. This is the first report on the detection of the subAB2-2 gene in a clinical STEC isolate.

DOI： 10.1139/cjm-2015-0519

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

Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4&#37; of tested samples. Among 25 STEC strains from meats, five strains (20&#37;) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80&#37;) were eae-negative. However, 28&#37; of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75&#37; of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32&#37; of these strains. The stx(2a) gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92&#37; of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30&#37; and 70&#37; strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8&#37;) and ehxA (96.7&#37;) genes and 92.5&#37; of them showed Stx productivity. Our study shows that some raw meat samples contain non-O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains.

DOI： 10.1111/1348-0421.12235

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

The study was conducted to examine the adhesion inhibition and antibacterial activities by combined use of some selected food additives such as Sucrose Fatty Acid Ester (SE) C18, Gardenia Yellow (GY), Monascus Pigment (MP), Protamine (PT), epsilon-polylysine (PL) and Milk Serum Protein (MSP), against Salmonella Enteritidis, Salmonella Typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. The adhesion of those pathogenic bacteria was reduced by several combination of food additives compared to that of each of the single use. The combinations decreased the relative adhesion more than 10&#37; compared to that of each of the single use were taken into consideration. The following combinations such as SE18 & GY, PT & MSP, and PL & MSP were effective in inhibiting the attachment of S. Enteritidis onto microtiter plate compared to that of each of the single use. In case of S. Typhimurium, the combination of MSP & MP, SE18 & GY, SE18 & PT, SE18 & PL, SE18 & MP, SE18 & MSP, MP & PT, MSP & PI, GY & PL, and MSP & PT were effective in adhesion inhibition. The combination of SE18 & GY, GY & MSP, and PL & MSP were effective in inhibiting the attachment of P. aeruginosa onto microtiter plate compared to that of each of the single use. The combination of GY & MSP, MSP & PT or PL and MP & PL were effective to inhibit the attachment of L monocytogenes. The adhesion of S. aureus was reduced by combined use of SE18 & PT, GY & PT, GY & PI, MSP & GY, MSP & PL, SE18 & GY, SE18 & MP, and MSP & PT compared to that of each of the single use. On the other hand, there were no such significant changes in viable counts of all pathogenic bacteria tested by using combination of food additives compared to that of each of the single use. However, the viable counts of S. Enteritidis and P. aeruginosa decreased drastically in the presence of PL (0.01&#37;) and PT (1&#37;), respectively and reached to the lower detection limit. In addition, the viable counts of L. monocytogenes decreased around 4 Log in the presence of PI. These results clearly showed that the combination of MSP and PT or PL effectively reduced adhesion of all the Gram-positive and negative pathogens tested on the microtiter plate. The pretreatment of cabbage leaves with combination of PL & MSP was also effective to reduce the viable counts of secondary-contaminated S. Enteritidis on the cabbage leaves by washing with water compared to that of untreated cabbage leaves. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodcont.2014.05.034

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

Mechanism of inhibitory action of 8 catechins and teaflavin was investigated at low concentration against Shiga-like toxin (Stx). Viability of Vero cells largely decreased in the presence of Stx1 and Stx2 preparations. Cytotoxicity of Stx1 decreased after preincubation with gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) at 100 mg/L. However, the cytotoxicity of Stx2 was not inhibited by the preincubation with catechins and teaflavin tested. The inhibitory activity of GCg and EGCg at 15 mg/L (0.0327 mM) was investigated against Stx preparations with various concentrations. The cytotoxicity of Stx1 at the concentration ranging from 1.6 to 50 ng/mL significantly reduced (p < 0.01) by the preincubation of Stx1 with GCg at 15 mg/L. Similarly, the cytotoxicity of Stx1 at the concentration ranging from 3.1 to 25 ng/mL was significantly reduced (p < 0.01) by the preincubation with EGCg at 15 mg/L. In contrast, GCg and EGCg did not inhibit cytotoxicity of Stx2 at any concentrations tested. On the other hand, EGC showed no significant effects on cytotoxicity of both Stx1 and Stx2 at the same concentrations tested. The pocket sizes formed at the center of the Stx1B and Stx2B pentamers were calculated to be. 778A(3) and 475 A(3), respectively. Docking simulations were conducted with EGCg positioned in the center of the pore of StxB pentamers. The docking models showed that EGCg formed 7 hydrogen bonds with side chains of amino acids faced inside the pocket of the Stx1B pentarmer with the lowest intramolecular energy (strain energy + electrostatic energy) of 0.1 kcal/mol. In contrast, in the case of Stx2B pentamer, EGCg formed 6 hydrogen bonds with the lowest intramolecular energy of 5.2 kcal/mol. In silico study suggested that EGCg forms more stable structure with Stx1B pentamer than Stx2B pentamer. These results indicated that both GCg and EGCg specifically inhibited cytotoxicity of Stx1 but not of Stx2. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodcont.2014.02.017

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

Surface plasmon resonance (SPR) biosensors were used to develop a rapid and simultaneous detection method for three important foodborne pathogens, i.e., Escherichia coli O157:H7 (0157), Salmonella Enteritidis (SE), and Listeria monocytogenes (LM). Bacterial homogenates prepared by sonication from bacterial suspensions at various cell concentrations were analyzed using SPR biosensors and sensor chips with polyclonal antibodies specific to each of the target pathogens. The precipitates from the homogenates were demonstrated to be suitable for specific and simultaneous detection of O157, SE, and LM. By using the precipitates and a custom-built multichannel SPR biosensor, the lower detection limit for O157, SE, and LM was determined to be 0.6 x 10(6), 1.8 x 10(6), and 0.7 x 10(7) CFU/mL, respectively, in the presence of non-target pathogens at concentrations of 10(5) to 10(8) CFU/mL.

DOI： 10.3136/fstr.20.317

Language：English  

Effects of Pretreatments on Detection of E. coli O157:H7 by SPR Biosensor

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

We have developed a method for quantifying gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) using a catechin-binding peptide (part of the 67-kDa laminin receptor). Using micro titer plates, we investigated various conditions, including the quantifiable range of EGCg concentrations, the optimal concentration of the catechin-binding peptide, and the optimal reaction conditions. In this microplate assay, after each well was coated with bovine serum albumin, sample containing GCg and EGCg was added at pH 8.0, and allowed to stand at 37 degrees C for 2 h. After washing, biotinylated-peptide solution was added at 1 mu g mL(-1) and allowed to react for 1 h at 37 degrees C. Each well was added with streptavidin -horseradish peroxidase conjugate, followed by chromogenic reaction for 25 min at room temperature. After the reaction, absorbance was measured at 405 nm. Our method is capable of quantifying EGCg in the range of approximately 0.1-2.0 mg L-1 with a high degree of sensitivity and a high correlation (R-2 = 0.98) between EGCg concentration and absorbance. The method was specific to GCg and EGCg and seems capable of estimating GCg and EGCg contents in the presence of other catechin compounds. The method is simple and highly sensitive for quantitative GCg and EGCg measurement that requires no special equipment or operation and can measure multiple samples simultaneously. (C) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodcont.2012.06.017

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

Tomatoes have recently been implicated as an important vehicle in outbreaks of produce-associated salmonellosis. Traceback reports suggested that pre-harvest contamination of Salmonella enterica might be the main reason for these outbreaks; however, the site of pathogen attachment remains unclear. Therefore, it is important to investigate the mechanisms of Salmonella contamination of fresh produce. To trace the presence of Salmonella in soil and plants, Salmonella Enteritidis transformed with a pEGFP plasmid vector (S. Enteritidis-EGFP) was used. Soil was artificially contaminated with S. Enteritidis-EGFP at 104, 106 or 108 CFU/g, followed by cultivation of tomato plants in the contaminated soil. Samples of the soil and each organ of the tomato (fruit, stems/leaves, and root) were assayed for Salmonella by plating onto Tryptic Soy Agar and using the MPN method. Salmonella levels in the soil gradually decreased over time, and soil persistence was dependent on the initial inoculation level. Salmonella levels were below the detection limit (< 100 CFU/g) in fruits and stems/leaves, regardless of the level of soil contamination. Moreover, S. Enteritidis-EGFP was not detected in the tomato fruits after root injury. These results indicate that the internalization of Salmonella in tomato fruits might not occur after cultivation in contaminated soil.

