Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Ken-ichi Honjoh Last modified date:2024.04.11

Associate Professor / Division of Food Science and Biotechnology / Department of Bioscience and Biotechnology / Faculty of Agriculture

1. Joh, T., Yoshimoto, M., Honjoh, K., Miyamoto, T., Hatano, S., Changes in translatable RNA population during hardening of Chlorella ellipsoidea C-27., J. Fac. Agr., Kyushu Univ., 37, 3-4, 257-263, 37(3,4), 257-263, 1993.02.
2. Ken Ichi Honjoh, Yuichi Oda, Ryoji Takata, Takahisa Miyamoto, Shoji Hatano, Introduction of the hiC6 gene, which encodes a homologue of a late embryogenesis abundant (LEA) protein, enhances freezing tolerance of yeast, J. Plant Physiol., 10.1016/S0176-1617(99)80046-7, 155, 4-5, 509-512, 1999.10, 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% to 66% survival rate) higher than that of the control yeast..
3. 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., Cryoprotective activities of group3 late embryogenesis abundant proteins from Chlorella vulgaris C-27, Biosci. Biotechnol. Biochem., 10.1271/bbb.64.1656, 64, 8, 1656-1663, 64(8): 1656-1663, 2000.08.
4. Ken Ichi Honjoh, Hideyuki Shimizu, Noriko Nagaishi, Hiroko Matsumoto, Koushirou Suga, Takahisa Miyamoto, Masayoshi Iio, Shoji Hatano, Improvement of freezing tolerance in transgenic tobacco leaves by expressing the hiC6 gene, Biosci. Biotechnol. Biochem., 10.1271/bbb.65.1796, 65, 8, 1796-1804, 65(8): 1796-1804, 2001.08, 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..
5. Suga, K., Honjoh, K., Furuya, N., Shimizu, H., Nishi, K., Shinohara, F., Hirabaru, Y., Maruyama, I., Miyamoto, T., Hatano, S., and Iio, M.,, 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, Biosci. Biotechnol. Biochem., 66, 6, 1314-1327, 66(6): 1314-1327, 2002.06.
6. Ken Ichi Honjoh, Koushirou Suga, Fuminori Shinohara, Isao Maruyama, Takahisa Miyamoto, Shoji Hatano, Masayoshi Iio, Preparation of protoplasts from Chlorella vulgaris K-73122 and cell wall regeneration of protoplasts from C. vulgaris K-73122 and C-27, J. Fac. Agr., Kyushu Univ., 47, 2, 257-266, 47(2): 257-266, 2003.02, 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% 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..
7. Ken Ichi Honjoh, Ayano Mimura, Eiko Kuroiwa, Takahiro Hagisako, Koushirou Suga, Hideyuki Shimizu, Rama Shanker Dubey, Takahisa Miyamoto, Shoji Hatano, Masayoshi Iio, Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27, Biosci. Biotechnol. Biochem., 10.1271/bbb.67.1888, 67, 9, 1888-1896, 67(9): 1888-1896, 2003.09, 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..
8. Kenichi Honjoh, Takeshi Machida, Takahiro Hagisako, Koushirou Suga, Madoka Yonekura, Hideyuki Shimizu, Naoto Ohashi, Takahisa Miyamoto, Shoji Hatano, Masayoshi Iio, Molecular cloning and characterization of a cDNA for low-temperature inducible cytosolic glucose 6-phosphate dehydrogenase gene from Chlorella vulgaris and expression of the gene in Saccharomyces cerevisiae, Plant Science, 10.1016/j.plantsci.2006.12.004, 172, 3, 649-658, 172, 649-658, 2007.03, A cDNA clone corresponding to the gene for glucose 6-phosphate dehydrogenase (E.C. 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..
9. Ken-ichi Honjoh, Takeshi Machida, Koutarou Nishi, Kanae Matsuura, Kevin Webby Soli, Takatoshi Sakai, Hiroya Ishikawa, Kiyoshi Matsumoto, Takahisa Miyamoto, Masayoshi Iio, Improvement of freezing and oxidative stress tolerance in Saccharomyces cerevisiae by taurine, Food Sci. Technol. Res., 10.3136/fstr.13.145, 13, 2, 145-154, 13,2, 145-154, 2007.05, 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..
10. Machida, T., Kato, E., Ishibashi, A., Ohashi, N., Honjoh, K., Miyamoto, T., Molecular characterization of low-temperaure-inducible NTR-C in Chlorella vulgaris, Nucleic Acids Symposium Series, No. 51, 463-464, 2007.11.
11. Takeshi Machida, Eri Kato, Akiko Ishibashi, Jun-ichi Sato, Shinji Kawasaki, Youichi Niimura, Ken-ichi Honjoh, Takahisa Miyamoto, Expression Pattern of a Chloroplast NADPH-Dependent Thioredoxin Reductase in Chlorella vulgaris during Hardening and Its Interaction with 2-Cys Peroxiredoxin, Biosci. Biotechnol. Biochem., 10.1271/bbb.80761, 73, 3, 695-701, 73(3), 695-701, 2009.03, 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..
12. Takeshi Machida, Ken-ichi Honjoh, Hideyuki Shimizu, Maiko Yamamoto, Masayoshi Iio, Takahisa Miyamoto, Expression of A Gene Encoding A Functional Glycosyl Hydrolase, Trehalase, from Nicotiana Tabacum in Saccharomyces Cerevisiae, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 54, 2, 297-303, 54, 2, 297-304, 2009.10, 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..
13. Takeshi Machida, Naoto Ohashi, Ayano Mimura, Ken-ichi Honjoh, Masayoshi Ho, Takahisa Miyamoto, Chloroplastic Glucose 6-Phosphate Dehydrogenase from Chlorella vulgaris Alleviates Freezing and Menadione-Induced Oxidative Stresses in Saccharomyces cerevisiae, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 55, 1, 29-38, 55, 1, 29-38, 2010.02, 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..
