Nanofibrils Formation Of Whey Protein Concentrate And Their Properties

The microstructure and functional properties on β-lactoglobulin and WPI at unconventionalconditions had already been studied, but a specific pattern of aggregation of whey proteinconcentrate （WPC） leading to nanofibrils was identified in unconventional conditions. WPCcould form long semi-flexible fibrils with diameters from24nm to28nm. Therefore, the projectwas aimd to study the formation of nanofibrils （the concentration of protein, pH, heat-time）, Inaddition, we studied the differences between nanofibrils and normal protein aggregates byanalyzing the major forces （the surface hydrophobicity, free sulfhydryl groups）, properties（turbidity, ThT, DSC, apparent viscosity and protein composition）, acid-responsive properties（adjusted pH repeatedly, reheating and ionic strength）, functional properties （gelation, foamingand emulsifying properties）. We hope to provide some theoretical basis to expand the applicationsof whey protein concentrate. Our main results were as follows:The preparation of nanofibrils and influence factors Transmission electron microscopy（TEM） was applied to investigate the morphology of nanofibrils.3.0wt%（protein basis） of WPCsolution with pH1.8could formed nanofibrils by heating10h at90℃, exploring the influence ofthe concentration of the protein, pH and heat-treatment time on the aggregation of whey proteinsconcentrate. We found that the nanofibrils was more coarse with the increase of the proteinconcentration and pH, if the protein concentration and the pH is too low, the nanofibrils was toothin. Heating3.0wt%（protein basis） of WPC solution with pH1.8at90℃was suitable to formnanaofbrils. In addition, with the increase of the heat treatment time, the aggregates is decreased inbranches of nanofibrils, the3.0wt%WPC was heated at90℃for10h could form longsemi-flexible fibrils.Major force The surface hydrophobicity and free sulfhydryl groups were determined.Compared with normal protein aggregates, the results indicated that nanofibrils have highersurface hydrophobicity, which were93.46±2.23and71.62±1.59, respectively. The free sulfhydrylof nanofibrils and normal protein aggregates were reduced from the initial22706.6（μmol/L）and21014（μmoL/L） to16606.66（μmol/L）and2325.19±59.01（μmoL/L）, respectively. It can beseen from the result that surface hydrophobicity interaction played a dominant role in theformation of fibrils aggregates, while the disulphide bonds after heating to form fibrils aggregatesat the acidic pH1.8was weaker than that of formation particulate aggregates at pH6.5.Properties The turbidity, ThT,apparent viscosity and DSC were determined. Compared with the normal protein aggregates, the results indicated that nanofibrils have lower turbidity,apparent viscosity and higher denaturation temperature. The protein structure of nanofibrils andnormal protein aggregates was different. After heating, the nanofibrils dispersion was the color ofdark brown while conventional protein aggregation dispersion was milky white. In addition, theamount of protein aggregation and protein composition were determined, the results indicated thatthere are obviously differences in process of forming nanofibrils and conventional proteinaggregation. The amount of nanofibrils and conventional protein aggregation were0.167±0.006g/L and0.786±0.011g/L, respectively. It was visible that the amount of nanofibrils was lowerthan that of conventional protein aggregation. The SDS-PAGE results indicated that the proteincomposition of nanofibrils was different from conventional protein aggregation, β-lactoglobulinwas aggregated into large aggregation at pH6.5by heating treatment, while β-lactoglobulin washydrolyzed into small peptides, and these peptides were aggregated into nanofibrils.Acid-responsive properties Nanofibrils were adjusted different pH（pH1.8, pH6.5） many times,and reheated treatment（90oC,10h）. The TEM, turbidity, aggregation amount, free sulfhydryl groups,surface hydrophobicity and secondary structure were determined, the results indicated thatnanofibrils have good acid-responsive, it could reform nanofibrils after adjusting back to pH1.8and have good thermal stability after reheating. Nanofibrils after reheating, the amount of proteinaggregation was not changed significantly, the amount of disulphide bonds was increased sightlyand surface hydrophobicity interaction was improved further after reheating nanofibrils, thesefactors leaded to nanofibrils form more branches structure. The ThT and CD were determined, theresults indicated that a small amount of α-helix converted into β-sheet structure lead to formationof nanofibrils after reheating. We proposed a model of acid-responsive aggregation pathway, a fewcontent of disulfide bonds and fibril fragments was dispersed in the pH6.5solutions, thesefragments was aggregated into large polymers through hydrophobicity interactions. Intermolecularrepulsion strengthened because of the increased in surface charges when the pH was changed backto pH1.8, large polymers of non-covalent dissociated lead to the reformation of fibrils, but thisdissociation was not completely which lead to fibrils formed more branched and amyloid structure.The solutions adjusted to pH6.5after reheating lost the ability of acid-responsive properties whichmainly due to much covalent bonds（disulfide bonds） formation accompanied by the aggregation offibril fragments, and the aggregations was not depolymerized changed back to pH1.8thus couldnot reform fibril structure.In addition, we analyzed the effect of ionic strength on acid-responsive,the TEM and ThT were determined, with the increasing concentration of Ca²⁺and Na⁺, the resultsindicated that nanofibrils became more shorter, thicker, and cluster aggregation finally, when theconcentration of Ca²⁺and Na⁺was10mM and20mM, respectively, nanofibrils maintained goodfibril structure. Nanofibrils have more acid-responsive and thermal stability in the presence of Na⁺than Ca²⁺.Functional properties The gelation was determined, adding whey protein concentrate wasbeneficial to improve yogurt gel, but adding nanofinrils was play a negative role in the formation of yogurt gel.With the increasing of add amount of nanofibrils, the results showed that thehardness and viscosity of yogurt gel were decreased. When adding0.45⁰.6wt%nanofibrils, thehardness and viscosity of yogurt gel were decreased significantly, and the microstructure ofyoghurts showed a more loose and less branches, and some whey was separated from yogurt gel.The foaming and emulsifying properties were determined. Compared with the conventional proteinaggregation, the results indicated that nanofibrils have higher foam property and foaming stability,there is obviously promotion in foaming stability with silent period prolonged. Compared with theconventional protein aggregation, the foam stability of nanofibrils was higher by approximately2times when the foam films silent120min. The same behaviors of nanofibrils in the emulsificationproperties, compared with the normal protein aggregates, the emulsifying stability of nanofibrilswas improved by20%when the solution silent30min. Nanofibrils after reheating have higherfoam property and foaming stability, compared with the nanofibrils, the foam property andfoaming stability of reheated nanofibrils was higher by approximately1.17and1.54times,respectively. The same behaviors of nanofibrils in the emulsification properties, compared withnanofibrils, the emulsion activity and emulsion sability of reheated nanofibrils was higher byapproximately1.11and1.25times, respectively. Foaming properties of nanofibrils were betterthan emulsion properties. It showed that the characteristics of the molecular structure ofnanofibrils are beneficial to improve interfacial properties of milk protein.