紅麴食品主成分分析技術之研究及有害物質之風險評估

Study of Analysis Technique for Major Components in Monascus Foods and Risk Assessment

蔡佳芬(Chia-Fen Tsai)

指導教授 ： 徐源泰

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.03340

摘要

本研究共分為四個章節，分別為「研究動機及背景」、「紅麴食品中HMG-CoA還原酶抑制物分析技術之研究及健康影響評估」、「紅麴食品中重金屬分析技術之研究及健康影響評估」及「紅麴食品中橘黴素分析技術之研究及健康影響評估」。
第一章簡述紅麴之應用歷史、生理活性物質及可能潛藏的健康風險，以其現階段之發展況狀。
第二章分別探討藥用statins與monacolins之檢驗方法，並據以進行市售紅麴產品中statins與monacolins之含量測定及健康影響評估。以半微量高效液相層析(semi-micro HPLC)同時檢測紅麴食品中3種statins (fluvastatin，lovastatin及simvastatin)。檢體經甲醇萃取，離心、過濾、濃縮後，以BAKERBOND speTM Silica Gel管柱淨化處理，以半微量HPLC分析，移動相為ACN：水：醋酸(70/30/0.5, v/v, pH 3.0)，分析管柱為LUNA C18 (2) (2.0×150 mm)，流速為0.15 mL/min，偵測波長為240 nm。方法之檢出限量均為2 ug/g。以高效液相層析法及Orbitrap LC/MS/MS分別對紅麴食品進行monacolin K lactone form (MKL)及acid form (MKA)之分析，將紅麴食品以乙醇萃取後，進行儀器分析。結果因方法中未涵蓋淨化步驟，在Orbitrap LC/MS/MS分析的部分，雖於標準溶液分析時可得到良好線性關係及偵測感度，惟檢液分析時仍因基質效應之影響，尚難直接進行分析。而以HPLC法分析時，以HOLA TM C18，2.7 um，4.6 × 150 mm層析管柱，於ACN：甲醇：0.1%磷酸(40/40/20, v/v)為移動相溶液，波長236 nm進行偵測，其標準曲線之線性關係良好，且檢出限量可達MKL 8 ug/g，MKA 20 ug/g。以建立之分析方法分別進行statins與monacolins之含量檢測，結果41件含紅麴原料之加工食品僅檢出lovastatin一種statin；24件膳食補充品之MKL含量為36 - 10,238 ug/g，MKA含量為＜20 - 3,399 ug/g；8件紅麴米MKL含量為＜8 - 113 ug/g，MKA均未檢出；8件紅麴醬MKL含量為＜8 - 14 ug/g，MKA均未檢出。現今發展出來的statins類藥物已有很多種，比較經查驗登記之藥品劑量與市售膠囊錠狀紅麴食品monacolins之檢驗結果發現，24件膳食補充品中其每粒膠囊含monacolins超過4 mg者有4件，超過2 mg者有7件，其劑量已為藥品之10 - 20%，因此在食用這些膳食補充品時，已有影響生理功能之可能。
第三章鑒於中國大陸係紅麴米之大宗來源，而大陸產稻米遭重金屬污染之報導時有所聞，本研究分別檢測紅麴產品之砷、鎘、鉻、銅、鉛、硒、鋅及汞含量，並進行健康影響評估。以ICP-MS進行台北地區販售之93件紅麴食品中微量元素(砷、鎘、鉻、銅、鉛、硒及鋅)之分析，以DMA進行汞之分析。分析結果其含量分別為：砷0.005 - 12.04 mg/kg，鎘為< 0.0005 - 2.22 mg/kg，鉻為0.014 - 6.95 mg/kg，銅為0.012 - 8.70 mg/kg，鉛為0.001-0.64 mg/kg，硒為< 0.001 - 1.29 mg/kg，鋅為0.020 - 67.02 mg/kg，汞為＜0.05 - 24.0 ug/kg。調查結果經劑量評估，以60公斤體重成年人來評估每週自各種紅麴食品攝入砷、鎘、鉻、銅，鉛、硒、鋅及汞之含量發現，國人自紅麴食品攝入的砷、鎘、鉻、銅，鉛、硒、鋅及汞含量，均遠低於FAO/WHO建議之暫定每週容許攝取量(PTWI：砷15、鎘7、鉛25、硒7、鉻23.3、銅3500、鋅7000及汞5 ug/kg)，因此尚不致造成人體健康之危害。而不同種類食品中所含微量元素含量差距大，研究推論原料係其微量元素之主要來源。同時本研究認為，ICP-MS及DMA為紅麴食品微量元素分析簡單快速之分析方法。
第四章以高效液相層析法及Orbitrap LC/MS/MS分別對紅麴食品進行citrinin之分析探討，將紅麴食品以乙醇萃取後，進行儀器分析。在Orbitrap LC/MS/MS分析的部分，雖於標準溶液分析時可得到良好線性關係及偵測感度，惟檢液分析時仍因基質效應之影響，尚難直接進行分析。而以HPLC法分析時，以HOLA TM C18，2.7 um，4.6 × 150 mm層析管柱，於ACN：甲醇：0.1%磷酸(40/40/20, v/v)為移動相溶液，於Em 330 nm及Ex 500 nm進行偵測，其標準曲線之線性關係良好，且檢出限量可達20 ng/g，已可據以進行食品中citrinin之分析。而24件膳食補充品之citrinin含量為0.08 - 466 ug/g；8件紅麴米citrinin含量為2.4 - 22.5 ug/g；8件紅麴醬citrinin含量為0.02 - 0.97 ug/g。其中有1件膳食補充品之citrinin含量高達466 ug/g，因此食品製造業者之原料管控仍有待加強。在健康影響評估方面，雖有報告指出citrinin對動物具肝臟毒性及腎毒性，但其對人類健康影響之研究仍嫌不足，且目前亦尚無直接證據及相關報告指出citrinin對人類具有致癌性。但鑑於台灣地處熱帶及亞熱帶，原為黴菌滋長之好發地區，而國人之健康又長期遭受腎病威脅，因此，避免食入有黴菌毒素之虞的食物仍是必要手段。