DOI： 10.3136/fstr.18.869

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

Mechanisms of recovery from heat injury in were elucidated. Recovery of the heat-injured cells in TSB resulted in full recovery after 3 h of incubation at 37A degrees C. The DNA microarray analysis of 30- and 60-min recovering cells resulted in an increase in transcription of 89 and 141 genes, respectively. Among them, 15 genes, with known function, seemed to be somewhat involved in recovery. They encoded proteins involved in branched-chain amino acid (BCAA) transport (, ), cell envelope integrity (), heat-shock response (, ), phage shock protein (), ribosome modulation factor (), virulence () transcriptional regulation (, , , , ) and ArcB signal transduction () and cytoplasmic membrane protein (). Among them, the effects of BCAA supplementation on recovery from heat injury were studied to confirm the importance of the BCAA transport genes during recovery. It was found that supplementation of TSB with 0.1&#37; BCAA resulted in an enhanced recovery of injured cells in comparison to those recovered in TSB without BCAA. Supplementation of BCAA at 0.1&#37; resulted in a cell count increase 4.4-fold greater than that of the control after 1 h incubation. It seems that BCAA promoted the recovery by promoting protein synthesis either directly through their use in translation or indirectly through stimulation of protein synthesis by activation of the Lrp protein.

DOI： 10.1007/s00726-011-0934-y

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

In this study, primer sets of L. monocytogenes virulence gene hlyA were designed for the L. monocytogenes-specific PCR assay. To evaluate the performance of these primer sets, the detection sensitivity and specificity were examined and the most suitable primer set was selected. Subsequently, it was subjected to compare the performance for detection of L. monocytogenes with commercially available kits (TaKaRa detection kit and ABI detection kit) on DNA level. Results of real-time PCR showed that the efficiency of this new primer set was 101.6&#37; while TaKaRa and ABI detection kit were 101.1&#37; and 107.4&#37;, respectively. The detection sensitivity of all three methods was equivalent to less than 1 copy per reaction using purified genomic DNA as template. Detection specificity were 100&#37; when testing L. monocytogenes isolates, and for other Listeria isolates were 15.4&#37;, 76.9&#37; and 100&#37; by the method using hlyA L. monocytogenes detection primer set 7, TaKaRa and ABI detection kit, respectively. In summary, this new PCR detection method is relatively sensitive and more specific to L. monocytogenes under certain conditions, could serve as a rapid detection method and has the potential for detection of L. monocytogenes from contaminated food.

DOI： 10.3136/fstr.18.47

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

Combined sequential treatment using 100 mg/L sucrose monopalmitate solution under microbubble generation and soaking in slightly acidic hypochlorous water containing 30 mg/L available chlorine for 5 min at 50 degrees C was tested for decontamination of ginger, Japanese ginger, perilla, parsley, Welsh onion and cucumber, and at 20 degrees C for strawberry. Viable bacterial count was reduced by about 2 log cfu/g in perilla, parsley, and Welsh onion. Ginger, parsley and Welsh onion maintained viable counts of less than 5 log cfu/g during 6 days of subsequent cold storage at 6 degrees C. Viable count for cucumber decreased by only 1 log cfu/g after combined treatment, and increased to 5.5 log cfu/g after storage for 6 days at 6 degrees C. For decontamination of strawberry, as 50 degrees C treatment with SAHW damaged the surface, the treatment was performed at 20 degrees C. After combined sequential treatment, viable bacterial count decreased from 4.5 to 2.0 log cfu/g, and increased slightly to 2.5 log cfu/g after storage at 6 degrees C for 6 days. Fungal count for strawberry also decreased from 4.9 to 2.3 log cfu/g immediately after treatment and did not increase after storage for 6 days. These results indicate the great potential of this approach in sanitization of fresh fruits and vegetables.

DOI： 10.3136/fstr.17.555

Language：English  

Language：Japanese  

Effects of Commercial Kitchen Detergents on Adhesion and Viability of Bacteria
Adhesion inhibition and antibacterial activities of 13 kitchen detergents were determined against S. Enteritidis IFO3313, P. aeruginosa NBRC 13275, P. fluorescens No. 3 and L. monocytogenes No. 185. All the detergents tested inhibited the adhesion of S. Enteritidis IFO 3313 to microplates more than 50&#37; at 0.025&#37; (W/V), without a decrease in viable counts. Adhesion of P. fluorescens No. 3 was inhibited more than 50&#37; by 2 detergents even at 0.0025&#37; (W/V). Four detergents completely inhibited the adhesion of tested bacteria, except for P. aeruginosa NBRC 13275, at a concentration lower than that recommended for washing vegetables. Adhesion inhibition activity appears higher in detergents containing linear alkylbenzenesulfonates and polyoxy ethylene alkyl ether sulfonates but not alkylamine oxides.

DOI： 10.3136/nskkk.58.127

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

Shiga toxin-producing Escherichia coli (STEC) can cause severe illnesses in humans such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, we carried out genotypic analysis of the Shiga toxin (stx) gene in 120 clinical isolates of STEC and enterohemorrhagic E. coli (EHEC) from patients in a southern district of Japan. We identified 88 stx(1)(+) and 103 stx(2)(+) strains. We further identified 12 stx(1)(+) and stx(2)(+) isolates expressing little or no Shiga toxin 1 (Stx(1)) and/or 2 (Stx(2)) by reversed passive latex agglutination (RPLA) and Vero cell toxicity assays. Among them, 1 strain could not produce Stx(1), 8 could not produce Stx(2), and 3 strains could produce neither. Two of the latter three strains were of the non-O157 serotype. Most of the Stx RPLA-negative strains belonged to the stx(1)/stx(2) subtype (11/12, [91.7&#37;]). Our quantitative reverse transcription PCR analysis indicated that the stx genes were not effectively transcribed in the RPLA-negative strains. This is the first report of the isolation of stx-positive strains showing Stx-negative phenotype from stx(1)-bearing strains and non-O157 strains. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.micpath.2010.06.005

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

The bactericidal effect of slightly acidic hypochlorous water (SAHW) on Salmonella Enteritidis, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, as well as some bacterial strains isolated from fresh lettuce was evaluated. Viable counts of all tested bacterial samples decreased immediately after treatment by SAHW Most bacterial cells with the exception of B. cereus, and S. aureus were not culturable on TSA after treatment by 1 to 30 mg/L SAHW. Likewise, Pseudomonas sp., and Flavobacterium or Xanthomonas sp., Kurthia sp., Micrococcus sp., and Corynebacterium or Microbacterium sp. were not culturable on TSA after treatment by 30 mg/L SAHW. Viable counts of S. aureus, E. coli, Flavobacterium or Xanthomonas sp., and Pseudomonas sp. showed a 5 to 6 log cfu/mL reduction at day 0 and maintained a count of less than 1 log cfu/mL from day 1 to day 7 following treatment by 30 mg/L SAHW. Sodium hypochlorite (NaOCl, 0.5-1.0 mg/L) decreased the viable counts of S. Enteritidis to less than the lower limit of detection, 1 log cfu/mL, from day 1 to day 7 following treatment by 1 mg/L. NaOCl was not sufficient at 0.5-0.75 mg/L in reducing viable counts of S. Enteritidis because of a 2 to 5 log cfu/mL increase from day 2 to day 5 due to recovery from injury. Initial counts of S. Enteritidis after hydrogen peroxide (H2O2, 1000-2000 mg/L) treatment slowly decreased over time to less than 1 log cfu/mL after day 2. Treatment by 1750 to 2000 mg/L H2O2, has sufficient bactericidal activity on S. Enteritidis cells, however, at a higher concentration compared to NaOCl or SAHW. SAHW decreased viability of S. Enteritidis immediately with higher reduction counts in 1, 5, and 30 mg/L from day 0 to clay 7 unlike NaOCl and H2O2.

