Studies On The Isolation And Purification Of Egg Peptides And Their Activity Of Reducing Blooding Fat

China is an agricultural country with rich resources of animal by-product. The yield of eggs ofthe poultry have a considerably proportion in the total output of the animal by-product. Theeggs of the poultry are the general cate on the dining-table in daily life. Egg is not only full ofnutrition such as protein and lipids but also the good resource of vitamin and mineralcomposition. Especially, the ratio of digestion of protein in egg is99.7%and the essentialamino-acid is rich enough. The traditional utilization for egg is to cook, product the power ofyolk and white. Thus, many active ingredients are lost and can not be taken fulladvantagement. Thus the common yolk protein is used as the experimental material in orderto make the resources exploited reasonable. The study emphases on the process of thehydrolysis of the yolk protein, the methods of the isolation and purification and the activity ofreducing the blooding fat of the peptides. The results of the research as follows:Single factor test and response surface methodology are employed to optimize theextraction condition for the protein in egg yolk. The best conditions of extracting proteinthose have been obtained in the experiment were egg yolk powder concentration30%, ethanolconcentration45%, pH4.4, NaCl concentration0.02mol/L. Under these conditions, theextraction rate of yolk protein is72.20%, rising20%compared to the other methods.2. The optimal hydrolytic conditions of Alcalase and Flavourzyme those the enzymeswhich have been chosen are assured according to the response surface methodology. Singlefactor test and response surface methodology are employed to optimize the hydrolysispretreatment conditions with two kinds of enzymes step by step. The best conditions for thehydrolysis are substrate concentration10%, the ratio of the amount of enzyme Alcalase andFlavourzyme0.8:0.4, pH6.5, the temperature55℃, hydrolysis time of Alcalase andFlavourzyme2h respectively. Under these conditions the yolk protein is hydrolyzed and thedegree of hydrolysis is up to13.233%and the peptide yield is up to45.573mg/ml.3. The ultrafiltration is used to separate preliminarily the egg yolk hydrolysate. Thehydrolysate that the molecular weigh less than10kD was obtained and it was powered underthe condition of freeze drying for the next step. Using Sephadex G-25gel chromatographycolumn to purify preliminarily the yolk peptides and the results show that the peptides can bepurified well when the purification sample yield3.5%of the gel volumn, sample thickness30mg/ml, the flow velocity1.3ml/min and the eluent distilled water with NaCl of0.25mol/L,so four parts of peptides were obtained:5810D EYP-1,1330D EYP-2,894D EYP-3and557D EYP-4according to the standard curve that is made by the standard protein.4. The three peptides1330D EYP-2,894D EYP-3and557D EYP-4were employed theexperiment of reducing the blooding lipid in vitro. The cholate that was not combined by thepeptide was detected on the HPLC. The experiment results showed that the557D EYP-4hasthe best effect to reduce the blooding lipid according to whether the peptide can combine the more cholate.5. The peptides of EYP-4is purified on the HPLC and optimized the purificationcondition. The better purification conditions are C18(5μm,4.6mm×150mm i.d), themobile phase methanol and ultrapure water, gradient elute(showed in Tab.4-3), columntemperature35℃, sample yield15uL, sample thickness60mg/ml and the flow velocity0.8ml/min,UV detector are get from the experimental results. And three ingredients A, B,andC are obtained on the above condition.6. HSCCC was used to prepare the monomer with the peptides of EYP-4that has beenget from the preliminary purification. The best preparation conditions are sample thickness40mg/ml, mobile phase the solution of PEG and PBS (There are150g of PEG and170g ogPBS in1000ml solution), the ration of K2HPO4and KH2PO43:1.7. The three monomers that have been obtained on HPLC are applied the massspectrometry by use of Nano-ESI-Ms/Ms. The order of amino acid of the three monomersare Lys-Glu-Pro-Ile, His-Ile-Pro-Leu and Lys-Glu-Tyr.8. The peptides of EYP-4were used to the small mice to apply the study of reducing theblooding lipid. The blood of the mice that was took from the eyeball and the cholesterol,triglycerides, low density lipoprotein, high-density lipoprotein and AI in the serum and thecholesterol, triglycerides and the total fat in the liver were detected by use of the kits. Theexperimental results showed that the cholesterol and triglycerides in the serum of the mice ofpouring the peptides into the stomach decreased very obviously, the high-density lipoproteinincreased very obviously, but the low density lipoprotein decreased obviously. They embodedas the weigh of the experimental groups is the76.99-90.44%of blank controller. Thecholesterol in the serum is49.68-79.90%of the blank controller,57.34-92.21%of the drugcontroller respectively. The triglyceride in the serum is51.20-87.08%of the blank controller,75.35-128%of the drug controller respectively. The low density lipoprotein in the serum is81.66-177%of the blank controller,80.23-174%of the drug controller respectively. Thehigh-density lipoprotein in the serum is116-140%of the blank controller,94.40-119%of thedrug controller respectively. The AI is0.31-55.62%of the blank controller,0.54-96.74%ofthe drug controller respectively. The cholesterol in the liver is29.83-92.89%of the blankcontroller,53.89-167%of the drug controller respectively. The triglyceride in the liver is43.10-87.76%of the blank controller,47.51-96.73%of the drug controller respectively.