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Detection of brewed soy sauce adulteration by analyzing levulinic acid with HPLC-UV
|Authors: ||Liu, Chung-Ching|
|Contributors: ||NTOU:Department of Food Science|
levulinic acid;HPLC;naturally brewed soy sauce;soy sauce adulteration
|Issue Date: ||2018-08-22T06:42:11Z
|Abstract: ||醬油 (soy sauce) 可依製造過程分類為：以食用蛋白經鹽酸水解成的酸水解植物性蛋白 (acid hydrolyzed vegetable protein, acid-HVP)，又稱水解醬油、釀造數個月製成的純釀造醬油 (naturally brewed soy sauce, NBS) 及以上兩種醬油按比例混合而成的調和醬油 (blended soy sauce)。傳統的釀造醬油製程中不會產生果糖酸，並且果糖酸在醬油中非常穩定及去除方法成本高，使得果糖酸為判斷是否為釀造醬油之適當指標。本研究發展以液液萃取 (liquid-liquid extraction) 及蛋白質沉澱 (protein precipitation) 萃取醬油中之果糖酸並以高效能液相層析儀 (high-performance liquid chromatograph, HPLC) 搭配紫外光偵測器 (UV detector) 測定其中果糖酸 (levulinic acid) 之含量，建立一個快速、簡易操作及高靈敏度之方法，以此判定市售標榜為純釀造之醬油是否摻入酸水解植物性蛋白醬油。 使用InertSustain® C18管柱進行果糖酸之分離，並以0.3% 磷酸水溶液：含0.3% 磷酸之甲醇 ＝ 95：5 (v/v) 做為移動相；偵測波長設為268 nm，果糖酸之滯留時間為21.17分鐘。 水解醬油中果糖酸含量之萃取法包括液液萃取法及蛋白質沉澱法。液液萃取法以乙酸乙酯 (ethyl acetate) 為萃取溶液，萃取三次，可得萃取量為1243 (± 20) mg/L；蛋白質沉澱法以乙酸鋅 (zinc acetate) 及亞鐵氰化鉀 (potassium hexacyanoferrate (II)) 作為沉澱劑，可除去其他波峰干擾，其萃取量為1557 (± 9) mg/L。蛋白質沉澱法所能萃取之量高於液液萃取法，且其步驟較簡單，能有效縮短萃取時間，因此以蛋白質沉澱法做為樣品分析時之萃取方法。 以本方法檢測E牌醬油中果糖酸之偵測極限為1.623 mg/L，回收率為 89.04 (± 0.04)~98.28 (± 0.01)%。調查市售樣品中，僅有E牌醬油中有果糖酸存在。|
Soy sauce can be classified by its manufacture process as: acid hydrolyzed vegetable protein (acid-HVP) whuch is made by soybeans hydrolyzed by hydrogen chloride; naturally brewed soy sauce (NBS) which is fermented for several months; blended soy sauce which is prepared as a blend of acid-HVP and NBS. NBS, which is produced according to traditional process does not contain levulinic acid (LV). LV is very stable. It is uneconomically to remove it from the acid-HVP. Therefore, LV is a proper index for determining whether the soy sauce is NBS or not. The purpose of this study is to explore the optimal method for extracting LV from soy sauce by the use of liquid-liquid extraction (LLE) and protein precipitation (PP). Meanwhile, a high performance liquid chromatography (HPLC) method is established for determining the content of LV in soy sauce and detecting whether the commercially available NBS is adulterated with acid-HVP or not. An InertSustain ® C18 column was used for separation. The UV detector wavelength was set at 268 nm. The mobile phase was 0.3% phosphoric acid in water：0.3% phosphoric acid in methanol = 95：5 (v/v). The retention time of LV was 21.17 minutes. The LV content of acid-HVP was extracted with different extraction methods. It was found that performing liquid-liquid extraction for three times by using ethyl acetate as the solvent had the yield of 1243 (± 20) mg/L, while performing protein precipitation method by using zinc acetate and potassium hexacyanoferrate (II) had the yield of 1557 (± 9) mg/L. The PP method had the higher yield and shorter extraction time. Its chromatogram showed less interference peaks than LLE method. Therefore, PP method was used for the later sample analysis. The limit of detection of LV of E brand soy sauce was 1.623 mg/L, and the recovery was 89.04 (± 0.04) to 98.28 (± 0.01)%. In all the samples we investigated, only E brand soy sauce had LV in it.
|Appears in Collections:||[食品科學系] 博碩士論文|
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