14. Ken-ichi Honjoh, Kanae Matsuura, Takeshi Machida, Koutarou Nishi, Miki Nakao, Tomoki Yano, Takahisa Miyamoto, Masayoshi Iio, Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine: co-expression of cDNA clones, from Cyprinus carpio, for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae, Amino Acids, 10.1007/s00726-009-0328-6, 38, 4, 1173-1183, 38(4): 1173-1183, 2010.04, 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..
15. Takeshi Machida, Ken-ichi Honjoh, Ayuko Aso, Maiko Yamamoto, Masayoshi Ho, Takahisa Miyamoto, Trehalose 6-Phosphate Synthase and Trehalose 6-Phosphate Phosphatase from Nicotiana tabacum Function in Trehalose Biosynthesis and Environmental Stress Tolerance of Yeast, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 55, 2, 261-268, 55, 2, 261-268, 2010.10, 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..
16. Takeshi Machida, Akiko Ishibashi, Ai Kirino, Jun-ichi Sato, Shinji Kawasaki, Youichi Niimura, Ken-ichi Honjoh, Takahisa Miyamoto, Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin, PLOS ONE, 10.1371/journal.pone.0045988, 7, 9, e45988, 2012.09, 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..
17. Ken-ichi Honjoh, Tomoko Mishima, Nozomi Kido, Misako Shimamoto, Takahisa Miyamoto, Investigation of Routes of Salmonella Contamination Via Soils and the Use of Mulch for Contamination Control during Lettuce Cultivation, Food Sci. Technol. Res., 10.3136/fstr.20.961, 20, 5, 961-969, 2014.09, [URL], 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..
18. Ken-ichi Honjoh, Yuri Iwazako, Yin Lin, Nobuyuki Kijima, Takahisa Miyamoto, Possibilities for Contamination of Tomato Fruit by Listeria monocytogenes during Cultivation, Food Sci. Technol. Res., 10.3136/fstr.22.349, 22, 3, 349-357, 2016.05, [URL], 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 (
19. Approaches of Improvement of Freezing-Stress Tolerance of Yeast.
20. 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..
21. Ken-ichi Honjoh, Yin Lin, Kiyomi Jo, Yuri Iwaizako, Masayuki Maeda, Nobuyuki Kjima, Takahisa Miyamoto, Possible Contamination Routes of Listeria monocytogenes in Leaf Lettuce during Cultivation, Food Sci. Technol. Res., 10.3136/fstr.24.911, 24, 5, 911-920, 2018.09, Outbreaks of foodborne illness caused by Listeria monocytogenes in or on fresh produce have been reported. Several contamination routes have been proposed and the cultivation process is one of the suspected routes. Leaf lettuce is one of the most important fresh produce products. In the present paper, we investigated the possible routes of L. monocytogenes contamination in leaf lettuce during cultivation. Leaf lettuce was cultivated in soils inoculated with cocktails of three isolates of L. monocytogenes belonging to different serotypes (1/2a, 1/2b, and 4b). The viability and injury state of L. monocytogenes in soils, and bacteria survival in or on leaf lettuce were investigated during 10 weeks of cultivation. Soils were artificially contaminated with L. monocytogenes at levels of 4, 6 or 8 log CFU/g, followed by cultivation of leaf lettuce in the contaminated soils. Populations of L. monocytogenes in the soil decreased to less than the detection limit (
22. Ken-ichi HONJOH, Hitomi OKANO, Aya KAWABATA, Masaru KUROKAWA, Taiki KIMURA, Takeshi MACHIDA, Yoshimitsu MASUDA and Takahisa MIYAMOTO, Freezing tolerance of Lactuca sativa and induction of CBF and GolS genes during cold treatment, Journal of Faculty of Agriculture, Kyushu University, 63, 2, 249-257, 2018.09.
23. Koushirou Suga, Ken-ichi Honjoh, Naoki Furuya, Hideyuki Shimizu, Koutarou Nishi, Fuminori Shinohara, Yoshie Hirabaru, Isao Maruyama, Takahisa Miyamoto, Two low-temperature-inducible chlorella genes for delta 12 and omega-3 fatty acid desaturase (FAD): isolation of delta 12 and omega-3 fad cDNA clones, expression of delta12 fad in Saccharomyces cerevisiae, and expression of omega-3 fad in Nicotiana tab・・・, Biosci. Biotechnol. Biochem., 10.1271/bbb.66.1314, 66, 6, 1314-1327, 2002.01, Two low-temperature-inducible chlorella genes for delta 12 and omega-3 fatty acid desaturase (FAD): isolation of delta 12 and omega-3 fad cDNA clones, expression of delta12 fad in Saccharomyces cerevisiae, and expression of omega-3 fad in Nicotiana tabacum.
24. Mohamed El-Telbany, Chen-Yu Lin, Marwa Nabil Abdelaziz, Aye Thida Maung, Ayman El-Shibiny, Tahir Noor Mohammadi, Mahmoud Zayda, Chen Wang, Su Zar Chi Lwin, Junxin Zhao, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Potential application of phage vB_EfKS5 to control Enterococcus faecalis and its biofilm in food, AMB Express, https://doi. org/10.1186/s13568-023-01628-6., 13, 130, 2023.11.
25. Ken–ichi HONJOH, Hitomi OKANO, Mika SASAKI, Masaru KUROKAWA, Taiki KIMURA, Kyosuke SHIBATA, Yoshimitsu MASUD A and Takahisa MIYAMOTO, Identification of Low Temperature Inducible Genes of Lactuca sativa by Using Suppression Subtractive Hybridization Method, J. Fac. Agr., Kyushu Univ.,, 69, 1, 11-23, 2024.02.
26. 宮本 敬久, 川畑 浩一, 本城 賢一, 波多野 昌二, Effects of Trehalose on Freeze Tolerance of Baker's Yeast, 九州大学大学院農学研究院紀要, 41, 1, 105-112, 41(1・2), 105-112., 1996.11, 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% 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% 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% trehalose medium retained to nearly 50% 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% trehalose was about 60% 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% and 30% of those of the unfrozen cells, respectively, after 7 days of frozen storage. When the cells cultured in the presence of 3% trehalose were frozen-stored, the CO_2 productivity of the cellfree extracts prepared from 7-days frozen-stored cells decreased to 50% 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..
