Fatal Imperfection of Enzymatic-HPLC Quantitative Method for Non-Digestible Oligosaccharides and Its Proposed Solution Strategy Newly Quantitative Method for Non-Digestible Oligosaccharides

The enzymatic-HPLC quantitative method is widely used for the determination of non-digestible oligosaccharide. However, the results obtained from the method are inconsistent with some reports. In the present study, we clarified the reason that the existent enzymatic-HPLC method could not accurately measure the quantity of non-digestible oligosaccharide, and then we developed the improved enzymatic-HPLC method for accurate quantification of non-digestible oligosaccharide. When some digestible and non-digestible oligosaccharides were measured by the existent enzymatic-HPLC method, 67% of isomaltooligosaccharide (IMO) was recovered as intact form, and 91% of sucrose and 95% of lactose were recovered as intact form, respectively. This result is questionable because IMO, sucrose and lactose were clearly measured as non-digestible oligosaccharides in this method. However, IMO was readily hydrolyzed and the intact form was not detected by the improved enzymatic-HPLC method which pig small intestinal enzymes were added to the hydrolyzing process in the existent enzymatic-HPLC method. Also, sucrose and lactose were completely hydrolyzed and the intact form were not recovered. The recovery rates of intact fructooligosaccharide (FOS) and resistant maltodextrin (RMD), which are not digested by small intestinal disaccharidase, were 92.1% and 78.8%, respectively. The contradiction appears to demonstrate that thermostable α-amylase and amyloglucosidase may not be able to hydrolyze the low molecular weight oligosaccharides, although they can hydrolyze spontaneously high molecular polysaccharides. In conclusion, the present study strongly suggests that the existent enzymatic-HPLC quantitative method cannot accurately measure non-digestible oligosaccharides, and that the improved enzymatic-HPLC method is suitable for the quantity of oligosaccharides.