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

Viable counts were enumerated in 36 raw samples of 19 different vegetables. Coliform, fecal coliform, and E. coli were determined in 31 vegetable samples. Tomato was found to have the lowest viable count of 2.12 log cfu/g while radish sprout had the highest count of 9.05 log cfu/g. Although E. coli was not detected in all the vegetables tested, most of these vegetables were positive for fecal coliform. Viable counts of the tenth leaves from the outside were lower by only 1 log cfu/g than that of the outermost leaves in cabbage and lettuce. Among viable counts of vegetable parts, celery leaves, lower sterns in radish sprout, and spinach were found to have the highest viable counts of 7.28 log cfu/g, 9.27 log cfu/g, and 6.10 log cfu/g respectively while lower stem in parsley had the lowest count of 5.10 log cfu/g. The microflora of the four vegetables, celery, parsley, radish, and radish sprout were determined by using biochemical methods. There were 105, 50, 48, 130, and 61 bacterial isolates from celery, lettuce, parsley, radish, and radish sprout, respectively. The predominant bacterium on the four vegetables was about 30-60&#37; Gram negative Flavobacterium or Xanthomonas. Other Gram negative bacteria isolated from the vegetables include 11&#37; Neisseria or Veillnella (celery), 18&#37; Moraxella (radish), 15&#37; Alcaligenes and 12&#37; Pseudomonas (radish sprout) while Enterobacteriaceae accounted for less than 5&#37; for each of the flora of celery, parsley, and radish sprout. On the contrary, parsley had 25&#37; Kurthia or Bacillus, and 13&#37; Micrococcus, both Gram positive.

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

Treatment by slightly acidic hypochlorous water (SAHW) in combination of pretreatment with sucrose fatty acid ester under microbubble generation was effective for decontamination of lettuce. Sufficient contact time of SAHW containing 30 mg/L of available chlorine on reduction of viable counts of lettuce was determined to be 5 min. For 5 min at 18-20 degrees C, treatment with 30 mg/L of available chlorine in SAHW appeared more effective in the reduction of bacteria on lettuce compared with 15 mg/L. The treatment of lettuce at 50 degrees C with SAHW at 30 mg/L of available chlorine showed a 2 log reduction of bacterial counts without injury in the tissue. The treatment at 50 degrees C, SAHW also delayed browning on cut lettuce for the first 5-6 days of subsequent storage at 6 degrees C. Among two sucrose fatty acid esters tested, sucrose monopalmitate at 100 mg/L had a higher efficacy for pretreatment under microbubble generation. After pretreatment for 5 min with 100 mg/L sucrose monopalmitate under microbubble generation and subsequent treatment with SAHW at 50 degrees C for 5 min, viable counts of lettuce were decreased by about 3-4 logs. After the same treatment, Pseudomonas sp. predominant on lettuce decreased drastically. These results indicate the effectiveness of the combined treatments of sucrose fatty acid ester under microbubble generation and SAHW at 50 degrees C for decontamination of fresh produce. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodcont.2010.02.009

Language：Japanese  

Application of Green Tea Extract as Food Preservative to Improve Shelf Life of Overnight Pickled Cucumber
Application of green tea extract (GTE) to food preservation was investigated. Viable counts of cucumber pickled overnight at 10&deg;C with seasoning solution containing acetic acid, NaCl, and/or GTE were determined during cold storage at 10&deg;C. Viable counts did not increase above the initial values for 7 days of treatment with seasoning solution containing 0.1&#37; acetic acid, 2&#37; NaCl, and 0.05&#37; GTE. Use of these seasoning components alone or in combination showed no bacteriostatic effects on the pickled cucumber. Viable counts of Escherichia coli O157 : H7 did not increase when the cucumber was artificially contaminated with this bacterium and then pickled overnight with the seasoning solution containing 0.1&#37; acetic acid. These results indicated that the addition of GTE to seasoning solution containing both acetic acid and NaCl prolongs the shelf life of overnight pickled cucumber.

DOI： 10.3136/nskkk.56.660

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

Salmonella is one of major food-poisoning bacteria. In food companies, controlling the bacterial growth is critically important to continue provision of good products to customers. At first, to Study the attachment of the bacteria to food materials and final products, we focused on biofilm formation that is thought to be one of the reasons of the attachment. Thus, the genes related in formation of biofilm were also focused on, and Delta agfA and Delta bcsA mutants of Salmonella Enteritidis were constructed by using 2-step PCR and pKOBEGA helper plasmid. Secondary, we focused on heat-injured Salmonella after sublethal heat treatment as previously reported (Kobayashi et al., 2005). The heat-injured Salmonella showed expression of several specific genes to recover the bacterial functional state as well as other bacteria. To clarify the mechanism of the recovery, several gene mutants (Delta ahpC, Delta ahpF, and Delta katG mutants of Salmonella Typhimurium, and of Delta rpoE, Delta rpoH, and Delta rpoS mutants of S. Enteritidis and S. Typhimurium) were constructed.

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

Simultaneous enrichment broth (SEB) was developed for the single-enrichment simultaneous screening of six, major food poisoning bacteria. After enrichment in SEB for 18 h at 37 degrees C, viable counts of six major food poisoning bacteria (Staphylococcus aureus, Salmonella spp., Escherichia coli O157:H7, Vibrio parahaemolyticus, Bacillus cereus, and Listeria monocytogenes) were sufficient for 5' nuclease multiplex real-time PCR assay using existing primers and probes. By labeling the probes with three different fluorescent dyes, the assay could be carried out in 2 tubes. The whole process, including enrichment and PCR, was completed within 24 h and the detection limits for the target bacteria from the food sample (boiled chicken) were 36 cfu/25 g for S. aureus, 5.3 cfu/25 g for Salmonella spp, 2.9 cfu/25 g for E. coli O157:H7, 2.0 cfu/25 g for V. parahaemolyticus, 5.5 cfu/25 g for B. cereus, and 6.2 cfu/25 g for L. monocytogenes. The results were comparable to conventional methods that require 4-6 days.

DOI： 10.3136/fstr.15.427

Language：Japanese  

Inhibitors of Adhesion Ability of Salmonella Enteritidis
Inhibitors of Salmonella Enteritidis adhesion to microplates were screened among 39 additives including natural and food additives known to be highly safe. S. Enteritidis adhered strongly to microplates after incubation in 0.1% Bacto-soytone for 24h. Adhesion was inhibited in the presence of pigments (Annatto pigment, Gardenia yellow, and Monascus pigment), food coloring products (Annatto AN, San-brown AC, San-yellow No. 2AU, San-red YM, San-red RCFU, and San-beet L), food additives (protamine, Chili extract, sucrose fatty acid esters, and monoglycerine fatty acid esters), and a flavonoid (Kaempferol). Among them, Annatto AN, Gardenia yellow, Monascus pigment, and sugar fatty acid esters inhibited adhesion at concentrations below antibacterial activity. In the case of sugar fatty acid esters of C8 to C18, the longer the fatty acid chain the stronger the inhibitory effects. Monascus pigment and sucrose monostearate were the strongest inhibitors of S. Enteritidis adhesion, even at 0.01% without antibacterial activity.