27. Hatano, S., Honjoh, K., Miyamoto, T., Rapid detection of Salmonella spp. in foods by combination of RAPD analysis and QCM technique., 4th International ANQUE chemistry conference., 1998.01.
28. Takahisa Miyamoto, Yo Ichiro Kuramitsu, Asumi Ookuma, Sudsai Trevanich, Ken Ichi Honjoh, Shoji Hatano, Rapid detection and counting of viable bacteria in vegetables and environmental water using a photon-counting TV camera, J. Food Prot., 10.4315/0362-028X-61.10.1312, 61, 10, 1312-1316, Vol.61, No.10, 1312-1316, 1998.10, 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% 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..
29. Rapid detection method of Salmonella-specific PCR product by DNA-immobilized quartz crystal microbalance. Jpn. J. Food Microbiol., 16(1) 57-63. (1999).
30. 宮本 敬久, Trevanich Sudsai, 本城 賢一, サルモネラ特異的gatDの遺伝子領域のPCR増幅によるサルモネラ迅速検出, 日本食品微生物学会雑誌, 10.5803/jsfm.16.99, 16, 2, 99-109, 第16巻2号,99-109., 1999.07.
31. Studies on Freezing Injury of Unwashed and Washed Cells of Escherichia coli O157:H7.
32. Takahisa Miyamoto, Kouji Sukimoto, Md Abu Sayed, Sam In Kim, Ken Ichi Honjoh, Shoji Hatano, Detection of penicillin-binding proteins in Bacillus cereus by using biotinylated β-lactams, J. Fac. Agr., Kyushu Univ., 44, 3-4, 299-307, 44(3,4): 299-307 (2000), 2000.02, 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..
33. Sudsai Trevanich, Takahisa Miyamoto, Yoko Harada, Ken Ichi Honjoh, Shoji Hatano, Rapid detection of enterotoxigenic Escherichia coli O6 in water by using monoclonal antibody and a photon-counting television camera, J. Food Prot., 10.4315/0362-028X-63.4.534, 63, 4, 534-538, Vol.63(4), 534-538, 2000.04, 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% 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..
34. Kim, S.-I., Miyamoto, T., Honjoh, K., Iio, M., and Hatano, S., Molecular cloning, overproduction and characterization of the Bacillus cereus IMP dehydrogenase, Biosci. Biotechnol. Biochem., 10.1271/bbb.64.1210, 64, 6, 1210-1216, Vol.64, No.6, 1210-1216, 2000.06.
35. Miyamoto, T., Ichioka, N., Sasaki, C., Kobayashi, H., Honjoh, K., Iio, M. and Hatano, S., Polymerase chain reaction assay for detection of Escherichia coli O157:H7 and Escherichia coli O157:H-, J. Food Protection, 65, 1, 5-11, 65(1),5-11 (2002), 2002.01.
36. Miyamoto, T., Sayed, Md. A., Sasahara, R., Sukimoto, K., Umezaki, A.,Honjoh, K., Iio, M. and Hatano, S., Cloning and overexpression of Bacillus cereus penicillin-binding protein 3 gene in Escherichia coli, Biosci. Biotechnol. Biochem., 66, 1, 44-50, 66(1): 44-50, 2002.01.
37. Hideyuki Shimizu, Naoki Furuya, Koushirou Suga, Ken Ichi Honjoh, Takahisa Miyamoto, Shoji Hatano, Masayoshi Iio, Freezing tolerance of transgenic tobacco with increased content of unsaturated fatty acid by expressing the CvFad2 or CvFad3 gene, J. Fac. Agr., Kyushu Univ., 47, 2, 307-317, 47(2): 307-317, 2003.02, 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% 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% 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..
38. Development of a Rapid and Simple Method for Measuring Total Viable Bacterial Counts by Flow Cytometry.
39. Takahisa Miyamoto, Hideaki Kamikado, Hiroshi Kobayashi, Ken Ichi Honjoh, Masayoshi Iio, Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk, Journal of Food Protection, 10.4315/0362-028X-66.7.1222, 66, 7, 1222-1226, 66(7):1222-1226, 2003.07, 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% 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..
40. Takahisa Miyamoto, Hideaki Kamikado, Chie Sasaki, Kiyoko Sadakari, Ken Ichi Honjoh, Masayoshi Iio, An attempt to identify Bacillus cereus by PCR, Biocontrol Sci., 10.4265/bio.9.69, 9, 3, 69-75, 9(3): 69-75, 2004.09, 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..
41. Miyamoto, T., Shimizu, Y., Kobayashi, H., Honjoh, K., 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., Sens. Mater., 15, 8, 453-466, 15 (8): 453-466, 2005.01.
42. Hiroshi Kobayashi, Takahisa Miyamoto, Yoshikazu Hashimoto, Madoka Kiriki, Ai Motomatsu, Ken Ichi Honjoh, Masayoshi Iio, Identification of factors involved in recovery of heat-injured Salmonella enteritidis, Journal of Food Protection, 10.4315/0362-028X-68.5.932, 68, 5, 932-941, 68 (5): 932-941, 2005.05, 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% 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..
43. Takeshi Machida, Hisako Murase, Eri Kato, Ken-ichi Honjoh, Kiyoshi Matsumoto, Takahisa Miyamoto, Masayoshi Iio, Isolation of cDNAs for hardening-induced genes from Chlorella vulgaris by suppression subtractive hybridization, Plant Sci., 10.1016/j.plantsci.2008.04.002, 175, 3, 238-246, 2008.09, 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..
