以逆相高效液相層析法分析甜味種楊桃鹽醃發酵過程中醃漬液所含有機酸之變化

Abstract

本論文之研究目的在以自然發酵條件下，探討鹽醃發酵甜味種楊桃汁於發酵過程中有機酸含量的變化，作為研究發酵行為之依據。 本研究針對鹽醃發酵楊桃中可能含有之八種有機酸：醋酸、乳酸、琥珀酸、蘋果酸、草酸、胡延索酸、酒石酸及檸檬酸進行分析，並建立定性及定量之分析方法。 實驗以7％鹽度醃漬馬來西亞種及紅龍種兩品種之甜味種楊桃，各發酵六十二天，於發酵期間取樣二十次測定有機酸含量。 楊桃富含草酸，其pKa值極低，選用99％ pH 2.70之移動相組成，1％甲醇，整體分離時間約12分鐘。利用此方法於不經萃取的實際樣品檢測，於UV 210 nm時基質效應明顯，影響部分波峰純度，以此法無法偵測酒石酸、乳酸、醋酸、檸檬酸、琥珀酸。抗壞血酸於醃漬楊桃汁中含量亦高，且滯留時間與乳酸相近，進而遮蓋乳酸訊號。 經此法鑑定醃漬甜楊桃汁發酵過程中有機酸的變化，馬來西亞種之草酸、蘋果酸、抗壞血酸及胡延索酸於醃漬第1天為1598 ppm、1249 ppm、87 ppm、6 ppm，於第20天達到最高，為10301 ppm、3800 ppm、678 ppm、26 ppm。之後慢慢下降至第62天為8580 ppm、2912 ppm、630 ppm、18 ppm。紅龍種之草酸、蘋果酸、抗壞血酸及胡延索酸於醃漬第1天為1813 ppm、1197 ppm、95 ppm、3 ppm，於第20天達到最高，為8149 ppm、4771 ppm、408 ppm、29 ppm。之後慢慢下降至第62天為8132 ppm、3969 ppm、366 ppm、28 ppm。 使用離子配對法改善波峰的分離，以期進一步分離酒石酸、乳酸、醋酸、檸檬酸、琥珀酸。以內含有機相10％（ACN：MeOH = 1：1），pH 7.00的移動相，加入對偶離子TBA 0.005 M，以UV 210 nm偵測，可增進有機酸的分離效果。但實際樣品分析條件仍需進一步測試。

We set up a qualification and quantification analytical method to test 8 organic acid in sweet carambola pickling brines, including acetic, lactic, succinic, malic, oxalic, fumaric, tartaric and citric acids. Malaysia and Red Dragon sweet carambola were pickled with 7% salt for 62 days; the exudates were taken and analyzed 20 times during the pickled process. The results showed that carambola has high content of oxalic acid，the pKa value is extremely low. The optimum analytical method of HPLC was 99% pH 2.70 phosphorus buffers as mobile phase and 1% methanol, and the analytical time was 12 min. We analyze real sample without purification found that strong matrix effect at UV210 nm, affect plurality of some peaks so we cannot detect tartaric, lactic, acetic, citric and succinic acids. Ascorbic acid also has high content in sweet carambola pickling brines; the retention time closed to lactic acid and covered the signal of lactic acid. After analyzed the organic acid in sweet carambola pickling brines during the pickled process; oxalic, malic, ascorbic and fumaric acids content of Malaysia carambola in the first day respectively is 1598 ppm, 1249 ppm, 87 ppm, 6 ppm. The highest content of organic acids in the 12th day respectively is 10301 ppm, 3800 ppm, 678 ppm, 26 ppm. The organic acids content decreased slowly to the 62th day respectively is 8580 ppm, 2912 ppm, 630 ppm, 18 ppm. Oxalic, malic, ascorbic and fumaric acids content of Red Dragon carambola in the first day respectively is 1813 ppm, 1197 ppm, 95 ppm, 3 ppm，. The highest content of organic acids in the 12th day respectively is 8149 ppm, 4771 ppm, 408 ppm, 29 ppm. The organic acids content decreased slowly to the 62th day respectively is 8132 ppm, 3969 ppm, 366 ppm, 28 ppm. We try to improve separation of tartaric, lactic, acetic, citric and succinic acid peaks by ion-pair method. The HPLC analytical method of ion-pair was 90% pH 7.00 phosphorus buffers as mobile phase and 10% organic phase (ACN : MeOH = 1 : 1), with 0.005 M TBA as counter ion. Detected organic acid at UV 210 nm, separation of acid peaks was improved by the ion-pair method. The proper ion-pair method in real sample needed the advanced testing.