DOI： 10.3136/nskkk.56.200

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

Combined effects of surfactants and preservatives on the antibacterial activity of green tea extracts

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

Chlorella vulgaris C-27 has developed freezing tolerance by hardening at 3 degrees C for 24 hour. It was reported that many genes which encode proteins related stress response, storage, protein synthesis and metabolism including zeta-carotene desaturase increase at transcriptional levels. We focus on the change of photosynthesis pigments, and assessed the effects of the cold acclimation on chlorophyll and carotenoid in the hardened and unhardened cells of C. vulgaris C-27 and C. vulgaris C-102 (a chilling-sensitive strain). Cold hardening at 3 degrees C for 24 hour caused a slight reduce of the chlorophyll content in C. vulgaris C-27, but did a significant reduction of that in C. vulgaris C-102. In chlorella cells, six major carotenoids, i.e., beta-carotene, alpha-carotene, lutein, zeaxanthin, violaxanthin and neoxanthin, were detected by an original method for carotenoid determination of C. vulgaris C-27 and C. vulgaris C-102. It was possible to separate lutein and zeaxanthin by HPLC using cholester column. The carotenoid composition was highly influenced by the cold hardening. It was elucidated that total carotenoid was little affected by hardening at 3 degrees C, but zeaxanthin content increased with decrease of violaxanthin content. On the other hand, incubation at 25 degrees C after freeze-thaw restored the altered levels of both pigments to pre-hardening levels. This result suggests that freezing tolerance of C. vulgaris C-27 induced during cold acclimation have a close involvement of change in xanthophyll cycle which plays a significant role to relieve oxidative stress at freezing and thawing.

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

To develop a new method for identification of Listeria monocytogenes genetically similar to clinical isolates, single-nucleotide polymorphism (SNP) typing and multi-locus sequence typing (MLST) of 126 isolates of L. monocylogenes from clinical and environmental samples were performed based on sequence analysis of parts of four genes (hlyA, clpC, inlA, and plcA). Based on the sequences of the isolates in this study, SNP typing showed that hlyA, clpC, inlA, and plcA genes were categorized into 9, 14, 17, and 21 types, respectively. MLST showed that the isolates were grouped into 35 types including 12 types of clinical isolates. Out of those, four MLST types were found in food or environmental and clinical isolates. In particular, all clinical isolates with serotype 1/2a were grouped into the same hlyA SNP A5 type. A method using real-time PCR combined with Cycling Probe Technology was developed for rapid identification of SNP type of L. monocytogenes genetically similar to the clinical isolates. By using this method, the 1/2a clinical isolates showing MLST-2 were successfully identified with a specific primer set and a cycling probe designed on the basis of sequence of hlyA. Furthermore, clinical isolates of serotype 4b showing MLST-4 or -35 were successfully identified by a method using cycling probes based on sequences of clpC and inlA.

DOI： 10.3136/fstr.14.557

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

Several organisms acquire freezing tolerance by altering intracellular reactions under sublethal low temperatures. Although many researchers have reported that organisms change their intracellular functions, such as transcript level and enzymatic activity, under stress conditions, acquisition mechanisms of freezing tolerance are still poorly clarified. In this study, we attempted to identify novel hardening-induced genes from Chlorella vulgaris C-27, a frost-hardy strain. A PCR-based suppression subtractive hybridization library was generated and the corresponding cDNA clones were isolated. A total of 263 unique cDNA clones were obtained and sequenced. Homology analysis showed that 64 distinct known proteins were encoded by the respective clones. The expression patterns of 29 of the genes were analyzed by using qPCR. Especially, six genes, which respectively encode two late embryogenesis abundant (LEA) proteins (HIC6 and HIC12), NADPH thioredoxin reductase (NTR), chlorophyll a/b-binding protein (Cab), zeta-carotene desaturase, and Nip7, showed remarkable increase in transcripts over 100 times greater than those of unhardened cells. We discuss the possible contribution of the genes, which showed remarkable transcriptional increases, in acquisition of freezing tolerance of Chlorella. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.plantsci.2008.04.002

Language：Japanese  

Effects of Food Additives on the Antibacterial Activity of Green Tea Extracts

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

The effect of Lunularic acid on the freezing tolerance of Chlorella vulgaris IAM C-27

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

An automated fluorescence microscopic method developed for the measurement of viable bacterial counts was applied to the detection and enumeration of viable bacteria in milk. In the automated analysis of viable bacteria, bacterial cells were recovered from milk after the treatments with EDTA and Triton X-100, stained with 6-CFDA, and the stained cells were counted with the automated microscopic method. Salmonella Enteritidis expressing the enhanced green fluorescent protein (S. Enteritidis-EGFP) was used to elucidate the relationship between the fluorescent bacterial counts by the automated microscopic method and the viable counts by the conventional plating method. A high correlation coefficient (r2 = 0.97) was obtained for the relationship between the counts by the microscopic method and those by the plating method. When S. Enteritidis-EGFP was recovered from sterile milk after treatments with EDTA and Triton X-100, the correlation coefficient was calculated to be r2 = 0.91. Viable counts by the plating method and the counts of 6-CFDA stained bacterial cells by the automated microscopic method were determined for various raw milk samples. Thirty-seven raw milk samples were measured and the correlation coefficient was calculated to be r2 = 0.73.

DOI： 10.4265/bio.10.147

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

Template DNA from Brevibacillus brevis, Geobacillus stearothermophilus, Paenibacillus macerans, P. polymyxa and 10 species of Bacillus was used with the commercially available RAPD Analysis Primers to generate DNA fragments by PCR. Different RAPD band patterns were generated from the DNA of 14 different Bacillus species and related genera with Primer 1 or 4. The result suggested that the single use of Primer 1 or 4 is effective for the simple identification of these strains of Bacillus and related genera by the RAPD analysis. By the RAPD analyses of the template DNA prepared from bacterial colonies formed on nutrient agar plates after cultivation at 35°C for 24 to 48 h, B. brevis, B. circulans, B. firmus, B. licheniformis, P. macerans, P. polymyxa, B. pumilus, B. sphaericus, B. subtilis and B. thuringiensis were identified by the combination of the present RAPD analysis using Primer 4 and morphological observations of colonies, vegetative cells and spores. For identification of B. coagulans and G. stearothermophilus, the RAPD analyses of the template DNA prepared from colonies formed on standard plate agar after cultivation at 55°C for 24 to 48 h might be effective though further improvements of the assay are required for identification of these Bacillus species. For simple identification of B. cereus and B. megaterium by the PCR-based method, the use of other RAPD primers or a specific PCR primer set should be developed in addition to morphological observations of colonies, vegetative cells and spores.