44. Effects of Food Additives on the Antibacterial Activity of Green Tea Extracts.
45. Ken-ichi Honjoh, Kumiko Fujihara, Takahiro Haraguchi, Yukari Ono, Hiroshi Kobayashi, Hiroshi Hiwaki, Hideaki Kamikado, Sung Sik Jang, Sangryeol Ryu, Takahisa Miyamoto, Subtyping of Listeria monocytogenes Based on Nucleotide Polymorphism in the clpC, inlA, hlyA, and plcA Genes and Rapid Identification of L. monocytogenes Genetically Similar to Clinical Isolates, Food Sci. Technol. Res., 10.3136/fstr.14.557, 14, 6, 557-564, Vol. 14, No.6, 557-564, 2008.11, 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..
46. Yuta Watanabe, Naotaka Yamada, Takeshi Machida, Ken-ichi Honjoh, Eiichi Kuwano, Influence of Cold Hardening on Chlorophyll and Carotenoid in Chlorella vulgaris, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 54, 1, 195-200, 54(1): 195-200, 2009.02, 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..
47. Combined effects of surfactants and preservatives on the antibacterial activity of green tea extracts.
48. Hiroshi Kobayashi, Jun Kubota, Kumiko Fujihara, Ken-ichi Honjoh, Masayoshi Ito, Naomi Fujiki, Mika Nakabe, Shun-ichi Oda, Toshiya Satoyama, Kazushige Takasu, Hisao Nakanishi, Takahisa Miyamoto, Simultaneous Enrichment of Salmonella spp, Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes by Single Broth and Screening of the Pathogens by Multiplex Real-time PCR, Food Sci. Technol. Res., 10.3136/fstr.15.427, 15, 4, 427-438, Vol. 15, No.4, 427-438, 2009.07, 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..
49. Ken-ichi Honjoh, Yoshikazu Hashimoto, Satoshi Shimotsu, Hsu-Ming Wen, Madoka Kiriki, Kimitaka Naito, Mio Tokugawa, Emiko Satake, Hiroshi Kobayashi, Takahisa Miyamoto, Construction of Several Deletion Mutants for Genes Involved in Biofilm Formation and Recovery of Heat-injured Salmonella: Delta agfA and Delta bcsA Mutants of Salmonella Enteritidis; Delta ahpC, Delta ahpF, and Delta katG Mutants of S. Typhimurium; and Delta rpoE, Delta rpoH, and Delta rpoS Mutants of S. Enteritidis and S. Typhimurium, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 54, 2, 421-431, 54, 2, 421-432, 2009.10, 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..
50. 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°C with seasoning solution containing acetic acid, NaCl, and/or GTE were determined during cold storage at 10°C. Viable counts did not increase above the initial values for 7 days of treatment with seasoning solution containing 0.1% acetic acid, 2% NaCl, and 0.05% 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% 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..
51. Kevin Webby Soli, Asako Yoshizumi, Ai Motomatsu, Mami Yamakawa, Masako Yamasaki, Tomoko Mishima, Natsumi Miyaji, Ken-Ichi Honjoh, Takahisa Miyamoto, Decontamination of fresh produce by the use of slightly acidic hypochlorous water following pretreatment with sucrose fatty acid ester under microbubble generation, Food Control, 10.1016/j.foodcont.2010.02.009, 21, 9, 1240-1244, 21(9) 1240-1244, 2010.09, 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..
52. Rui Li, Takaaki Harada, Ken-ichi Honjoh, Takahisa Miyamoto, Phylogenetic analysis and Shiga toxin production profiling of Shiga toxin-producing/enterohemorrhagic Escherichia coli clinical isolates, Microb. Pathog., 10.1016/j.micpath.2010.06.005, 49, 5, 246-251, 49: 246-251, 2010.11, 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%]). 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..
53. Kevin Webby Soli, Ai Motomatsu, Asako Yoshizumi, Mami Yamakawa, Tomoko Mishima, Ken-ichi Honjoh, Takahisa Miyamoto, Comparison of the Bactericidal Effect of Slightly Acidic Hypochlorous Water with That of Conventional Sterilizers, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 55, 2, 275-280, 55, 2, 275-280, 2010.10, 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..
54. Kevin Webby Soli, Asako Yoshizumi, Mami Yamakawa, Tomoko Mishima, Ken-ichi Honjoh, Takahisa Miyamoto, Bacterial Contamination and Microflora of Several Fresh Produce, JOURNAL OF THE FACULTY OF AGRICULTURE KYUSHU UNIVERSITY, 55, 2, 269-273, 55, 2, 269-273, 2010.10, 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% Gram negative Flavobacterium or Xanthomonas. Other Gram negative bacteria isolated from the vegetables include 11% Neisseria or Veillnella (celery), 18% Moraxella (radish), 15% Alcaligenes and 12% Pseudomonas (radish sprout) while Enterobacteriaceae accounted for less than 5% for each of the flora of celery, parsley, and radish sprout. On the contrary, parsley had 25% Kurthia or Bacillus, and 13% Micrococcus, both Gram positive..
55. 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% at 0.025% (W/V), without a decrease in viable counts. Adhesion of P. fluorescens No. 3 was inhibited more than 50% by 2 detergents even at 0.0025% (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..
56. Miyamoto, T., Kawagishi, J., Oishi, A., Shimotsu, S., Mishima, T., Kobayashi, H., Honjoh, K., Inhibition of adhesion of several bacteria onto microtiter plate by selected food additives, Jpn. J. Food Microbiol., 28, 3, 157-166, 2011.10.
57. Kevin Webby Soli, Asako Yoshizumi, Mami Yamakawa, Tomoko Mishima, Ken-ichi Honjoh, Takahisa Miyamoto, Application of a Combined Decontamination Method for Fresh Produce Using SAHW, Sucrose Fatty Acid Ester and Microbubbles, Food Sci. Technol. Res., 10.3136/fstr.17.555, 17, 6, 555-559, 17, 6, 555-559, 2011.11, 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..
