{"created":"2023-12-20T06:19:34.872826+00:00","id":2000036,"links":{},"metadata":{"_buckets":{"deposit":"926c2910-5b95-4aed-9074-16c071019a9e"},"_deposit":{"created_by":17,"id":"2000036","owner":"17","owners":[17],"pid":{"revision_id":0,"type":"depid","value":"2000036"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:02000036","sets":["370:15:392"]},"author_link":[],"control_number":"2000036","item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2023-08-31"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"32701","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(獣医学)"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"Cow’s milk is a highly nutritious food and has supported human health. However, because the cow’s milk is an animal-derived food, it is not completely free from bacterial toxins. Cow’s milk contaminated by cereulide (CRL) produced by Bacillus cereus and staphylococcal enterotoxin type A (SEA) produced by Staphylococcus aureus caused　extensive outbreaks of gastroenteritis. The individual methods for identifying and quantifying CRL and SEA had their own problems and needed to be improved. Furthermore, there is a need for an analytical method capable of measuring two bacterial toxins that require differentiation. The aim of this study was to reliably identify the presence of CRL and SEA in cow’s milk under identical conditions using liquid chromatography-tandem mass spectrometry (LC-MS/MS). An accurate quantitative method was also developed with easy and simple sample preparations.\nThe presence of CRL in cow’s milk was identified and quantified using our validated method with LC-MS/MS. CRL was concentrated using protein acid-precipitation and extracted from the precipitate by using acetonitrile twice. The combination of protein acid-precipitation and extraction sufficiently eliminated the matrix compounds from the milk and a further clean-up step utilising solid-phase extraction could be omitted. For robustly measuring the samples and keeping the MS devices clean, the extraction solution was diluted 10-fold using methanol. Owing to the minimisation of the interferences caused by fragmentation patterns, multiple reaction monitoring information-dependent acquisition-enhanced product ion spectra enabled the characterisation and identification of CRL. Besides the matrix effect (−4%), an external solvent calibration curve was adapted for accurate quantification. The method was validated using fortified recovery tests, at two concentrations (10 and 50 ng/g), using three samples daily on five different days based on the Japanese guidelines. This new method exhibited good accuracy ranging from 91% to 94%. The relative standard deviations (RSD) of repeatability ranged from 2% to 5%, and the RSD of within-laboratory reproducibility ranged from 5% to 6%. These standard deviations satisfied the criteria for the Japanese validation guidelines. The limit of quantification (LOQ) was estimated to be 2 ng/g. On the product ion spectra at the LOQ level, the library match was satisfactory with a purity fit value of >70%. Thus, this study developed a practical screening method for quantifying CRL and proposed an operation model with conventional individual methods for verifying the trueness.