關鍵字

紅麴 ； 生理活性 ； HMG-CoA還原酶抑制物 ； monacolins ； 橘黴素 ； 重金屬 ； 健康影響評估

並列摘要

This study includes four parts: (1) research motive and background, (2) content and risk assessment of the HMG-CoA reductase inhibitor in foods containing red mould rice (Monascus), (3) survey and risk assessment of As, Cd, Cu, Cr, Hg, Pb, Se, Zn in foods containing red mould rice (Monascus), (4) content and risk assessment of citrinin in foods containing red mould rice (Monascus).
In the first study, sketching the application history, physiological active substance, health risk that may be hiding of Monascus.
In the second study, the detection method of statins and monacolins were established. Statins (fluvastatin, lovastatin and simvastatin) were analyzed by semi-micro HPLC. The principal steps involved extraction in methanol followed by a BAKERBOND speTM Silica Gel clean up step. In semi-micro HPLC, a reversed phase C18 (LUNA C18) column was used and compounds were separated by a mobile phase of acetonitrile/H2O/acetic acid (70:30:0.5, v/v, pH 3) at a flow rate of 0.15 mL/min. A wavelength of UV detector was set at 240 nm. The minimum detectable quantity of the method was found to be 2.0 μg/g. Monacolin K lactone form (MKL) and acid form (MKA) were analyzed by HPLC and Orbitrap LC/MS/MS. The steps involved extraction in ethanol followed by HPLC or Orbitrap LC/MS/MS analysis. In HPLC, a reversed phase HOLA TM C18 column was used and compounds were separated by a mobile phase of acetonitrile/methanol/0.1% phosphoric acid (40/40/20, v/v). A wavelength of UV detector was set at 236 nm. The minimum detectable quantity of the method was found to be 8 μg/g for MKL and 20 μg/g for MKA. Only lovastatin was found in 41 foods containing red mould rice. The contents of MKL in 24 dietary supplement samples were ranged from 36 to 10238 μg/g, MKA were ranged from < 20 to 3399 μg/g, 8 red mold rice contained MKL ranged from < 8 to 113 μg/g, 8 sauces contained MKL ranged from < 8 to 14 μg/g. The contents of monacolins in dietary supplement were very closer to statin drugs, about 10 - 20% of drugs.
In the third study, the concentrations in 93 red mold rice food samples of As, Cd, Cr, Pb, Se, Cu and Zn were determined by inductively coupled plasma - mass spectrometry (ICP-MS), Hg was determined by DMA. The results, calculated in mg/kg (wet weight) for each sample, revealed the general scenario of food safety in Taiwan: As (0.005 - 12.04), Cd (< 0.0005 - 2.22), Cr (0.014 - 6.95), Cu (0.012 - 8.70), Pb (0.001 - 0.64), Se (< 0.001 - 1.29), Zn (0.020 - 67.02), and calculated in μg/kg (wet weight) for each sample: Hg (< 0.00005 - 0.024). This study suggests that the estimated intakes of these 8 elements from the consumption of foods containing Monascus pose little risk, as the contents in the majority of samples were lower than the permissible/tolerable intakes per week according to the guidelines recommended by FAO/WHO. Moreover, their concentrations in foods containing Monascus differ widely for different food varieties, suggesting that external contaminants and raw materials are the main sources of these elements.
In the forth study, citrinin was analyzed by HPLC and Orbitrap LC/MS/MS. The steps involved extraction in ethanol followed by HPLC or Orbitrap LC/MS/MS analysis. In HPLC, a reversed phase HOLA TM C18 column was used and compounds were separated by a mobile phase of acetonitrile/methanol/0.1% phosphoric acid (40/40/20, v/v). A wavelength of fluorescene detector was set at Em 330 nm and Ex 500 nm. The minimum detectable quantity of the method was found to be 20 ng/g. The contents of citrinin in 24 dietary supplement samples were ranged from 0.08 to 466 μg/g, 8 red mold rice were ranged from 2.4 to 22.5 μg/g, 8 sauces were ranged from 0.02 to 0.97 μg/g. One dietary supplement contained so higher citrinin, the manufacturer must more care about the quality of raw materials.

並列關鍵字

Monascus ； physiological activity ； HMG-CoA reductase inhibitor ； monacolins ； citrinin ； heavy metals ； health risk assessment

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