Extraction And Purification Of Sialic Acid From Edible Bird’s Nest Fragment And Its Effect On Learning And Memory Abilities Of Young Rats

Edible bird’s nest is one of the Chinese favorite health-care products. It is found in large quantities in nature, whose annual global total output is more than 2000 t. A large amount of fragment comes from the processing, storage and transportation stage of edible bird’s nest. Due to the difficulty in shaping of edible bird’s nest fragment, the commodity value decreases, leading to lower utilization rate. There is great significance for full utilization of edible bird’s nest fragment since it has no difference with edible bird’s nest in terms of nutritional value, likewise the abundant content of sialic acid, up to about 10%, which is widely used in pharmaceutical and food industry. The process of extraction and purification of sialic acid were investigated in this study, using edible bird’s nest fragment as the raw material. Effect of sialic acid sample obtained by this process on learning and memory abilities of young rats were also identified. The main contents and results are showed as follows:First of all, sialic acid was extracted by ultrasound-assisted water extraction, hot water extraction, enzymatic extraction and acidolysis extraction respectively, and the percent content of free sialic acid in supernatant was evaluated. The results showed that the content of free sialic acid in supernatant with ultrasound-assisted water extraction, hot water extraction and enzymatic extraction were all lower than that of acidolysis extraction, especially the percent contents of free sialic acid with ultrasound-assisted water extraction and hot water extraction were only 0.23% and 2.74%, respectively, which were markedly lower than 96.06% of acidolysis extraction. It suggested that sialic acid of binding form in edible bird’s nest fragment were almost converted into free sialic acid by acidolysis extraction. Therefore, acidolysis extraction was selected as the extraction method of sialic acid in edible bird’s nest fragment.In order to compare the extraction effect of sialic acid from edible bird’s nest fragment with acetic acid, hydrochloric acid, sulfuric acid and citric acid, the percent content of free sialic acid was mainly investigated. The distribution of major components of edible bird’s nest fragment in supernatant and the color, water-holding capacity of precipitation were also investigated. On this basis, single factor experiment was applied to investigate the factors of particle size of edible bird’s nest fragment powder, acid concentration, the ratio of solid-liquid and extraction time. The optimum extraction conditions were that extraction with citric acid, the particle size of edible bird’s nest fragment powder was 120 μm, the concentration of citric acid was 0.05 mol/L, the ratio of solid-liquid was 1:100, the extraction temperature was 100 oC, and the extraction time was 20 min. Under these conditions, the percent content of free sialic acid in supernatant was about 95%.To obtain sialic acid sample of high purity, the sialic acid extract need to be purified. First of all, a preliminary purification was conducted by ultra filtration to remove macromolecular proteins. By the comparison of ultra filtration separation performance of different ultra filtration membrane, operating pressure, operating time and concentration multiples, the optimum operating conditions were obtained as follows: using ultra filtration membrane whose molecular weight cut offs was 3 kDa, at room temperature, under the initial pH, pressure was 40 psi, and concentration multiples was 4~6. Under the optimum condition, the protein removal rate was 98.87% and the sialic acid transmittance was 92.68%. Then ion exchange column chromatography was used to conduct secondary purification. By the screening of different resins, static and dynamic adsorption experiment, the optimum process conditions were obtained as follows: D301 anion exchange resin was adopted, the concentration of citric acid was 9.76 mg/mL, the volume of sample was 9.5 BV, and the flow rate of load column was 1 mL/min. By the process of D301 anion exchange gum purification, the removal rate of citric acid was 99.62% and the recovery of sialic acid was 85.50%. After the purification of ultra filtration and dynamic column chromatography, condensation, freeze-drying, free sialic acid product was obtained which had a purity of 73.47%.To evaluate the effect of sialic acid sample obtained by the process of extraction and purification on the learning and memory abilities of young rats and compare with the sialic acid standard and the binding sialic acid, the age of 21-days Wistar rats were selected as study object. After gastric complement of sialic acid sample solution, sialic acid standard solution, stewed edible bird’s nest fragment solution(sialic acid mainly in the form of protein-bound), edible bird’s nest fragment hydrolysates(sialic acid mainly in the form of polypeptide-bound), Morris water maze test was conducted. The results suggested that the exogenous supplement of different form of sialic acid in developmental stage is likely to enhance the spatial learning and memory ability of the rats. The efficacy enhancement of sialic acid sample obtained by citric acid extraction and purification was better than those of the sialic acid standard and sialic acid in the form of protein-bound or polypeptide-bound.