\nSEA contaminant was quantified in cow’s milk by LC–MS/MS with the use of a stable isotope-labelled peptide of SEA as an internal standard. SEA was cleaned up in a two-step process that included pH control and trichloroacetic acid (TCA) precipitation. The pH control phase eliminated other proteins. TCA precipitation cleaned up SEA without special equipment. An appropriate enzyme-to-protein ratio maximised tryptic digestion. A desalting process guaranteed the stable retention of SEA-digested peptides. The coverage of amino-acid sequences (>10%) clearly identified the toxin’s presence. SEA was accurately quantified using LC-MS/MS based on a multiple-reaction monitoring mode. The developed method was validated based on spiked recovery tests at 50 and 100 ng/g conducted with three samples collected on a daily basis for five days based on Japanese validation guidelines. The new method exhibited good accuracy which ranged from 80% to 82%. The RSD of repeatability were 13–14% and the RSD of within-laboratory reproducibility were 13–18%. These standard deviations satisfied the criteria of the Japanese validation guidelines. The LOQ was estimated to be 10 ng/g. By optimizing a sample preparation method for peptide analysis by LC-MS/MS, this study established an accurate reliable method for SEA quantification according to the criteria of the guideline.\nTo verify the applicability of the developed methods to raw milk, a fortified test using a special milk sample as an alternative was tested. In the fortified tests using the milk samples with 10 ng/g CRL (n = 3), the recovery and RSD were 81.1% and 1.6%, respectively. In fortified tests using the milk samples with 50 ng/g SEA (n = 3), the recovery and RSD were 78.8% and 9.1%, respectively. The suitability of the methods was tested and gave satisfactory results. Additionally, a survey of the actual residues of CRL and SEA was conducted. The developed method was applied to 14 raw milk and 8 milk samples pasteurised using the low-temperature, long-time process and collected in Tokyo. None of the samples was found to contain the target toxin. \nIn this study, an analytical method that can measure the two bacterial toxins, CRL and SEA, was developed using the same condition via LC-MS/MS. An accurate and reliable quantitative method was also developed with easy and simple sample preparations. The developed method was applied to a survey in Tokyo, and it was confirmed that there were no residues of CRL and SEA in cow’s milk samples. The developed method is expected to contribute to the identification of causative agents of food poisoning and the prevention of health hazards. Furthermore, it is expected that this study has contributed to the quality assurance of cow’s milk and the strengthening of the inspection system in food hygiene."}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"牛乳は古くから高栄養食品として人々の健康を支えてきた。しかしながら、牛乳は動物由来食品であるため、細菌性毒素の汚染を完全に防除することができず、セレウス菌が産生するセレウリド（CRL）や黄色ブドウ球菌が産生するエンテロトキシンA（SEA）が牛乳に残留したことで、大規模な集団食中毒事件が発生してきた。CRLとSEAを同定かつ定量する個別試験法にはそれぞれの課題があり、改良が必要であった。特に、鑑別が必要な2つの細菌性毒素を測定できる信頼性の高い分析法が求められていた。本研究の目的は、液体クロマトグラフ-タンデム型質量分析計（LC-MS/MS）を用いて、牛乳中CRLとSEAを同一の条件で連続して測定し、再現性高く定量するとともに、簡単かつシンプルな前処理法を構築することとした。\n　LC-MS/MSによる牛乳中のCRLの同定および定量法を開発した。酸性化によるタンパク質沈殿法によって牛乳中のCRLを濃縮し、アセトニトリルによる2回の抽出で回収した。これらの組み合わせにより、牛乳から夾雑成分を除去し、固相精製を用いたクリーンアップステップを省略した。さらに、再現性の良い測定と検出器を清浄に保つために、抽出溶液をメタノールで10倍に希釈した。その結果、フラグメンテーションへの干渉を最小限に抑え、プロダクトイオンスペクトルによるCRLの同定が可能となった。加えて、夾雑成分の影響を-4%とすることで、より簡便な外部標準検量線を採用し、正確な定量値を得た。開発した本法について、牛乳を対象試料に妥当性評価を行った。CRLを2濃度（10、50 ng/g）で添加し、1試験者が1日3併行5日間の回収試験を実施した。その結果、本法は充分な選択性を有しており、真度91-94%、併行精度2-5%、室内精度5-6%であった。定量下限値（LOQ）は2 ng/gに設定できた。各パラメータは農薬等の試験法開発に使用される厚生労働省ガイドライン基準に適合し、本法が試験検査に適応できる分析性能を有していることを確証した。さらに、LOQのプロダクトイオンスペクトルにおいて，標準品との一致率は70%以上であり、LOQまで確実かつ精確に同定・定量する本法の有用性を確証した。本研究は再現性の高いCRLスクリーニング法を開発し、従来の個別法と併用することで真度の検証といった運用を提案した。\n(Koike, H., et al. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 35:2424-2433. 2018)\n　SEAの安定同位体標識ペプチドを内部標準物質として用い，LC-MS/MSにより牛乳中のSEA定量法を開発した。本研究ではトリプシン消化したSEA由来ペプチドを測定することで、牛乳中のSEAを確実かつ精確に同定・定量する。まず、SEA由来ペプチドを3つ選定し、SEA全体の10%以上のアミノ酸配列を網羅することで、LC-MS/MSを使用して未知タンパク質を同定するための基準を満たす同定性能を確保した。