Study On The Application Of Electrophoresis Technology In The Identification Of Edible Brds' Nest

Objective:Edible birds'nest is a kind of precious medicinal materials, which can be used as both food and drug, because it's rare and expensive the market demand is massive, lots of counterfeits in the market, so there are urgent need to make rules for its quality and habitat. Edible birds'nest is mainly compose of asialoglycoprotein, there are many water soluble proteins in its extract, previous research show that isoelectric focusing electrophoresis and SDS polyacrylamide gel electrophoresis can reveal the difference between variety edible birds'nests and counterfeits, so the technology of electrophoresis is a ideal method to analysis protein extract from edible birds'nest. Study on factors which influence protein extraxt rate and degradation degree, explore optimum protein extract conditions suitable for electrophoresis. Improve existing methods of electrophoresis to analysis bands of variety edible birds'nest and counterfeits, determining characterristic bands to identify certified product and counterfeits, meanwhile, discuss relationship between quality of Edible birds'nest and its habitat, nature environment and processing. Explore two-dimensional electrophoresis conditions to separate protein of edible birds'nest, using PDQuest software to discuss the sopts on the gel, find out the differential expression spots of certified products of edible birds'nest and preliminarily intercompary them.Method:Using Single factor experiment, based on protein content and electrophoresis map, study on the influence of solid-liquid ratio,extract time an temtemper and different extract to edible birds'nest. Study different methods to prepare gels and staining liquid, and how effect on special protein and definition of electrophoresis map, comparing the maps and based on this idenfity variety commercial grades of edible birds'nest and its counterfeits. Improve the methods of regular two-dimensional electrophoresis, such as remove impurities and ions by salt bridges. Result:The optimum extract process is 1:45 solid-liquid ratio, extract temperature 70℃, extract time 3h by pure water, or extract by the lysis buffer contain 7Murea/2M thiourea and 1M Tris(pH8.8) in room temperature for one hour. In addition, there was no effect by using ultrasound to extract protein of edible birds'nest, ammonium sulfate can depodite protein of edible birds'nest, but the saltions remained influence the maps of electrophoresis. Determing the protein concentration of edible birds'nest by the method of Bradford, the operation process of this method is easy relatively, precision and stability is better then other methods. Determing monosaccharide, oligosaccharide and total polysaccharides of edible birds'nest by the method of phenol-concentrated sulfate, there shows some differences between varieties edible birds'nest and its counterfeits, but not high enough to identity them. The optimum electrophoresis condition is:10% separation gel,6%concentratio gel,sample amount 15μL,Tricine-SDS system,coomassie brilliant blue-silve and Schiff reagent staining methods. Nine varieties of edible birds'nests shows obvious difference in the map of polyacrylamide gel electrophoresis, there are characteristic protein bands can distinguish common counterfeits such as gelatin, tremella and pigskin. Glycoprotein dyeing show clearly difference between certified product and counterfeits. Using centrifugal column to concentrate and purify protein of edible birds'nest, then by putting salt-bridage and removaling filter-paper to shorten the time and two-dimensional electrophoresis, meanwhile improving the numbers and resolution of edible birds'nest protein spots.Discuss:In the privious research, we collect sixteen batches of edible birds'nest form four habitats, based on this, experiments of electrophoresis carried out successfully. We got higher-concentration protein of edible birds'nest and low-degradation degree by optimum extract method. Polyacrylamide gel electrophoresis show characteristic protein bands of edible birds'nest, it is possible to identify certified product and its counterfeits by this method. This method is accurate reliable and showed high reproducibility, can provide reference for production and quality control of medicinal material snake, artificial calculus bovis, calculus bovis and its preparation. Optimizing method of two-dimensional electrophoresis suitable for edible birds'nest, and laied a foundation for further seprate protein of edible birds'nest.