螯合性食用色素於生物分子檢測之應用

Abstract

幾丁聚醣（Chitosan）是近年來重要的生物性材料，其中去乙醯率（Degree of deacetylation, DD%）的程度高低對於幾丁聚醣的生物性質有顯著的影響。在過去檢測去乙醯率的公認方法，都是仰賴於核磁共振（Nuclear Magnetic Resonance, NMR）與傅立葉轉換紅外光譜（Fourier Transform Infrared Spectroscopy, FTIR）的檢測，但這兩者的費用昂貴且耗時，並需要專業人員操作才可以完成。 本研究發現日落黃（Sunset yellow FCF）、胭脂紅（Ponceau 4R）、檸檬黃（Tartrazine）三種螯合性食用色素與幾丁聚醣反應後吸光值會有下降的表現，以吸光值變化量最大的日落黃作為實驗指試劑，日落黃吸光值變化量（△A500）與幾丁聚醣的去乙醯率有高度正相關（R2=0.9718）的表現，不受幾丁聚醣分子量影響，經過最佳化後以pH 4.0、20 mM 醋酸緩衝溶液，幾丁聚醣濃度10 mg/L為本研究的實驗條件，能有穩定實驗結果表現。 最後以不同去乙醯率與分子量（10-1000 kDa）的幾丁聚醣共25個樣品製成檢量線（R2=0.9417），並以未知去乙醯率的幾丁聚醣樣品進行實測，並與13C-NMR檢測結果進行比對，去除一個幾丁聚醣的顏色因素造成誤判，其餘樣品的檢測誤差均小於2%。整體檢測流程僅需1分鐘，是有潛力作為一種新的去乙醯率的快速檢測方法。

Chitosan is an important biomaterial used widely in tissue engineering and environmental application. Degree of deacetylation (DD%) is the most important quality parameter of chitosan. Several methods have been developed to determine DD% for chitosan. NMR (Nuclear Magnetic Resonance) and FTIR (Fourier Transform Infrared Spectroscopy) are typical method for detecting DD%. However, these methods are both expensive and time-consuming. In this study, we developed a rapid and low-cost method to detect DD% by UV-VIS spectroscopy. We found that Sunset yellow FCF (SY), Ponceau 4R (P4R) and Tartrazine can chelate with chitosan, and have absorbance change. Because SY absorbance change is higher than P4R and Tartrazine, we chose SY as our indicator. The result showed that SY absorbance change had positive correlation (R2=0.9718) with DD%. In addition, it is not influenced by Chitosan molecular weight. After method optimization, pH 4.0 20 mM Acetic buffer and 10 mg/L chitosan are the best for DD% determination. Finally, We use 25 chitosan samples(MW=10-1000 kDa) to produce a calibration curve (R2=0.9417). Compared our mehod with 13C-NMR results to detect unknown DD% chitosan samples and their results had an error in less than 2 %, except for one sample which its yellow color had the effect on our detection. The process takes only 1 min and it has the potential for DD% rapid screening in the future.