| Comparative research on ammonium sulfate precipitation with gel chromatography and high salt, low pH methods with dialyze-method to separate theβ-lactoglobulin which researched recovery and purity. Optimized the condition of enzymatic hydrolysis by determining the inhibitory ratio of ACE.Desalted the enzymolysis liquid ofβ- lactoglobulin by exchange resin of cationic and DA201-C macroporous resin. Obtained the ACE inhibition peptides fromβ-lactoglobulin with Sephadex G-25 and Sephadex G-15 in Series.1. Ammonium sulfate precipitation with gel chromatography separatedβ- lactoglobulin. Whey power were used as raw materials, separated whey protein with ammonium sulfate precipitation which concentration was 70%.Used gel chromatography column of SephadexG-50 and phosphate buffer of eluent which pH was 6.8 and concentration was 0.2mol/L , elute velocity was 1.0rpm , collected target peak by these conditions . The result showed that the concentration was 1mg/mL,the purity was more than 90% by using ammonium sulfate precipitation with gel chromatography.2. High salt, low pH methods with dialyze-method separatedβ- lactoglobulin. Whey power were used as raw materials, used NaCl which concentration between 7% to 10% in this condition which pH was 2,separated whey protein primary .Separating it by high speed centrifuge ,time was 20min and gravity was 10000g. Getted the isolate in the dialytic-bag , and took the dialytic-bag to the distilled water about 20 hours. The result showed that the concentration of theβ-lactoglobulin was 11mg/mL,the purity resched 90% by using high salt, low ph methods with dialyze-method.3. Optimized the condition of enzymatic hydrolysis to prepare the high ACE inhibition peptides fromβ-lactoglobulin. The test used trypsin, neutral protease,and pepsin to hydrolyzeβ- lactoglobulin to obtain the ACE inhibition peptides ,the inhibitory ratio of ACE of measurement which used by detection method in vitro.The result was that the trypsin hydrolysate of inhibitory ratio of ACE was the maximum.Used twice rotation test by three factors to optimized the condition of enzymatic hydrolysis about trypsin enzymaticβ-lactoglobulin, study on substrate concentration,temperature and enzyme to substrate ratio effected the ACE inhibitor activity. Established regression equation: Y=50.62-2.33X1-1.97X2+5.81X3-3.36X2X3-6.56X22-1.96X32 .Analysis on every factors which effected inhibitory ratio of ACE , the best hydrolysis condition of determination : hydrolysis temperature was 40℃, mass concentration of substrate was 60g/L , enzyme to substrate ratio was 3%.Under the best hydrolysis condition of trypsin ,the best time was hydrolyze 6 hours .4. Separation and purification of ACE inhibition peptides fromβ-lactoglobulin. The test used cation exchange resin and macroporous resin DA201-C to desalt the higher ACE inhibition peptides ofβ- lactoglobulin.The result showed that desalinization ratio of cation exchange resin was 86.67% , desalinization ratio of macroporous resin DA201-C was 83.33%.Howerer, macroporous resin DA201-C had greater effect for inhibition activity of ACE and the inhibition rate was improved about half . Therefore , macroporous resin DA201-C desalted the hydrolysate ofβ- lactoglobulin was an ideal method; Separation and purification of ACE inhibition peptides fromβ-lactoglobulin with Sephadex G-25 and Sephadex G-15 in Series. Selecting three kinds of ionic strength of phosphate buffer and washing out and separation when elution flow rate was 0.7mL/min , obtained the best condition of the higher ACE inhibition peptides ofβ- lactoglobulin which was separated. The result showed that use phosphate buffer that ionic strength was 0.03mol/L and pH8.3 eluted hydrolysate which could get four components peak , fourth of these had great effect for inhibition activity of ACE , inhibition rate was 87.54% .
|
PDF Full Text Request