58. Pei Liu, Hiromi Mizue, Kumiko Fujihara, Hiroshi Kobayashi, Hideaki Kamikado, Takashi Tanaka, Ken-ichi Honjoh, Takahisa Miyamoto, A New Rapid Real-Time PCR Method for Detection of Listeria monocytogenes Targeting the hlyA Gene, Food Sci. Technol. Res., 10.3136/fstr.18.47, 18, 1, 47-57, 2012.01, 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% while TaKaRa and ABI detection kit were 101.1% and 107.4%, 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% when testing L. monocytogenes isolates, and for other Listeria isolates were 15.4%, 76.9% and 100% 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..
59. Wen Hsu-Ming, Kimitaka Naito, Yoshimasa Kinoshita, Hiroshi Kobayashi, Ken-ichi Honjoh, Kousuke Tashiro, Takahisa Miyamoto, Changes in transcription during recovery from heat injury in Salmonella typhimurium and effects of BCAA on recovery, Amino Acids, 10.1007/s00726-011-0934-y, 42, 6, 2059-2066, 42, 6, 2059-2066, 2012.06, 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% BCAA resulted in an enhanced recovery of injured cells in comparison to those recovered in TSB without BCAA. Supplementation of BCAA at 0.1% 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..
60. Tomoko Mishima, Nozomi Kido, Satoko Nakashima, Mami Yamakawa, Natsumi Miyaji, Kevin Webby Soli, Ken-ichi Honjoh, Md Latiful Bari, Takahisa Miyamoto, Investigation of Possible Situation of Internalization of Salmonella Enteritidis in Tomato Fruits and Bacterial Survival during Tomato Plant Cultivation, Food Sci. Technol. Res., 10.3136/fstr.18.869, 18, 6, 869-877, 2012.11, 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 (
61. Takahisa Miyamoto, Youko Mekada, Masahiro Kurahachi, Mai Umeno, Motokazu Nakayama, Naofumi Shigemune, Takashi Tsugukuni, Hajime Tokuda, Hirofumi Tachibana, Ken-ichi Honjoh, A highly sensitive method for quantifying gallocatechin gallate and its epimer using a catechin-specific peptide, Food Control, 10.1016/j.foodcont.2012.06.017, 29, 1, 162-166, 29, 1, 162-166, 2013.01, 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..
62. Zhang Xiaoguang, Tsuji Sachiko, Kitaoka Hayato, Tamai Mitsuru, Kobayashi Hiroshi, Honjoh Ken–ichi, Miyamoto Takahisa, Honjo Ken-ichi, 張 暁光, 辻 祥子, 北岡 勇人, 玉井 充, 小林 弘司, 本城 賢一, 宮本 敬久, Effects of Pretreatments on Detection of E. coli O157:H7 by SPR Biosensor, 九州大学大学院農学研究院紀要, 58, 2, 307-312, 58, 2, 307-312, 2013.09, Escherichia coli (E. coli) O157:H7 was detected by using a surface plasmon resonance (SPR) biosensor and two antibodies with different characters. The lower limit of detection of E. coli O157:H7 samples after pretreatments was determined by SPR. Seven pretreatment methods for preparing E. coli O157:H7 samples for SPR detection; beads disruption, sonication, and heat shock, osmotic shock, lysozyme, alkali, and boiling treatments were compared for SPR signal with untreated cells as a control. In the case of the antibody raised against intracellular substance, β–D–galactosidase (β–gal), was used for the detection, the lower limit of detection was 4.9×10^5 CFU/ml for both sonicated and alkali treated samples. The lower limit of detection was 8.3×10^6 CFU/ml for beads disrupted samples, and 8.2×10^8 CFU/ml for both lysozyme treated and untreated samples. In contrast, significant SPR signal was not obtained for heat shocked, osmotic shocked and boiled samples even at 8.2×10^8 CFU/ml. Sonication pretreatment improved the lower limit of detection for E. coli O157:H7 by three orders of magnitude compared with that of untreated sample when anti-β-gal antibody was used for detection by SPR biosensor. In the case of antibody raised against lipopolysaccharide (LPS), the cell surface substance, was used, sonicated E. coli O157:H7 sample was detected by SPR at 1.3×10^5 CFU/ml. The lower limit of detection was 1.1×10^6 CFU/ml for heat shocked, lysozyme treated, alkali treated, boiled and untreated samples, and 7.7×10^7 CFU/ml for beads disrupted samples, respectively. After the osmotic shock treatment, E. coli O157:H7 was not detected by SPR even at 2.1×10^8 CFU/ml. These results show that sonication was the most effective pretreatment method for the detection of E. coli O157:H7 by SPR using both antibodies recognizing intracellular β–gal, and cell surface LPS..
63. Xiaoguang Zhang, Hayato Kitaoka, Sachiko Tsuni, Mitsuru Tamai, Hiroshi Kobayashi, Ken-ichi Honjoh, Takahisa Miyamoto, Development of a Simultaneous Detection Method for Foodborne Pathogens Using Surface Plasmon Resonance Biosensors, Food Sci. Technol. Res., 10.3136/fstr.20.317, 20, 2, 317-325, 2014.03, 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..
64. Takahisa Miyamoto, Seiyo Toyofuku, Narumi Tachiki, Etsuko Kimura, Ting Zhou, Tadahiro Ozawa, Motokazu Nakayama, Naofumi Shigemune, Kanami Shimatani, Hajime Tokuda, Ken-ichi Honjoh, Specific inhibition of cytotoxicity of Shiga-like toxin 1 of enterohemorrhagic Escherichia coli by gallocatechin gallate and epigallocatechin gallate, Food Control, 10.1016/j.foodcont.2014.02.017, 42, 263-269, 2014.08, 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
65. Md Tariqul Islam, Akinobu Oishi, Chikako Machida, Aya Ogura, Shoken Kin, Ken-ichi Honjoh, Takahisa Miyamoto, Combined effects of selected food additives on adhesion of various foodborne pathogens onto microtiter plate and cabbage leaves, Food Control, 10.1016/j.foodcont.2014.05.034, 46, 233-241, 2014.12, 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% 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%) and PT (1%), 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..
