Preparation Of N-acetylneuraminic Acid From Eggshell Membrane And Its Effect On Learning And Memory Abilities Of Young Rats

This study is based on “twelfth five-year” Project of National Key TechnologyResearch and Development Program “enzymatic hydrolysis process of egg yolkpowder and eggshell membrane protein and quality and structure changes duringprocessing”(2012BAD28B08-5).This study is mainly engaged in hydrolysistechnology of the egg shell membrane. This study takes the egg shell membrane asraw material, and N-acetylneuraminic acid (Neu5Ac) as study object.First of all, in order to study the N-acetylneuraminic acid in eggshell membrane,we established two determination methods: ninhydrin colorimetric method andO-phenylendiamine derivative method. The standard curve equations of the twomethods are: y=0.0017x+0.0469, R2=0.9983and y=10407x+80629, R2=0.9994. Aftercompleting the standard curves, the evaluation indexes of method were determined,the conclusion is that ninhydrin colorimetic method is suitable for the rapiddetermination and o-phenylendiamine derivative method is more suitable for theaccurate determination of the content of N-acetylneuraminic acid.In order to effectively extract n-acetyl neuraminic acid from egg shell membrane,the extraction method of N-acetylneuraminic acid was screened and optimized. Usesingle factor experiment to investigate the factors of hydrolysis method, hydrolysisacid concentration, hydrolysis temperature and hydrolysis time. The optimumextraction conditions is that hydrolysis with sulfuric acid, the concentration of sulfuricacid is0.05mol/L, the hydrolysis temperature is80℃, the hydrolysis time is2h.In order to further study the properties of the N-acetylneuraminic acid in eggshell membrane, we need to purify the egg shell membrane N-acetylneuraminic acidhydrolysate. First of all, according to the previous experimental conclusion, Usingethanol precipitation method to conduct a preliminary purification to remove proteins, add30%ethanol of the volume fraction to remove the main impurity proteins. So weobtain preliminary purification egg shell membrane N-acetylneuraminic acidhydrolysate. Later in the pH of4, under the condition of high concentration ofN-acetylneuraminic acid, add the hydrolysate to732cation exchange resin tosecondary purification.To evaluate the impact of exogenous egg shell membrane N-acetylneuraminicacid on young rats learning and memory ability, we choose the age of21-days Wistarrats and feed N-acetylneuraminic acid egg shell membrane to them by gastriccomplement. Then use Morris water maze test to evaluate. Morris water maze test isdivided into three parts: spacial acquisition, spacial reversal and spatial workingmemory test.24h after the end of the spacial acquisition and spacial reversal test, theprobe trial was carried out. The results showed that the egg shell membraneN-acetylneuraminic acid supplement group performed better than the control group,they were able to be better and faster to adapt the Morris water maze task. It suggestedthat the exogenous supplement of N-acetylneuraminic acid is likely to improve thespatial learning and memory ability of the rats.