DOI： 10.4265/bio.10.45

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

Proteins and genes involved in the recovery of heat-injured Salmonella Enteritidis were investigated. Salmonella Enteritidis cells cultured overnight in tryptic soy broth (TSB; nonselective medium) were suspended in citric acid-disodium hydrogen phosphate buffer (pH 6). After heat treatment at 55°C for 15 min, the culturable counts measured by tryptic soy agar (TSA; nonselective medium) decreased from 108 to 107 CFU/ml. On the other hand, culturable counts measured by desoxycholate-hydrogen sulfite-lactose (DHL) agar (selective medium) were decreased from 108 to 104 CFU/ml by the same treatment. The results suggest that 99.9&#37; of Salmonella Enteritidis detected on TSA were injured but recoverable. When injured Salmonella Enteritidis was incubated in TSB, the culturable count measured by TSA did not increase for 2 h, whereas that by DHL agar increased after incubation for 30 min. After incubation for 2 h, the culturable count measured by DHL agar reached a similar level with that by TSA, indicating that Salmonella Enteritidis had recovered. The two-dimensional polyacrylamide gel electrophoresis analysis revealed that elongation factor G (FusA) and pyruvate kinase (PykF) specifically increased in the cells just after heat treatment and in the recovery cells. The levels of transcription of 86 stress-inducible genes were also investigated by reverse transcription PCR. Nineteen heat-inducible (clpB, clpX, degP, dnaJ, fkpA, ftsJ, gapA, hflB, hslJ, hslU, hslV, htpG, htrA, lon, mopA, mopB, mreB, rpoE, and ppiD), and 12 oxidative-stress and DNA damage-inducible (ahpC, ahpF, fldB, fur, grxA, dinF, katG, mutM, recA, soxR, trxC, and zwf) genes were transcribed extensively during recovery in TSB. The results obtained in this study will be used to develop the media or culture conditions that will promote recovery for the detection of food poisoning bacteria, including injured cells from food products. Copyright ©, International Association for Food Protection.

DOI： 10.4315/0362-028X-68.5.932

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

Miyamoto, T., Shimizu, Y., Kobayashi, H., Honjoh, K., and Iio, M., Studies of collagen binding with immobilized Salmonella enteritidis and inhibiton with synthetic and naturally occurring food additives by a surface plasmon resonance biosensor. Sensors and Materials., 15 (8): 453-466 (2003).

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

The DNA band patterns generated by PCR using Primer 4 and template DNAs from various Bacillus strains were compared. Sequence analysis of the 3.2 kb PCR product detected only in B. cereus revealed that the DNA sequence from base 11 to 2962 of the DNA seemed to contain a sequence specific to B. cereus. The primers BCF and BCR that amplify the region between base 1168 and 2320 of the 3.2 kb DNA generated the 1153 bp PCR product with DNAs from B. cereus type strain JCM 2152, 3 cereulide-positive B. cereus strains and 20 strains among 49 B. cereus isolated from dairy products and processing factories. The same band was not amplified with those from 5 strains of B. thuringiensis, other 11 Bacillus species and 12 different bacteria which were not Bacillus.

DOI： 10.4265/bio.9.69

Language：Japanese  

Development of a Rapid and Simple Method for Measuring Total Viable Bacterial Counts by Flow Cytometry

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

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10&#37; of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.

DOI： 10.4315/0362-028X-66.7.1222

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

Two fatty acid desaturases were independently expressed in tobacco plants and freezing tolerance of the transgenic plants was investigated: the two desaturases were CvFAD2, which desaturates microsomal C18:1 (oleic acid) to C18:2 (linoleic acid), and CvFAD3, which desaturates microsomal C18:2 to C18:3 (linolenic acid). Fatty acid composition analysis showed that the C18:2 contents in the plants transformed with pBE2113/CvFad2 did not increase compared with that of the wild-type plant. However, an increase in the C18:3 content by 2.4&#37; was observed in the line of No. 19 although the increase was not statistically significant. In the plant transformed with pBE2113/CvFad3, the C18:3 content increased by 6.4&#37; compared with that of the wild-type plant. Measurement of the electrolyte leakage of the leaves showed that freezing tolerance of the CvFad2 plant was a little higher than that of the wild-type plant at all temperatures investigated (from -1 to -4°C). Contrary to the case of the CvFad2, the freezing tolerance of the CvFad3 plant tended to be slightly lower than that of the wild-type at all temperatures tested despite the unsaturation levels of the plant were higher than those of the CvFad2 plants.

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

Polymerase chain reaction assay for detection of Escherichia coli O157:H7 and Escherichia coli O157:H- , Miyamoto, T., Ichioka, N., Sasaki, C., Kobayashi, H., Honjoh, K., Iio, M. and Hatano, S. , J. Food Protection, 65(1),5-11 (2002)

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

Cloning and overexpression of Bacillus cereus penicillin-binding protein 3 gene in Escherichia coli, Miyamoto, T., Sayed, Md. A., Sasahara, R., Sukimoto, K., Umezaki, A.,Honjoh, K., Iio, M. and Hatano, S., Biosci. Biotechnol. Biochem., 66(1): 44-50 (2002)

Language：Japanese  

Studies on Freezing Injury of Unwashed and Washed Cells of Escherichia coli O157:H7

DOI： 10.5803/jsfm.17.127

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

Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase, Kim, S.-I., Miyamoto, T., Honjoh, K., Iio, M., and Hatano, S. , Biosci. Biotechnol. Biochem., 64(6) 1210-1216 (2000)

DOI： 10.1271/bbb.64.1210

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

Monoclonal antibodies (MAbs) raised against Escherichia coli O6:H16 were screened against 15 strains of E. coli and 19 non-E. coli bacteria. A MAb-luminescence assay using MAb-5.8, which shows no cross-reactions with non-E. coli bacteria, and a photon-counting television camera were developed for rapid enumeration of E. coli O6:H16 in water. The membrane filter that retained bacteria was boiled for 5 min in a buffer and incubated with biotinylated MAb-5.8. After incubation with streptavidin-peroxidase conjugate, it was reacted with luminol-based reaction mixture. Luminous image and light intensity of the filter was recorded with a Biocell Counter. Levels of E. coli O6 higher than 7 x 103 CFU were detected by the MAb-luminescence assay when E. coli O6 was spotted onto the membrane filter. The sample that contained E. coli O6:H16 was filtered through a membrane filter, and the filter that retained bacteria was incubated on a filter paper soaked with nutrient broth supplemented with 0.5&#37; NaCl at 37°C for 6 h. The number of light emission points on the filter correlated well with initial E. coli O6:H16 counts within the range of 1 to 3 x 102 CFU. The correlation coefficient was 0.89.

DOI： 10.4315/0362-028X-63.4.534

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

A new method for detection of penicillin-binding proteins from bacterial membranes has been developed in this study. The method, that employed biotin-ampicillin conjugate (Bio-PCA), is very rapid and can be a significant alternative of hazardous and time consumimng conventional radiometric method for detection of PBPs in cell membranes. PBPs from B. cereus were examined by this technique. In vegetative and sporulating cells, 8 major PBPs were detected. Comparing with standard marker proteins, these PBPs were estimated for their molecular weight as 106, 83, 75, 72, 63, 46, 40, and 32 kDa. Affinity of cephalexin, cefoxitin, and cefotaxime to PBPs was measured indirectly by competition for subsequent binding of Bio-PCA. PBPs 2, 3, 4, and 7 were decreased or disappeared in the electrophoregram by prebinding with these β-lactams. Two PBPs, PBP 3 and PBP 4, which were predominant in vegetative and sporulating cells, respectively, showed strong affinity for cephalexin.

