Structrual Characterisation Of Low-density Lipoproteins Purified From Hen Egg Yolk And Emulsifying Properties Of Yolk Protein

Hen egg yolk plays a key role in producing a variety of food as food emulsions, such as mayonnaises, creams and salad dressings. Particularly, it contributes to the formation and the stability of these emulsions by constituting an interfacial film between oil and water. Eggs are rich in quality protein, which include low-density lipoprotein, high-density lipoprotein, livetins and phosvitins. low-density lipoprotein is the main component of egg yolk, it also contributes to the nature of emulsification. Low-density lipoproteins carry the most lipoprotein cholesterol and are reaponsible for transporting cholesterol to the liver to synthesize bile acids, It is easy to get atherosclerosis and coronary heart disease as a result of high levels of levels of low-density lipoprotein. Therefore, researching low-density lipoprotein have an important biological significance. The first part of the work in this paper is abstracting and purifying egg yolk low-density lipoprotein, making use of atomic force microscope to observe the surface morphology by nano-imaging technique, and analysis molecular functional groups of low-density lipoprotein by raman spectroscopy and fourier near-infrared spectroscopy.(1) The extraction and purification of low-density lipoprotein:During purifying low-density lipoprotein, Yolk was diluted with an equal volume of a 0.17 M NaCl solution and then centrifuged in the light of difference particle-density to remove excess proteins; re-use ammonium sulfate gradient were used to further purification protein, later protein solution dialysis 24 hours; After dialysis, protein solution pass through Sephadex G-200 (5,000-600,000 Da) column chromatography, which can separate protiens with difference molecular weight to get high purity low-density lipoprotein solution.(2) SDS-polyacrylamide gel electrophoresis:SDS-polyacrylamide gel electrophoresis detect protein concentration and composition, we found that this method can extract high purity low-density lipoprotein, and confirmed that protein contains 5 kinds of apoprotein, whose molecular weight are 130,80,65,60,15 kDa.(3) Atomic force microscopy observe surface morphology of low-density lipoprotein:low-density lipoprotein was diluted with distilled water and solution concentrations turn into 5μg/mL, 10μg/mL,30μg/mLm, respectively, we observed three difference concentration solution by a highp-resolution contact. showing that atomic force microscopy nano-imaging technology was associate with its purity and concentration. When concentration 5μg/mL, it is easy to be get a single egg yolk low-density lipoprotein molecular, that the scale range between 50 nm to 80 nm. As the concentration (10μg/m) increasing, low-density lipoprotein-scale larger, we canjecture which is small amount of aggregate; When the concentration was increased to 30μg/mL we observed that low-density lipoprotein in the form of polymer by way of intertwining. We guess the formation of aggregates may be related to the nature of lipoprotein emulsion properties.(4) Laser Raman and fourier near-infrared spectroscopy detection functional groups of low-density lipoprotein:Raman spectra characteristic peaks not fully test out becaue of impaction of oil in low-density lipoprotein solution (which may wasn't complete during vacuum drying, there is water), but the infrared spectrum has played a supplementary role. After two Spectral analysis, we founded that a low-density lipoprotein molecular has co-symmetric CH2 and CH3 asymmetric stretching vibration. In addition, fourier near-infrared spectroscopy also detected lipid chain P=O stretching vibration and N+(CH3)3 asymmetric vibration in Phosphatidyl choline aggregates.The second part of this paper studies the effect of pH on interface composition of oil-in-water emulsions made with hen egg yolk. We detected concentratioin and adsorption at protein interface(oil/water) with difference pH (0.2M glycine-HCl buffer for pH=3; 0.2 M imidazole-HCl buffer for pH=6; 0.1 M Tris-HCl buffer for pH=9). Bovine serum albumin produced using standard protein curve, after anti-calculating three yolk protein concentration in solutions, three kinds of yolk protein solutions'adsorption ratio at the oil/water interfaces as following:there were 42% protein adorptied at pH=6, nearly half. However, only 10.2% and 6.0% protein were adorptied at pH=3 and pH=9; Similarly, three kinds of yolk proteins in solution, Interfacial protein concentrationΓis 1.073mg/m-2 at pH=6, which higher than Interfacial protein concentrationΓ0.376mg/m-2 at pH=3 and Interfacial protein concentrationΓ0.306mg/m-2 at pH=9. These data supported that yolk protein was easy to absorb at pH=6.Through this research, we were not only purified high-purity low-density lipoprotein, but used atomic force microscope observing its surface morphology and detected structural functional groups with the help of laser Raman and infrared spectroscopy, which established experiment foundation for linking molecular structure of low-density lipid protein and emulsion function; Troungh studing effect of pH on protein emulsifying properties at oil/water interface, obtained the beat condition for forming film.