Effect Of Structural Modification On The Fibrils Formation Of Milk Protein And Their Mechaism

Different from the aggregation formed at normal pH(pH 6.5)condition,p-lg and whey protein could form amyloid like fibrils at low ionic strength,low pH,90? after long time heating through non-covalent interaction,the fibrils play very important role in improving protein's functional proterties.For now few researchers studied on the structural modification on the fibrils formation of protein.This paper seclected four proteases to modify the whey protein concentrate(WPC)at low degree of hydrolysis(DH)0.1%,0.2% and 0.3% before adjusting to pH 2.0 and heating at 90? in order to gain insight into the influence of proteolysis on fibril formation.Also it discussed the effect of different protein mix to the ?-lg fibrils formation.It is speculated that some molecular structure character of ?-lg was the precondition of its fibrils formation.Some structural modification could destrory the structure,which is very important to reveal the mechanism of fibrils formation.In addition,we could achieve mixed protein fibrils through controlling the aggregation pathway.The main results are below:Fibrils formation of WPC modified by different proteases The ability of fibrils formation was quite different from to modified whey protein concentrate(WPC)by four proteases at low degree of hydrolysis(DH)0.1%,0.2% and 0.3% before adjusting to pH 2.0 and heating at 90 ?.The native WPC and WPC modified by trypsin formed fibrils while WPC modified by the other proteases showed worse amyloid fibrils morphology.Kinetic parameters for fibril formation The content of aggregation and Th T kinetic data indicated that the abilities of fibrils formation were apparently different for WPC modified by the four proteases.WPC modified by trypsin aggregated more during heating and the aggregation rate constant was 1.62 times than the native WPC,Ea was lower thus easy to aggregate;WPC modified by protease A and pepsin had the same aggregation rate constant as native WPC,the aggregation rate constant of WPC modified by protease M was 19.22% lower than native WPC.Ea was increased 5.86% thus difficult to aggregate.In addition,WPC modified by trypsin had the same tlag as native WPC,(df/dt)max increased 4.44%~19.51 %,fmax increased 2.21 %~8.82%,while the results of WPC modified by other proteases were different.Secondary structural analyses Compared to the native WPC,the structure of WPC changed differently after being modified by proteases.The state of a-helix structure for modified WPC played the most important role in the formation of fibrils.Under the mild conditions used in this work the a-helix structure of WPC modified by trypsin caused little destruction and resulted in fibrils with good morphology;the content of a-helix for WPC modified by other proteasesdecreased to 36.19%~50.94% thus fibrils formation was inhibited.Analysis of different protein aggregationThen WPC and SP were selected as materials to separate and purify the ?-la??-lg and K-casein and their aggregating properties(the morphology?Th T fluorescence intensity?applied force and secondary structure)were analyzed during heating at low ionic strength,low pH2.0,90 C.The results showed that the surface hydrophobic first increased and then decreased,the content of-SH decreased slightly,the secondary strcture of ?-lg undergoes the transform from a-helix to(3-sheet thus could form fibrils.While the secondary structure of a-la and K-casein changed unconspicuously thus no fibrils obseversed.Effect of protein mixture on B-lactoglobulin fibrils formationa-lactalbumin or K-casein with different concentration added to ?-lactoglobulin(?-lg:a-la/K-casein 2: 1;3: 1;6: 1)at different period could form fibrils with different morphology.The results of TEM and partical size showed the adding a-la could overstriking the fibrils when the concentration are ?-lg: a-la 3 : 1 & 6: 1,the earlier added,the more overstriking.But increase a-la concentration to 2:1,a-la especially added in lag time was hard to form fibrils;when concentration of K-casein is low,it had little effect to fibrils formation,when increase the concentration of K-casein to 2: 1 & 3 : 1 especially added in lag time,it formed amorphous aggregates not fibrils.Kinetics of aggregation The variation of aggregaion ainic h aggregation and aggregate rate increased when ?