The Study On Purifying β-lactoglobulin And Hypocholesterolemic Active Peptides Derived From The Prepared β-lactoglobulin

The present study was designed to purify and prepareβ-lactoglobulin from whey protein , the degree of hydrolysis(DH) was chosen as index to optimize the condition parameter of trypsin.For screening right mode and condition parameter of hydrolysis producing the target biological active peptides, detecting the activity of HMG-CoA reductase in vitro was taken as the criterion. The target biological active peptides were separated and the range of its molecular weight was determined also. Cultivation of CaCo-2 cells was arranged for estimating the practical value—the inhibition of cholesterol absorption of the biological active peptides separated.1.β-Lactoglobulin was purified from whey protein concentrate by a combination of Ammonium sulfate precipitation and gel-filtration chromatography .The content of protein was detected by using SDS-PAGE,the SDS-PAGE results showed duplicate protein bands aboutβ-lactoglobulin andα-lactalbumin by using 20%-30%or70%-80% Ammonium sulfate precipitation ,single band coincidence to the standard ofβ-lactoglobulin was showed by using gel-filtration chromatography (SephadexG-50) after the pre-processing of precipitation that the puristβ-lactoglobulin obtained.2. In order to prepare a large amount ofβ-lactoglobulin,pepsin was added to whey protein incubated for 1.5 hours,the protein fraction was collected by ammonium sulfate precipitation ,and the precipitate was either dialyzed against water using a dialysis membrane or filtered using an UF membrane to procure the purierβ-lactoglobulin. The content of protein was detected by using SDS-PAGE. The SDS-PAGE results showed the purierβ-lactoglobulin was procured.3. In order to determine the right mode and condition parameter of hydrolysis for producing the biological active peptides derived fromβ-lactoglobulin we wanted, Singl factor experiments design and Center combination experimental design were used,then the effect of hydrolysate in different phases of trypsin hydrolysis mode on HMG-CoA reductase activity were measured.The result indicated that the hydrolysate in 9 hours phase of trypsin represents comparatively high restraining ability to HMG-CoA reductase activity,the percentage of HMG-CoA reductase activity inhibited is 66.67% .4. The SepHadexG-25 was designated as chromatography material for separating the target biological active peptides from the mixture. The result showed that the hydrolysate could be successfully detached four independent ingredient by SepHadexG-25 with pH8.0 and 30mmol/L phosphate buffer ion density, and the percentage of HMG-CoA reductase activity inhibited by the fourth constitutes could reachs 43.75%. The molecular weight range of target biological active peptides was identified by Tricine-SDS-PAGE,the result exhibited that the molecular weight range of target biological active peptides between 2000 and 4100.5.The results of Caco-2 cells culture in vitro indicated: Cholesterol uptake from experimen -tal groups containing the target biological active peptides (0.27±0.021,0.25±0.019,0.21±0.014,0.17±0.02 respectively) was significantly lower than that from the experimental gro -up containingβ-lactoglobulin(0.47±0.017)or whey protein(0.59±0.023),as well was was significantly lower than that from the blank experimental group(0.709).