Determination Of The Contents Of Sialic Acid In Milk And Milk Powder

There are two series in this experiment, in seriesⅠ, We established a new high performance liquid chromatography method that was accurate, simple and quick for determining the concentration of Sialic acid in milk; In seriesⅡ,We researched the dynamic changes on Sialic acid in different period Milk and determined the concentration of Sialic acid in Milk powder.SeriesⅠ: The establishment of the method of detecting the concentration of Sialic acid in milkThe column: Symmetry C18 column(250 mm×4.6mm,5μm). The mobile phase:1.0%tetraphdrofuran(containing0.2%phosphoricacid)-acetonitrile(92:8).The flow rate:1.0ml.min-1; The column temperatue: 35℃;The detection wave length:230nm,The response condition was optimized from the following several influence factors separately: the scanning wave, time, temperature and so on. The results illustrated that the absorption wave is 230nm, added 15mL trichloroacetic acid into 10mL milk to make the protein precipitate completely, and then respectively added 35mL and 40mL 0.1mol/L sulfuric acid at 80℃for 120 minutes to make the protein-bound and oligosaccharide-bound Sialic acid precipitate completely. Adjusted the PH of the sample solution to 5, rotary evaporation and metered volume for 10 mL. After collation, took 1mL sample solution , and then was derived with 1ml O-pheny-lenediamine.2HCL(10mg/mL)at 80℃for 50 minutes. The result was shown as follows: calibration on curves for Sialic acid is linear from 20 to 500μg/mL; linear formula is Y=63754X-277658;r=0.9993,the coefficient of variation in the same group is 1.49%,the coefficient of variation in the different group is 1.98%;Added low ,middle and high concentration of three different standard solutions into the sample solution, all low increment average returns-ratio is above 93.98%;the middle increment average returns-radio is above 96.38%;and the high increment average returns-ratio is above 100.97% .Therefore, we could draw a conclusion from the experiment that the method is simple, quick and efficient and that the method was suit for determining the concentration of Sialic acid in milk and milk powder.SeriesⅡ: The dynamic change mode of content of SA in the milk of different periods and the determination of the content of SA in milk powder. We determined the concentration of Sialic acid in the milk of different periods ,including colostrums, transitional milk and ultimate milk, the total concentration of Sialic acid is 760.83±13.42μg/mL in colostrums; The total concentration of Sialic acid is 111.43±2.06μg/mL in transitional milk; The total concentration of Sialic acid is 79.36±2.41μg/mL in ultimate milk; the concentration in milk powder is 476.4μg/g. The results showed that the concentration of the protein-bound sialic acid in colostrums, transitional milk and ultimate milk followed a downward trend in turn, the free and oligosaccharide-bound sialic acid in the milk also presents a downward trend , but the concentration of the total sialic acid in colostrums is the highest.