-la/K-casein added at lag time,the aggregation mixed K-casein aggregated faster than a-la,the effect of K-casein was stronger than a-la for fibrils formation,t0.5max is longer when mixed K-casein,it was 1.467 times as ?-lg aggregated.fmax decreased 1.6 times and(df/dt)max decreased 66.5%,thus it inhibited to form fibrils.The content of aggregation and aggregate rate less increased when a-la/K-casein added at growing time.ulyylo-grgated toApplied force The surface hydrophobicity and free sfdr f ?lg agge a-la/K-casein with different concentration adding at different phase showed that the surface hydrophobicity of ?-lg aggregated to a-la was similar to ?-lg aggregated itself,but the surface hydrophobicity of ?-lg mixed a-la decreased slowly aftering heating 6 hours.The surface hydrophobicity of ?-lg mixed ?-casein increased slightly,which inhibited aggregation through hydrophobic interaction.The content of-SH for ?-lg mixed K-casein decreased a lot,which more than ?-lg aggregated with a-la.When a-la/K-casein was 2: 1,a-la mixed at lag time,the content of-SH decreased 12.37%,K-casein mixed at lag time,the content of-SH decreased 19.26%Zeta-potential It determined the zeta-potential of ?-lg mixed a-la/K-casein at different period,the results showed that compared to mix during lag phase,the zeta-potential of ?-lg mixed K-casein at growing phase showed no change,the zeta-potential of ?-lg mixed a-la mix at growing phase decreased obviously,thus different protein mixture showed different effect on the structure of ?-lg.Secondary structure We determined and analyzed the secondary structure and the content of ?-helix for the ?-lg mixed ?-la??-casein or ?-lg.It showed that the decreased a-helixtent of ?-lg mixed a-la was similar to the ?-lg aggregation,it did not inhibit the transform from a-helix to ?-sheet thus could not effect fibrils formation.During the aggregation of ?-lg and?-casein,the content of ?-helix decreased.When heating 2 hours,content of a-helix decreased24.29%,especially ?-casein mixed during lag phase,the structure of a-helix destroyed thus if could not transform to ?-sheet and inhibited to form fibrils.The route of hybrid protein aggregation We prparated mixed protein according to control the aggregate route in order to decreased the effect of several protein mix on the fibrils formation.We speculated two main route of aggregation: 1,coexist copolymerization,the protein mixed at the lag phase,which is suitable for the small protein(for example a-la)could not destroy the characteristic region of ?-lg fibrils formation,such as a-helix;2,subsequent self-assembly control technology,it controlled the hybrid phase of mixed protein,a-la and K-casein mixed at growing phase and stable phase,which had little effect on the fibrils formation.Properties of modified milk protein The functional properties of modified milk protein The EAI and ESI of aggregation formed at pH 2.0 were lower than pH 6.5.While the foaming and foaming stability of aggregation formed at pH 2.0 were higher than pH 6.5.The functional properties of modified WPC by proteases were better than the native WPC.Especially the foaming property increased 70%~90%and foaming stability increased 2.85 times.The functional properties of modified ?-lactoglobulin by different protein(?-la or ?-casein)were determined and the results showed that the foaming and foaming stability of ?-lg fibrils and a-la mixture were higher than that of ?-casein mixture and aggregation at pH6.5,also the EAI and ESI of different protein mixture aggregation were much lower than that of ?-lg aggregation.The digestion properties of different aggregation The degree of hydrolysis(DH)for native WPC and WPC modified by different proteases was determined at pH 7.5,45?,the results showed that the digestion properties of WPC aggregated at pH 2.0 was higher than the WPC aggregated at pH 6.5.The digestion of fibrils increased 64.90% after hydrolyzed 60 min.The calcium absorption ablitity of modified ?-lactoglobulin by different protein The calcium absorption ablitity of ?-lactoglobulin fibrils was 2.68 times than aggregated at pH6.5.The P-lg mixed a-la at lag phase calcium absorption decreased 30.5%?1.45%,the ?-lg mixed a-la at growing phase it increased 13.91%.The ?-lg mixed ?-casein at lag phase inhibited to calcium absorption,it decreased 37.69%? 20.72%,the ?-lg mixed ?-casein at growing phase it decreased2.37%.