Study on Sialic Acid Binding State in Stewed Bird's Nest and Optimization of Enzymatic Extraction of Free and Oligosaccharide-Bound Sialic Acid

BACKGROUND: As a traditional Chinese health food, edible bird's nest (EBN) has high medicinal value, which is mostly attributed to the high content of sialic acid (SA). SA mainly exists in protein-bound, oligosaccharide-bound, and free forms and the binding forms of SA are closely related to the functions of EBN. OBJECTIVE: To establish a simple but robust method to distinguish and determinate the free and oligosaccharide-bound SA content and the protein-bound SA content, and investigate the changes to SA binding states in EBN during different processing. METHODS: Protein-bound SA in EBN was separated from other forms of SA by trichloroacetic acid precipitation, and SA content was determined by HPLC. The effects of stewing conditions on the distribution of SA in EBN were investigated and response surface methodology was used to explore the optimal conditions for enzymatic extraction of free and oligosaccharide-bound SA from EBN. RESULTS: The average recoveries of free and oligosaccharide-bound SA and protein-bound SA were 97.82-98.92% and 94.67-95.75%, respectively. The content of free and oligosaccharide-bound SA in stewed EBN was proportional to the stewing temperature, stewing time, and liquid to material ratio, while that of protein-bound SA was inversely proportional to those factors. Through response surface analysis, we found that the optimum technological parameters were as follows: liquid to material ratio, 60:1; enzymolysis time, 2 h; enzyme dosage; 12 000 U/g (alkaline protease); pH, 11; enzymolysis temperature, 60°C. CONCLUSION: This method can not only distinguish between free and oligosaccharide-bound SA and protein-bound SA effectively, but can also determine the contents of them. The results of the investigation on stewing conditions and response surface analysis can be used as the theoretical basis for further pharmacological research of EBN, and can also provide theoretical guidance for the development of EBN products. HIGHLIGHTS: A method for the determination of free and oligosaccharide-bound SA and protein-bound SA in EBN by HPLC was established, and the extraction process of free and oligosaccharide-bound SA was optimized.