66. Xiaoguang Zhang, Sachiko Tsuji, Hayato Kitaoka, Hiroshi Kobayashi, Mitsuru Tamai, Ken-ichi Honjoh, Takahisa Miyamoto, Simultaneous Detection of Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes at a Very Low Level Using Simultaneous Enrichment Broth and Multichannel SPR Biosensor, JOURNAL OF FOOD SCIENCE, 10.1111/1750-3841.13843, 82, 10, 2357-2363, 2017.10, 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..
67. Takahisa Miyamoto, Xiaoguang Zhang, Yuuki Ueyama, Kitichalermkiat Apisada, Motokazu Nakayama, Yasuto Suzuki, Tadahiro Ozawa, Asako Mitani, Naofumi Shigemune, Kanami Shimatani, Koji Yui, Ken-ichi Honjoh, Development of novel monoclonal antibodies directed against catechins for investigation of antibacterial mechanism of catechins, JOURNAL OF MICROBIOLOGICAL METHODS, 10.1016/j.mimet.2017.03.014, 137, 6-13, 2017.06, 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..
68. Xiaowen Cui, Hsu-Ming Sherman Wen, Yoshimasa Kinoshita, Shota Koishi, Chika Isowaki, Liushu Ou, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Role of Phage Shock Protein in Recovery of Heat-injured Salmonella, Biocontrol Sci., 10.4265/bio.23.17, 23, 1, 17-25, 2018.03, 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..
69. Duc Hoang Minh, Son Hoang Minh, Ken-ichi Honjoh, Takahisa Miyamoto, Isolation and bio-control of Extended Spectrum Beta-Lactamase (ESBL)-producing Escherichia coli contamination in raw chicken meat by using lytic bacteriophages, LWT-Food Sci. Technol., 10.1016/j.lwt.2016.04.013, 71, 339-346, 2016.09, Extended Spectrum Beta-Lactamase-producing Escherichia coli (ESBLEC) were isolated from 12/13 (92.3%) raw chicken meat samples by using selective culture and PCR. Of the 27 ESBLEC analyzed, 33.3% (9/27) of isolates were positive for ESBL of CTX-M group 1 followed by TEM (22.2%), SHV (22.2%), CTX-M group 2 (11.1%), and CTX-M group 9 (11.1%). 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%) and 20 (74%) of 27 ESBLEC isolates examined, respectively. The remaining phages lysed less than 50% 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..
70. Hoang Minh Son, Hoang Minh Duc, Ken-ichi Honjoh, Takahisa Miyamoto, Identification of the newly identified subtilase cytotoxin-encoding gene (subAB2-2) among clinical Shiga toxin-producing Escherichia coli isolates, Can. J. Microbiol., 10.1139/cjm-2015-0519, 61, 12, 990-994, 2015.12, 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%) eae-negative STEC strains isolated from patients. Most subAB-positive STEC strains (80%) 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% and 100% 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..
71. Son Hoang Minh, Etsuko Kimura, Duc Hoang Minh, Ken-ichi Honjoh, Takahisa Miyamoto, Virulence characteristics of Shiga toxin-producing Escherichia coli from raw meats and clinical samples, Microbial. Immune., 10.1111/1348-0421.12235, 59, 3, 114-122, 2015.03, 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% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae-negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% 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% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% 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..
72. Hoang Minh Duc, Hoang Minh Son, Ken-ichi Honjoh, Takahisa Miyamoto, Isolation and application of bacteriophages to reduce Salmonella contamination in raw chicken meat, LWT-Food Sci. Technol., 10.1016/j.lwt.2018.01.072, 91, 353-360, 2018.05, 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
73. Md Tariqul Islam, Aya Ogura, Chikako Machida, Noriko Morinaga, Ken-ichi Honjoh, Takahisa Miyamoto, Effects of epsilon-polylysine and Milk Serum Protein on the Attachment and Decontamination of Salmonella Enteritidis on Lettuce and Radish Sprouts, Food Sci. Technol. Res., 10.3136/fstr.22.703, 22, 5, 703-711, 2016.09, To understand the effects of pretreatment of lettuce during cultivation with 0.001% epsilon-polylysine (PL) in combination with 0.25% 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% 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
74. Munenori Furuta, Takayuki Nasu, Kouichi Umeki, Duc Hoang Minh, Ken-Ichi Honjoh, Takahisa Miyamoto, Characterization and Application of Lytic Bacteriophages against Campylobacter jejuni Isolated from Poultry in Japan, Biocontrol Sci., 10.4265/bio.22.213, 22, 4, 213-221, 2017.12, [URL], 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%) 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% 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..
75. Xiao-guang Zhang, Sachiko Tsuji, Hayato Kitaoka, Mitsuru Tamai, Hiroshi Kobayashi, Ken-ichi HONJOH, and Takahisa MIYAMOTO, Preparation and characterization of monoclonal antibodies suitable for detection of foodborne pathogens by biosensor, Journal of Faculty of Agriculture, Kyushu University, 63, 2, 319-330, 2018.09.
76. Hoang Minh Son, Hoang Minh Duc, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Application of bacteriophages in simultaneously controlling Escherichia coli O157:H7 and extended-spectrum beta-lactamase producing Escherichia coli, Appl. Microbiol. Biotechnol., 10.1007/s00253-018-9399-1, 102, 23, 10259-10271, 102, 10259-10271, 2018.12, 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..
77. Aye Thida Maung, Tahir Noor Mohammadi, Satoko Nakashima, Pei Liu, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Antimicrobial resistance profiles of Listeria monocytogenes isolated from chicken meat in Fukuoka, Japan, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 10.1016/j.ijfoodmicro.2019.05.016, 304, 49-57, 2019.09, 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%) were positive for L. monocytogenes in 2012, while 12 among 50 samples in 2017 (24%) 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%), 1/2b (73.9%), 1/2c (1.5%), and 4b/4e (3.1%). In contrast, the 2017 isolates showed 1/2a (48.3%) and 1/2b (51.7%) 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% and 100% of the isolates were resistant to cefoxitin, 57.3% and 95.7% to fosfomycin, 72.0% and 82.6% to oxacillin, 8.0% and 17.4% to clindamycin, respectively. In addition, 2.7% of the isolates in 2012 were resistant to flomoxef and 4.3% of the isolates in 2017 to linezolid. Multidrug resistance (MDR) to 3 or more antimicrobials was observed in 35/75 (46.7%) isolates of 2012 and 19/23 (82.6%) in 2017. Detection of antimicrobial resistance (AMR) genes by PCR showed that the resistant isolates of 2012 were positive for mecA (96.3%) and ermC (83.3%), whereas the resistant isolates in 2017 screened positive for mecA (94.7%) and mefA (25.0%). 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..