Language：English  

Rapid Detection of Salmonella spp.by PCR Amplification of Salmonella Specific Region in gatD gene

DOI： 10.5803/jsfm.16.99

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

Rapid detection method of Salmonella-specific PCR product by DNA-immobilized quartz crystal microbalance. Jpn. J. Food Microbiol., 16(1) 57-63. (1999)

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

A bioluminescence assay carried out with a photon-counting TV camera was evaluated for rapid enumeration of viable bacterial counts. The test sample was filtered through a membrane filter, and the membrane filter retaining bacteria was incubated at 37°C for 6 h on a filter paper soaked with nutrient broth supplemented with 0.5&#37; NaCl. The membrane filter was then subjected to a bioluminescence reaction, and the intensity of light and numbers of light emission points on the filter were measured with a photon-counting TV camera. The light intensity measured on seven different bacteria correlated with initial viable counts; the correlation coefficient was calculated to be 0.89. The number of light emission points measured on Escherichia coli also correlated with the initial viable counts (r = 0.81) in a range from 1 to 100 CFU. Presumptive bacterial counts by the present bioluminescence assay determined on 79 samples of vegetables and 122 samples of environmental water correlated well with the viable counts obtained by the conventional plating method, with correlation coefficients of 0.87 and 0.82, respectively.

DOI： 10.4315/0362-028X-61.10.1312

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

The random amplified polymorphic DNA (RAPD) band patterns from 23 Salmonella spp. produced by use of an oligonucleotide primer (called du primer) designed on the basis of the N-terminal sequence of dulcitol 1-phosphate dehydrogenase (5'-GTGGTGACCCAGGATGGCCAGGTG-3') were different from those from 16 non-Salmonella spp. The bands at 460 and 700 bp were produced in all Salmonella strains tested. These RAPD fragments obtained from Salmonella typhimurium strongly hybridized with the corresponding RAPD bands from the other strains of Salmonella, but not with those from non-Salmonella spp. in Southern blot analysis. The RAPD bands were detected by ethidium bromide staining even when genomic DNA prepared from as few as 2.8 X 103 cells was used. The minimum detectable cell number in the initial inoculum of S. typhimurium was 4 X 10-1 CFU/25 g of raw beef after the preenrichment in Enterobacteriaceae enrichment mannitol (EEM) broth for 6 h and the selective enrichment in dulcitol-magnesium chloride-pyridinesulfonic acid-brilliant green-novobiocin (DMPBN) medium for 18 h at 42°C. Seven raw foods inoculated with S. typhimurium at numbers from 4 X 10-1 to 2.6 X 102 CFU/25 g of food were positive in both the RAPD analysis and the conventional culture method.

DOI： 10.4315/0362-028X-61.7.785

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

IMP dehydrogenase was purified from a crude extract of B. cereus cells. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE and 225 kDa by gel filtration. The optimum pH of the enzyme was about 9.5. The first seven residues at N-terminus of the enzyme was determined to be Met-Trp-Glu-Ser-Lys-Phe-Val. The enzyme showed a significant specificity for inosine nucleotides among 15 purines and pyrimidines tested, but not acted on other purines and pyrimidines including inosine. Among 11 metal ions and 3 enzyme inhibitors tested, A13+ activated the IMP dehydrogenase. The enzyme activity was strongly inhibited by Zn2+ and Fe3+.

DOI： 10.1016/S0944-5013(98)80017-7

Language：English  

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

VH (heavy-chain variable region) and VL (light-chain variable region) genes were amplified by PCR from hybridomas producing MAb-11 and MAb-18 which inhibited Japanese radish acid phosphatase. Nucleotide sequencing of the V genes demonstrates that the MAbs contained similar VH and identical VL domains. Initially, the VH and VL genes were expressed in Escherichia coli as single-chain Fv (ScFv) fragments. Fragments ScFv-11 and ScFv-18, named for MAb-11 and MAb-18, respectively, inhibited the enzyme activity to the same extent as the intact MAbs. Both of the antibody fragments widely cross-reacted with other phosphatases, including some phosphomonoesterases and phosphodiesterases from different sources. ScFv-18 also inhibited acid phosphatase from a different origin, but stimulated the activity of alkaline phosphatase from calf intestine. The PCR-amplified VH and VL genes were subsequently expressed separately in Escherichia coli as fusion products with glutathione S-transferase. The fusion proteins had little effect on Japanese radish acid phosphatase. Furthermore, a large number of recombinant ScFv fragments specific to the acid phosphatase were generated by using a bacteriophage expression system and a mouse ScFv gene library. These ScFv fragments had a range of effects on the enzyme activity, including inhibition, stimulation, and none. Among them, an ScFv fragment, designated ScFv-G7, inhibited more strongly than ScFv-11 and ScFv-18. © 1998 Taylor & Francis Group, LLC.

DOI： 10.1271/bbb.62.1041

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

Cephalexin, cefaclor, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereus ts-4 at 1 μg/ml. Cephalexin was most inhibitory on the sporulation of B. cereus when the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4',6-diamidino-2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [3H]penicillin, eight penicillin-binding proteins (PBPs) were detected from the cells of B. cereus ts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin G. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells.

DOI： 10.1016/S0944-5013(97)80032-8

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

IMP dehydrogenase activity of B. cereus increased parallel to cell growth in YE-EMM, where B. cereus did not sporulate. When B. cereus was cultured in a modified G medium, a sporulation medium, the activity reached the highest level at 6 hr and decreased thereafter. After induction of sporulation by nutritional shift down in 1/100 G medium, the enzyme activity decreased to about 5&#37; compared with exponentially growing cells at 1 hr of resuspension. The sporulation rate of B. cereus was over 90&#37; in the modified G medium and 1/100 G medium. Sporulation was strongly inhibited by mycophenolic acid at 1 mM, when the drug was added at 0 and 1 hr of resuspension in 1/100 G medium. Intracellular GTP concentration of B. cereus decreased to the lowest level about 1 hr of resuspension. Although GTP increased to about 50&#37; of the exponentially growing cells at 2 hr of resuspension in control cells, the concentration did not increase in the presence of 1 mM mycophenolic acid.

DOI： 10.1016/S0944-5013(97)80040-7

Language：English  

Effects of Trehalose on Freeze Tolerance of Baker's Yeast
A conventional baker's yeast strain D incorporated trehalose into its cells from a YPG medium supplemented with trehalose. Cells cultured in a medium containing 3 to 5&#37; trehalose increased to nearly 5 times the trehalose content of cells cultured in the absence of trehalose. After 1 day of frozen storage at -2O"C, cells cultured in 3&#37; trehalose medium experienced a lesser decrease in both viability and CO, productivity than cells cultured in the absence of trehalose. Even after 10 days frozen storage, the strain D cultured in the 3&#37; trehalose medium retained to nearly 50&#37; of the viability and CO, productivity of the unfrozen cells. Although the freeze-tolerant yeast strains DFT and S. cerevisiae MAFF lo-03056 showed, during freezing, smaller decreases in viability than strain D, the large decreases in CO_2 productivity were comparable among all three strains. The CO_2 productivity in both freeze-tolerant strains cultured in the presence of 3&#37; trehalose was about 60&#37; of that in the unfrozen cells, even after 10 days of frozen storage. The CO_2 productivity and actin content of the cell-free extracts prepared from the strain D cultured in YPG medium decreased significantly to about 15&#37; and 30&#37; of those of the unfrozen cells, respectively, after 7 days of frozen storage. When the cells cultured in the presence of 3&#37; trehalose were frozen-stored, the CO_2 productivity of the cellfree extracts prepared from 7-days frozen-stored cells decreased to 50&#37; of that from the unfrozen cells. The actin content, however, did not decrease after the same frozen storage. In eukaryotic cells, the activities of some glycolytic enzymes are increased by association with actin. It seems that the native structure of actin is necessary for yeast CO, productivity after frozen storage.

