Glycation Of β-lactoglobulin And Inhibitory Effect Of Genistein On It

P-lactoglobulin,widely used in food industry such as dairy products,baked goods and sports drinks for its good biological functions as a food additive,usually produce a series of advanced glycation end products(AGEs)with reducing sugars during food processing and storage period.Glycation will affect the activity of protein by changing the structure of β-lactoglobulin and causing cross-linking between proteins.The change of β-lactoglobulin structure which was caused by glycation was detected by UV,Circular Dichroism,and polyacrylamide gel electrophoresis and size exclusion chromatography.The generation of AGEs was detected by fluorescence spectrophotometry.Genistein,a natural edible glycation inhibitor,was chosen to analysis its inhibition activity of cross-linking and AGEs induced by glycation.1.A fast method to purify β-lactoglobulin in large quantity was established:firstly combine salting out with isoelectric precipitation to extract whey protein crudely,and then use Sephadex G-50 to purify β-lactoglobulin.The purity of(3-lactoglobulin was 92.9%with a recovery rate of 87.1%.2.The content of dicarbonyl compounds produced by β-lactoglobulin glycation were detected by gas chromatography and the results showed that:the content of MGO and GO all reached the highest at 207μg/g and 180μg/g respectively after heating 30 minutes.In the subsequent 30 minutes,the quantity of dicarbonyl compounds began to decline,and remained at around 150μg/g.3.β-lactoglobulin-methylglyoxal/glyoxal,β-lactoglobulin-sugar reaction system were modeled to analysis its structure change during glycation.(1)Ultraviolet absorption value of the reaction system at 280 nm significantly increased with the extension of reaction time within 48 hours,indicating that the dicarbonyl compounds can change the structure of tyrosine and tryptophan in β-lactoglobulin.The activity of ribose was strongest,followed by MGO,lactose and GO.(2)The ultraviolet absorption intensity of circular dichroism increased significantly with the extension of reaction time.The reducing of β-sheet and increasing of α-helix suggested glycation may transform β-sheet to a-helix.In addition,the random coil structure had a certain degree of reducing.The secondary structure changed which caused by MGO most obviously with 8%increase of a-helix and 6.4%increase ofβ-sheet.(3)Glycation caused cross-linking of β-lactoglobulin during storage time detected by SDS-PAGE.With the extension of storage time,the cross-linking degree increased.Genistein can inhibit the cross-linking,and inhibition effect is proportional to the concentration.PAS glycoprotein staining method proved that the glycation makesβ-lactoglobulin link sugar chains.(4)A new absorption peak was detected between 120～190 min via Sephadex G-75 size exclusion chromatography.With the extension of glycation processing time,the absorption between 48～72 min increased.4.The effects of temperature,time,pH,substrate concentration,substrate types and genistein on the glycation of β-lactoglobulin were detected by fluorescence spectrum method.The results showed that:Glycation of β-lactoglobulin easily occurred in neutral alkali environment.High temperature,long processing time and high concentration of substrate will promote glycation of β-lactoglobulin.The activity of dicarbonyl compounds was higher than sugars.In the meantime,ribose most active followed by Lactose,fructose,galactose,arabinose,rhamnose,glucose.5.The inhibition effect of genistein on β-lactoglobulin glycation reducing the generation of AGEs was significant.Its Inhibitory effect had a positive correlation with concentration and action time within a certain scope.Different reaction system had a different inhibition rate of genistein.