膠囊錠狀食品中蝦紅素之檢驗方法探討   全文下載

Method Evaluation for Astaxanthin in Foods in Capsule or Tablet Form

蝦紅素（Astaxanthin）天然存在於藻類（如雨生紅球藻）及蝦蟹殼，亦可由微生物合成，具抗癌與抗發炎之潛在能力。衛生福利部訂定之「以基因改造大腸桿菌（Escherichia coli）Ast12菌株發酵生產之食品原料蝦紅素之使用限制及標示規定」中，每日食用限量以蝦紅素計為2 mg。未經水解之蝦紅素產品以高效液相層析儀配合光二極體陣列檢出器（HPLC-PDA）分析，可鑑別其為酯化型及游離型，惟酯化型蝦紅素難以精確定量，故本研究探討以酵素水解方式將酯化型蝦紅素水解成游離型，搭配內部標準品以HPLC-PDA分析建立膠囊錠狀中蝦紅素之檢驗方法。檢體以含0.1%2,6-二丁基對甲酚（BHT）之丙酮溶液均勻分散，於超音波振盪萃取離心後，取上清液經膽固醇酯酶水解及石油醚萃取，收集上層液後以氮氣吹乾，經含0.1% BHT之丙酮回溶後，以YMC-Carotenoid C30（5μm, 4.6×250 mm）管柱，搭配甲醇、甲基第三丁基醚及1%磷酸水溶液作為移動相，以流速1.0 mL/min於波長478 nm採用光二極體陣列偵測器檢測。結果顯示，酯化型蝦紅素經酵素作用可完全水解成游離型蝦紅素（包括13-cis、all-trans及9-cis）。確效試驗結果顯示，於粉狀及油狀之空白基質中分別添加以游離型蝦紅素計為2.5 mg/g、5.0 mg/g及12.5 mg/g之酯化型蝦紅素，其同日間平均回收率為92.9-111.2%，變異係數為2.5-4.8%；異日間平均回收率為90.2-107.2%，變異係數為4.4-7.0%，均符合衛生福利部食品藥物管理署食品化學檢驗方法之確效規範，顯示本方法之精密度與準確度均良好。以本方法檢測8件含蝦紅素之市售膠囊食品，檢驗結果皆符合「包裝食品營養標示應遵行事項」中營養標示值誤差允許範圍≥標示值之80%之規範。

Astaxanthin naturally exists in the algae such as Haematococcus pluvialis and the shells of shrimp and crab. It can also be synthesized by microorganisms, considered as a potential activity of anti-cancer and anti-inflammatory. Furthermore, astaxanthin derived from food materials fermented from genetically modified Escherichia coli Ast12 strains has a daily consumption limit of 2 mg, as stipulated by the using restrictions and labeling regulations announced by the Ministry of Health and Welfare (MOHW) in Taiwan. Astaxanthin products, whether in ester for free form, can be identified by high performance liquid chromatography (HPLC) with a photodiode array detector (PDA) without the need for hydrolysis. Esterified astaxanthin is difficult to quantify accurately, therefore, we established an analytical method for astaxanthin in foods in capsule or tablet form by enzymolysis for esterified astaxanthin to yield free astaxanthin with an internal standard by HPLC-PDA in this study. The sample was uniformly dispersed and extracted with 0.1% 2,6-butylated hydroxytoluene (BHT) in acetone under ultrasonication. After centrifugation, the supernatants were hydrolyzed by cholesterol esterase and extracted with petroleum ether. The combined petroleum ether layer was evaporated to dryness using a stream of nitrogen, and dissolved in acetone containing 0.1% BHT. After filtration, the filtrate was analyzed by a YMC-Carotenoid C30 (5μm, 4.6×250 mm) column at 25℃using methanol, methyl tert-butyl ether and 1% phosphate solution as the mobile phase by a gradient elution of 1.0 mL/min and detected at 478 nm by HPLC-PDA. The results showed that enzymolysis completed converted esterified astaxanthin into free astaxanthins, including 13-cis, all-trans and 9-cis. The method validation was performed by spiking astaxanthin ester at the levels of 2.5 mg/g, 5.0 mg/g and 7.5 mg/g as free form into the blank sample, respectively. The average recoveries of astaxanthin ester in intra-day were between 92.9 and 111.2%, and the coefficients of variation were between 2.5 and 4.8%. The average recoveries of astaxanthin esters in inter-day were between 90.2 and 107.2%, and the coefficients of variation were between 4.4 and 7.0%. The above results showed that this method could provide high precision and accuracy, and comply with the validation guideline of the chemical testing method published by the Taiwan Food and Drug Administration (TFDA). Eight products with astaxanthin labeling were assayed by this method. The results showed that the ratios of the detected values to the labeled values of astaxanthins were all in compliance, meeting the regulation that the allowable error range of nutrition label values should be≥80% of the labeled value, as specified in the Regulations on Nutrition Labeling for Prepackaged Food Products.