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

A rapid-detection method was developed for food-borne dulcitol-positive Salmonella spp. in foods that involves a new preenrichment and selective enrichment system and a sandwich ELISA using two monoclonal antibodies against dulcitol 1-phosphate dehydrogenase. Preenrichment and selective enrichment were in Enterobacteriaceae enrichment mannitol (EEM) broth at 42°C for 6 h and in a new dulcitol-magnesium chloride-pyridinesulfonic acid- brilliant green-novobiocin (DMPBN) medium at 42°C for 27 h, respectively. The cells were collected from the selective enrichment culture and suspended in 0.1 ml of 1 N NaOH for 2 min. The solution was neutralized with 0.1 ml of 2 M Tris-HCl buffer (pH 7.5) and the mixture was used as a sample for ELISA. The detection sensitivity of the ELISA was 105 CFU of Salmonella spp. per ml of culture. Competing non-Salmonella organisms in raw food did not interfere with the detection of Salmonella cells even when present at 107:1 (non- Salmonella: Salmonella ratio) in food. Nonmotile Salmonella gallinarum was detected by the ELISA. The minimum detectable number of initial inoculum of Salmonella typhimurium was 0.69 CFU/25 g of raw chicken after the preenrichment in EEM broth and the selective enrichment in DMPBN medium. The present ELISA method required a total analysis time of 36 h including the preenrichment and selective enrichment periods. The ELISA method was compared with a conventional cultural method for the detection of Salmonella cells in 130 samples of raw foods. Of the samples tested, 16 were Salmonella-positive and 114 samples were negative by both methods. False positive and false negative results were not encountered.

DOI： 10.4315/0362-028X-59.11.1158

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

After frozen storage for 7d, the viability and CO2productivity of a conventional baker’s yeast strain D greatly decreased. The viability of a freeze-tolerant strain, DFT, used for the frozen dough method slightly decreased after the same storage period, while the CO2productivity greatly decreased. The CO2productivity and DNase I inhibitory activity of actin of the cell-free extracts prepared immediately after thawing from 7-d frozen-stored cells markedly decreased in both strains. In DFT, however, the productivity and the inhibitory activity of the cell-free extract increased when the extract was prepared after incubation of the frozen-thawed cells at 30°C. The increase in the inhibitory activity first occurred and then the increase in the CO2productivity. Gel filtration patterns of actin and glycolytic enzymes were compared between cell-free extracts of both strains. Peaks of actin and activity peaks of hexokinase and pyruvate kinase decreased in the strain D after frozen storage, but only slightly in the strain DFT. After frozen storage, phosphofructokinase activity peak shifted to a lower molecular weight in strain D. © 1996, Taylor & Francis Group, LLC. All rights reserved.

DOI： 10.1271/bbb.60.61

Event date： 2023.10

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Venue：福岡市（九州大学西新プラザ）   Country：Japan  

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Venue：京都市（京都女子大学）   Country：Japan  

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Event date： 2022.11

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Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

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Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

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Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

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Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

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Event date： 2021.8

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Event date： 2021.8

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Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

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Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

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Event date： 2020.12

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Venue：中村学園大学、福岡市   Country：Japan  

Event date： 2020.11

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Venue：九州大学西新プラザ、福岡市   Country：Japan  

Event date： 2019.11

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Venue：沖縄県青年会館（那覇市）、琉球大学（沖縄県中頭郡）   Country：Japan  

Event date： 2019.8

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Venue：藤女子大学北16条キャンパス（札幌市）   Country：Japan  

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Venue：マリンメッセ福岡、福岡市   Country：Japan  

Event date： 2019.3

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Event date： 2018.11

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Event date： 2018.6

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Event date： 2017.8

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Venue：日本大学藤沢キャンパス（藤沢市）   Country：Japan  

Event date： 2016.9

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Venue：九州大学、福岡市   Country：Japan  

Event date： 2013.10

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Venue：九州大学、福岡市   Country：Japan  

Event date： 2013.10

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Venue：九州大学、福岡市   Country：Japan  

Event date： 2013.9

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Venue：MAX ATRIA   Country：Singapore  

Event date： 2013.8

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Venue：実践女子大学（日野市）   Country：Japan  

Event date： 2013.8

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Venue：実践女子大学（日野市）   Country：Japan  

Event date： 2013.8

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Venue：実践女子大学（日野市）   Country：Japan  

Event date： 2013.7

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Venue：北九州国際会議場（北九州市）   Country：Japan  

Event date： 2012.9

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Venue：鹿児島大学   Country：Japan  

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Venue：九州大学箱崎キャンパス、福岡市   Country：Japan  

Event date： 2011.9

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Venue：九州大学箱崎キャンパス、福岡市   Country：Japan  

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Venue：九州大学農学部、福岡市   Country：Japan  

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Venue：崇城大学   Country：Japan  

Event date： 2010.7

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Venue：北九州国際会議場   Country：Japan  

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Venue：北九州国際会議場   Country：Japan  

Event date： 2009.10

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Venue：琉球大学   Country：Japan  

Event date： 2009.10

Language：Others   Presentation type：Oral presentation (general)  

Venue：琉球大学   Country：Japan  

Event date： 2009.10

Language：Others   Presentation type：Oral presentation (general)  

Venue：琉球大学   Country：Japan  

Event date： 2009.7

Language：Others   Presentation type：Oral presentation (general)  

Venue：北九州国際会議場   Country：Japan  

Event date： 2009.3

Language：Others  

Venue：マリンメッセ福岡、福岡市   Country：Japan  

Event date： 2009.3

Language：Others  

Venue：マリンメッセ福岡、福岡市   Country：Japan  

Event date： 2009.3

Language：Others  

Venue：マリンメッセ福岡、福岡市   Country：Japan  

Event date： 2008.11

Language：Others  

Venue：Chungnum National University, Daejeon   Country：Korea, Republic of  

Event date： 2008.10

Language：Others   Presentation type：Oral presentation (general)  

Venue：東京ビッグサイト   Country：Japan  

Event date： 2008.9

Language：Others   Presentation type：Oral presentation (general)  

Venue：長崎大学   Country：Japan  

Event date： 2008.8

Language：Others   Presentation type：Symposium, workshop panel (public)  

Venue：Tampere   Country：Finland  

Event date： 2008.8

Language：Others   Presentation type：Symposium, workshop panel (public)  

Venue：Tampere   Country：Finland  

Event date： 2008.7

Language：Others  

Venue：北九州国際会議場   Country：Japan  

Event date： 2007.10

Language：Others   Presentation type：Oral presentation (general)  

Venue：Tsukuba, Ibaraki   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：名城大学   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：鹿児島大学（鹿児島市）   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：鹿児島大学（鹿児島市）   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：福岡市、九州大学 農学部   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：福岡市、九州大学 農学部   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：福岡市、九州大学 農学部   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：千里ライフサイエンスサンター、大阪府豊中市千里東町1-4-2   Country：Japan  

Language：Others   Presentation type：Oral presentation (invited, special)  

Venue：SUSONI,   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：石川県地場産業センター（金沢市）   Country：Japan  

Language：Others   Presentation type：Symposium, workshop panel (public)  

Venue：九州大学   Country：Japan  

Language：Others  

Venue：北九州国際会議場   Country：Japan  

Language：Others  

Venue：北九州国際会議場   Country：Japan  

Language：Others  

Venue：北九州国際会議場   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：佐賀大学   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：佐賀大学   Country：Japan  

Language：Others  

Venue：Aiina Hall, Morioka, Iwate   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：東京農業大学   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：東京農業大学   Country：Japan  

Language：Others  

Venue：北九州国際会議場   Country：Japan  

Language：Others  

Venue：Kyusyu University   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：山口大学，山口市   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：中村学園大学，福岡市   Country：Japan  

Language：Others   Presentation type：Symposium, workshop panel (public)  

Venue：The University of Tokyo, Yasuda Auditorium   Country：Japan  

Language：Others   Presentation type：Symposium, workshop panel (public)  