78. Pham Thi Vinh, Yui Shinohara, Akifumi Yamada, Hoang Minh Duc, Motokazu Nakayama, Tadahiro Ozawa, Jun Sato, Yoshimitsu Masuda, Ken-Ichi Honjoh, Takahisa Miyamoto, Baicalein Inhibits Stx1 and 2 of EHE: Effects of Baicalein on the Cytotoxicity, Production, and Secretion of Shiga Toxins of Enterohaemorrhagic Escherichia coli, TOXINS, 10.3390/toxins11090505, 11, 9, 2019.09, 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..
79. Yue Guo, Xiaowen Cui, Liushu Ou, Chika Isowaki, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Effects of Sucrose on Heat Resistance and Gene Expression in Salmonella Typhimurium, Food Sci. Technol. Res., 10.3136/fstr.25.903, 25, 6, 903-913, 2019.11, 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 % (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 % 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 % 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..
80. Hoang Minh Duc, Hoang Minh Son, Pham Hong Ngan, Jun Sato, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Isolation and application of bacteriophages alone or in combination with nisin against planktonic and biofilm cells of Staphylococcus aureus, Appl. Microbiol. Biotechnol., 10.1007/s00253-020-10581-4, 104, 11, 5145-5158, 2020.06, 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..
81. Hoang Minh Duc, Hoang Minh Son, Hazel Pang Shu Yi, Jun Sato, Pham Hong Ngan, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Isolation, characterization and application of a polyvalent phage capable of controlling Salmonella and Escherichia coli O157:H7 in different food matrices, Food Res. Int., 10.1016/j.foodres.2020.108977, 131, 108977-108977, 2020.05, 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..
82. Cunkuan Shen, Md Tariqul Islam, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Transcriptional changes involved in inhibition of biofilm formation by epsilon-polylysine in Salmonella Typhimurium, Appl. Microbiol. Biotechnol., 10.1007/s00253-020-10575-2, 104, 12, 5427-5436, 2020.06, 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% epsilon-polylysine in 0.1% 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..
83. Xiaowen Cui, Chuanqi Hu, Liushu Ou, Yumiko Kuramitsu, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Transcriptional analysis on heat resistance and recovery from thermal damage in Salmonella under high salt condition, LWT-Food Sci. Technol., 10.1016/j.lwt.2019.02.056, 106, 194-200, 2019.06, 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% (TSB), 4% (4SC) and 8% (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..
84. Mahmoud Ge Zayda, Yoshimitsu Masuda, Ahmed M. Hammad, Ken-ichi Honjoh, Abdelrahman M. Elbagory, Takahisa Miyamoto, Molecular characterisation of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus isolated from bovine subclinical mastitis and Egyptian raw milk cheese, INTERNATIONAL DAIRY JOURNAL, 10.1016/j.idairyj.2020.104646, 104, 2020.05, 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% 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..
85. Apisada Kitichalermkiat, Masahiro Kurahachi, Ai Nonaka, Motokazu Nakayama, Kanami Shimatani, Naofumi Shigemune, Takashi Tsugukuni, Jun Hitomi, Jun Sato, Takumi Sonoda, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Effects of Epigallocatechin Gallate on Viability and Cellular Proteins of Staphylococcus aureus, Food Sci. Technol. Res., 10.3136/fstr.25.277, 25, 2, 277-285, 2019.03, 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..
86. Ayumi Musou-Yahada, Ken-ichi Honjoh, Kenta Yamamoto, Takahisa Miyamoto, Hideaki Ohta, Utilization of Single Nucleotide Polymorphism-based Allele-specific PCR to Identify Shiikuwasha (Citrus depressa Hayata) and Calamondin (Citrus madurensis Lour.) in Processed Juice, Food Sci. Technol. Res., 10.3136/fstr.25.19, 25, 1, 19-27, 2019.01, 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..
87. Apisada Kitichalermkiat, Mao Katsuki, Jun Sato, Takumi Sonoda, Yoshimitsu Masuda, Ken ichi Honjoh, Takahisa Miyamoto, Effect of epigallocatechin gallate on gene expression of Staphylococcus aureus, J. Glob. Antimicrob. Resist., 10.1016/j.jgar.2020.06.006, 22, 854-859, 2020.09, © 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..
88. Yoshimitsu Masuda, Erika Sakamoto, Ken Ichi Honjoh, Takahisa Miyamoto, Role of toxin-antitoxin-regulated persister population and indole in bacterial heat tolerance, Appl. Environ. Microbiol., 10.1128/AEM.00935-20, 86, 16, 1-10, 2020.08, © 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..
89. Cunkuan Shen, Chikako Machida, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Effect of Selected Food Additives on Biofilm Formation by Foodborne Pathogens on Stainless Steel, J. Fac. Agr., Kyushu Univ., 10.5109/4363550, 66, 1, 45-52, 2021.04.
90. Yu Zhang, Hung-Hsin Huang, Hoang Minh Duc, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto , Endolysin LysSTG2: Characterization and application to control Salmonella Typhimurium biofilm alone and in combination with slightly acidic hypochlorous water
, Food Microbiology , 10.1016/, 98, 103791, 2021.09.
91. Khin Zar Linn, Munenori Furuta, Motokazu Nakayama, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Characterization and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from chicken and pork
, International Journal of Food Microbiology, 360, 109440, 2021.10.