次に、CRLと同じLC条件の適用を検証した結果、これらペプチドを十分に分離できることから、2つの細菌性毒素を効率よく迅速に鑑別できることとなった。前処理では、牛乳中のSEAをpH調整とトリクロロ酢酸（TCA）沈殿法の2段階のプロセスで採集した。pH調整によってカゼインを取り除き、SEAを選択的に濃縮した。TCA沈殿法では、特別な器具を使用せずにSEAと他のタンパク質を分別した。また、酵素とタンパク質の比率を最適化することで、トリプシン消化効率を最大かつ安定化した。さらに、脱塩操作によって、分離を阻害する夾雑成分を取り除き、SEA由来ペプチドの保持時間を保証し再現性を高めた。開発した本法について、牛乳を対象試料に妥当性評価を行った。SEAを2濃度（50、100 ng/g）で添加し、1試験者が1日3併行5日間の回収試験を実施したところ、充分な選択性を有しており、真度80-82%、併行精度13-14%、室内精度13-18%であった。定量下限値は10 ng/gに設定できた。各パラメータは農薬等の試験法開発に使用される厚生労働省ガイドライン基準に適合し、本法が試験検査に適応できる分析性能を有していることを確証した。本研究はLC-MS/MSによるペプチド測定に特化した前処理法を開発することで、先行研究よりも高精度かつガイドライン基準を満たす信頼性の高いSEA定量法を確立した。\n(Koike, H., et al. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 36:1098-1108. 2019)\n　開発した分析法を生乳に応用するため、生乳の代替となる特別牛乳を用いて添加回収試験を実施した。CRL 10 ng/gを添加した結果（n=3）では、回収率81.1%、RSD1.6%であった。SEA 50 ng/gを添加した試験（n=3）では、回収率およびRSDはそれぞれ78.8%および9.1%であった。本法の妥当性を検証した結果、十分な結果が得られた。さらに、CRLおよびSEAの実残留量の調査を実施した。東京都内で採取または購入した生乳14検体および低温殺菌牛乳8検体に本法を適用した。いずれの試料からも対象毒素であるCRLとSEAを検出しなかった。\n　以上のことから、本研究はCRLとSEAの2つの細菌毒素を同一のLC-MS/MS条件で連続して測定できる分析法を開発した。また、簡便な前処理法による精確かつ再現性の高い定量が可能となった。開発した本法を東京都内の実態調査に適用し、流通する牛乳類に定量下限値以上のCRLとSEAの残留がないことを確証した。開発した本法が食中毒の病因物質の同定や健康被害の防止に寄与するとともに、本研究が牛乳の品質保証や食品衛生における検査体制の強化に貢献することが期待される。"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第444号"}]},"item_10006_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.14944/0002000036","subitem_identifier_reg_type":"JaLC"}]},"item_10006_textarea_23":{"attribute_name":"Rights","attribute_value_mlt":[{"subitem_textarea_value":"本論文の一部は以下に公表した(Part of this dissertation has been published as follows. )\n1. Koike, H., Kanda, M., Hayashi, H., Matsushima, Y., Ohba, Y., Nakagawa, Y., Nagano, C., Sekimura, K., Hirai, A., Shindo, T., Kamiie, J., Sasamoto, T., Hashimoto, T.: Identification and quantification of cereulide in cow’s milk using liquid chromatography-tandem mass spectrometry. Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment, 35:2424-2433, 2018. \n2. Koike, H., Kanda, M., Hayashi, H., Matsushima, Y., Ohba, Y., Nakagawa, Y., Nagano, C., Sekimura, K., Hirai, A., Shindo, T., Otsuka, K., Kamiie, J., Sasamoto, T., Hashimoto, T.: Quantification of staphylococcal enterotoxin type A in cow’s milk by using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry. Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment, 36:1098-1108, 2019."}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"小池, 裕"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-12-20"}],"filename":"diss_dv_otsu444.pdf","filesize":[{"value":"1.3 MB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"url":"https://az.repo.nii.ac.jp/record/2000036/files/diss_dv_otsu444.pdf"},"version_id":"6ae1f3c3-940b-4b9a-b1ae-824664c84a98"},{"accessrole":"open_access","date":[{"dateType":"Available","dateValue":"2023-12-20"}],"filename":"diss_dv_otsu444_jab&rev.pdf","filesize":[{"value":"196 KB"}],"format":"application/pdf","mimetype":"application/pdf","url":{"url":"https://az.repo.nii.ac.jp/record/2000036/files/diss_dv_otsu444_jab&rev.pdf"},"version_id":"ae768240-5a5f-4416-b6ef-65eea1d1ca2a"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis"}]},"item_title":"牛乳中の細菌性毒素の分析法開発と実態調査","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"牛乳中の細菌性毒素の分析法開発と実態調査","subitem_title_language":"ja"},{"subitem_title":"Development of the novel quantification for bacterial toxins in cow’s milk","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"17","path":["392"],"pubdate":{"attribute_name":"PubDate","attribute_value":"2023-12-20"},"publish_date":"2023-12-20","publish_status":"0","recid":"2000036","relation_version_is_last":true,"title":["牛乳中の細菌性毒素の分析法開発と実態調査"],"weko_creator_id":"17","weko_shared_id":-1},"updated":"2023-12-20T09:58:46.689657+00:00"}