Venue：札幌コンベンションセンター   Country：Japan  

Language：Others   Presentation type：Oral presentation (general)  

Venue：崇城大学   Country：Japan  

Event date： 2024.11

Language：English  

Venue：Virtual Meeting   Country：Other  

Event date： 2024.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎   Country：Japan  

Event date： 2024.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎   Country：Japan  

Event date： 2024.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎   Country：Japan  

Event date： 2024.10

Language：English   Presentation type：Oral presentation (general)  

Venue：Palazzo dei Congressi / Palazzo degli Affari   Country：Italy  

Event date： 2024.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：埼玉大学   Country：Japan  

Event date： 2024.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：埼玉大学   Country：Japan  

Event date： 2024.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：佐賀大学   Country：Japan  

Event date： 2024.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名城大学、名古屋   Country：Japan  

Event date： 2024.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名城大学、名古屋   Country：Japan  

Event date： 2024.8

Language：English   Presentation type：Oral presentation (general)  

Venue：名城大学、名古屋   Country：Japan  

Event date： 2024.7

Language：English   Presentation type：Poster presentation  

Venue：Cairns Australia  

Event date： 2024.6

Language：English   Presentation type：Poster presentation  

Venue：北九州市   Country：Japan  

Event date： 2024.6

Language：English   Presentation type：Poster presentation  

Venue：北九州市   Country：Japan  

Event date： 2023.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：English   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都市（京都女子大学）   Country：Japan  

Event date： 2023.7

Language：English   Presentation type：Oral presentation (general)  

Venue：CCH - Congress Center Hamburg   Country：Germany  

Event date： 2023.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：CCH - Congress Center Hamburg   Country：Germany  

Event date： 2023.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：CCH - Congress Center Hamburg   Country：Germany  

Event date： 2023.7

Language：English   Presentation type：Oral presentation (general)  

Venue：COEX   Country：Korea, Republic of  

Event date： 2023.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：COEX   Country：Korea, Republic of  

Event date： 2023.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：COEX   Country：Korea, Republic of  

Event date： 2023.7

Language：English   Presentation type：Oral presentation (general)  

Venue：COEX   Country：Korea, Republic of  

Event date： 2022.11

Language：English   Presentation type：Oral presentation (general)  

Venue：Ho-Chi Mihn City University   Country：Viet Nam  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

Language：English   Presentation type：Oral presentation (general)  

Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島大学郡元キャンパス（鹿児島市）   Country：Japan  

Event date： 2022.9

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：⻑崎⼤学医学部ポンぺ会館（長崎市）   Country：Japan  

Event date： 2022.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：WASHINGTON   Country：United States  

Event date： 2022.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：WASHINGTON   Country：United States  

Event date： 2021.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン開催   Country：Japan  

Event date： 2021.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン開催   Country：Japan  

Event date： 2021.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島県医師会館４F大ホール（鹿児島市）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：English   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：オンライン、一部ハイブリッド（発信場所：福岡市、九州大学）   Country：Japan  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2021.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2020.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：九州大学西新プラザ、福岡市   Country：Japan  

Event date： 2020.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2020.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2020.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2020.6

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：オンライン   Country：United States  

Event date： 2019.11

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：タワーホール船堀（東京都江戸川区）   Country：Japan  

Event date： 2019.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：沖縄県青年会館（那覇市）、琉球大学（沖縄県中頭郡）   Country：Japan  

Event date： 2019.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：米子コンベンションセンター BIG SHIP（米子市）   Country：Japan  

Event date： 2019.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：米子コンベンションセンター BIG SHIP（米子市）   Country：Japan  

Event date： 2019.9

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：千里ライフサイエンスセンター（大阪府豊中市）   Country：Japan  

Event date： 2019.9

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：千里ライフサイエンスセンター（大阪府豊中市）   Country：Japan  

Event date： 2019.8

Language：Japanese  

Venue：藤女子大学北16条キャンパス（札幌市）   Country：Japan  

Event date： 2019.8

Language：Japanese  

Venue：藤女子大学北16条キャンパス（札幌市）   Country：Japan  

Event date： 2019.8

Language：Japanese  

Venue：藤女子大学北16条キャンパス（札幌市）   Country：Japan  

Event date： 2019.8

Language：Japanese  

Venue：藤女子大学北16条キャンパス（札幌市）   Country：Japan  

Event date： 2019.8

Language：English   Presentation type：Oral presentation (general)  

Venue：九州大学西新プラザ（福岡市）   Country：Japan  

Event date： 2019.8

Language：English   Presentation type：Oral presentation (general)  

Venue：九州大学西新プラザ（福岡市）   Country：Japan  

Event date： 2019.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：北九州国際会議場（北九州市）   Country：Japan  

Event date： 2019.7

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：北九州国際会議場（北九州市）   Country：Japan  

Event date： 2019.7

Language：English   Presentation type：Oral presentation (general)  

Venue：SEC Centre（グラスゴー）   Country：Other  

Event date： 2019.7

Language：English   Presentation type：Oral presentation (general)  

Venue：SEC Centre（グラスゴー）   Country：Other  

Event date： 2019.7

Language：English   Presentation type：Oral presentation (general)  

Venue：SEC Centre（グラスゴー）   Country：Other  

Event date： 2019.7

Language：English   Presentation type：Oral presentation (general)  

Venue：SEC Centre（グラスゴー）   Country：Other  

Event date： 2019.6

Language：English  

Venue：Duke Energy Convention Center（オハイオ州シンシナティ）   Country：United States  

Event date： 2019.6

Language：English  

Venue：BITEC（バンコク）   Country：Thailand  

Event date： 2018.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：ホテルナゴヤキャッスル／名城大学天白キャンパス（愛知県名古屋市）   Country：Japan  

Event date： 2017.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：タワーホール船堀（東京都江戸川区）   Country：Japan  

Event date： 2017.11

Language：English  

Venue：タワーホール船堀（東京都江戸川区）   Country：Japan  

Event date： 2017.11

Language：English  

Venue：タワーホール船堀（東京都江戸川区）   Country：Japan  

Event date： 2017.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：徳島県郷土文化会館（徳島市）   Country：Japan  

Event date： 2017.9

Language：Japanese  

Venue：千里ライフサイエンスセンター（大阪府豊中市）   Country：Japan  

Event date： 2017.9

Language：Japanese  

Venue：千里ライフサイエンスセンター（大阪府豊中市）   Country：Japan  

Event date： 2017.9

Language：Japanese  

Venue：千里ライフサイエンスセンター（大阪府豊中市）   Country：Japan  

Event date： 2017.8

Language：English   Presentation type：Oral presentation (general)  

Venue：熊本大学（熊本市）   Country：Japan  

Event date： 2017.8

Language：English   Presentation type：Oral presentation (general)  

Venue：熊本大学（熊本市）   Country：Japan  

Event date： 2017.7

Language：Japanese  

Venue：北九州国際会議場（北九州市）   Country：Japan  

Event date： 2017.7

Language：English  

Venue：北九州国際会議場（北九州市）   Country：Japan  

Event date： 2017.6

Language：Japanese  

Venue：東京都江東区豊洲シビックセンター   Country：Japan  

Event date： 2017.6

Language：English  

Venue：Ernest N. Morial Convention Center   Country：United States  

Event date： 2017.6

Language：English  

Venue：Ernest N. Morial Convention Center   Country：United States  

Event date： 2017.6

Language：English  

Venue：Ernest N. Morial Convention Center   Country：United States  

Event date： 2017.6

Language：English  

Venue：Ernest N. Morial Convention Center   Country：United States  

Event date： 2017.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：ウェスティン都ホテル京都／京都女子大学（京都府京都市）   Country：Japan