92. 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..
93. Ken Ichi Honjoh, Takeshi Machida, Koutarou Nishi, Kanae Matsuura, Kevin Webby Soli, Takatoshi Sakai, Hiroya Ishikawa, Kiyoshi Matsumoto, Takahisa Miyamoto, Masayoshi Iio, Improvement of freezing and oxidative stress tolerance in Saccharomyces cerevisiae by taurine, Food Science and Technology Research, 10.3136/fstr.13.145, 13, 2, 145-154, 2007.05, The effect of taurine on the survival of Saccharomyces cerevisiae after freezing and oxidative stress was investigated. Proline and NaCI 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 cryoprotectant and/or an antioxidant in yeast..
94. Takahisa Miyamoto, Yasuko Murata, Hiroshi Kobayashi, Masaaki Shimoyae, Hideaki Kamikado, Naohiro Noda, Kouji Maruyama, Ken Ichi Honjoh, Masayoshi Iio, Enumeration of viable counts in raw milk using the automated fluorescence microscopic method, Biocontrol Sci., 10.4265/bio.10.147, 10, 4, 147-154, 2005.12, 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..
95. Hung-Hsin Huang, Yu Zhang, Nanami Asoshima, Hoang Minh Duc, Jun Sato, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Complete Genome Sequence of Campylobacter coli Bacteriophage CAM-P21, Microbiol. Resour. Ann., 10, 15, e00223-21, 2021.04.
96. Yoshimitsu Masuda, Shun Kawabata, Tatsuya Uedoi, Ken-ichi Honjoh, Takahisa Miyamoto, Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting E. coli and Neighboring Gram-Positive Pathogens, Food Microbiol., 10.1128/Spectrum.00141-21, 9, 1, 2021.09.
97. Huang, Hung-Hsin; Furuta, Munenori; Nasu, Takayuki; Hirono, Miku; Pruet, Jaroenkolkit; Hoang Minh Duc; Zhang, Yu; Masuda, Yoshimitsu; Honjoh, Ken-ichi; Miyamoto, Takahisa, Inhibition of phage-resistant bacterial pathogen re-growth with the combined use of bacteriophages and EDTA, Food Microbiol., 10.1016/, 100, 103853, 2021.12.
98. Zhang, Yu; Huang, Hung-Hsin; Duc, Hoang Minh; Masuda, Yoshimitsu; Honjoh, Ken-ichi; Miyamoto, Takahisa, Application of endolysin LysSTG2 as a potential biocontrol agent against planktonic and biofilm cells of Pseudomonas on various food and food contact surfaces, Food Control, 10.1016/j.foodcont.2021.108460, 131, 108460, 2022.01.
99. Tahir Noor Mohammadi, Cunkuan Shen, Yuncheng Li, Mahmoud Gamaleldin Zayda, Jun Sato, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Characterization of Clostridium perfringens bacteriophages and their application in chicken meat and milk, International Journal of Food Microbiology, 361, 109446, 2022.01.
100. Trang Nguyen Phan, Takahisa Miyamoto, Yoshimitsu Masuda, Ken-ichi Hohjoh, Anh Ngoc Tong Thi, Occurrence, antimicrobial resistance, and genetic diversity of Listeria monocytogenes at fish-processing plants in Vietnam, Food Sci. Technol. Res., 10.3136/fstr.FSTR-D-21-00195, 28, 2, 141-149, 2022.01.
101. Cunkuan Shen, Yunzhi Lin, Tahir Noor Mohammadi, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Characterization of novel antimicrobial peptides designed on the basis of amino acid sequence of peptides from egg white hydrolysate, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 10.1016/j.ijfoodmicro.2022.109802, 378, 2022.06.
102. Zhang, Y., Huang, H.-H., Ma, L.Z., Masuda, Y., Honjoh, K.-I., Miyamoto, T., Inactivation of mixed Escherichia coli O157:H7 biofilms on lettuce by bacteriophage in combination with slightly acidic hypochlorous water (SAHW) and mild heat treatment, Food Microbiol., 10.1016/, 104, 2022.06.
103. Tahir Noor Mohammadi, Cunkuan Shen, Yuncheng Li, Mahmoud Gamaleldin Zayda, Jun Sato, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Characterization and optimization of bacteriophage cocktails to control Clostridium perfringens in vitro and in curry roux, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 10.1016/j.ijfoodmicro.2022.109886, 380, 2022.08.
104. Trang Nguyen Phan, Anh Ngoc Tong Thi, Yoshimitsu Masuda, Ken-ichi Hohjoh, Takahisa Miyamoto, Slightly acidic hypochlorous water effective against dual-species biofilm of Listeria monocytogenes and Escherichia coli strains isolated from Pangasius fish-processing plants, Food Sci. Technol. Res., 10.3136/fstr.FSTR-D-22-00074, 28, 6, 521-527, 2022.09.
105. Hoang Minh Duc, Yu Zhang, Hoang Minh Son, Hung-Hsin Huang, Yoshimitsu Masuda, Ken-ichi Honjoh, Takahisa Miyamoto, Genomic characterization and application of a novel bacteriophage STG2 capable of reducing planktonic and biofilm cells of Salmonella, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, 10.1016/j.ijfoodmicro.2022.109999, 385, 2023.01.
106. Yuan L, Nakamichi R, Hirata Y, Matsuda A, Shinohara Y, Yamada A, Masuda Y, Honjoh KI, Miyamoto T., Mechanism for inhibition of cytotoxicity of Shiga toxin by luteolin, Toxicol. Vitro, 10.1016/j.tiv.2022.105537, 87, 2023.01.
107. Phan Nguyen Trang, Tong Thi Anh Ngoc, Yoshimitsu Masuda, Ken-ichi Hohjoh, Takahisa Miyamoto, Biofilm Formation From Listeria monocytogenes Isolated From Pangasius Fish-processing Plants, JOURNAL OF FOOD PROTECTION, 10.1016/j.jfp.2023.100044, 86, 3, 2023.03.
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