Functionality Modification Of Whey Proteins Induced By The Binding With Phytophenolics At Neutral PH

Whey protein has a balanced amino acid distribution,excellent water solubility and functionality.What's more,it plays a role in glycemic and lipid metabolism,and immune system in vivo,which confer whey protein biological functionality for human.With the raise of health awareness and development of dairy products,whey protein became one of the most important functional food protein ingredients.Phenolic compounds are also impoartant food additives,which are natural antioxidant and have numerous health benefits.The protein–phenolic interaction attracted much attention.Phenolic compounds were designed to be added into food system to contribute to the antioxidant activity and biological functionality.Protein was always considered to be a good carrier for phenolic compounds.High temperature and alkali derivations are widely applied to induce attachment of phenolics to proteins.However,high temperature and alkaline conditions could be detrimental to the stability of phenolic compounds and their derivatives,including discoloration and anthocyanin degradation,generate undesirable side-products such as quinone polymers,and decrease the throughput of micronutrients in food processing.Because of the perceived low reactivity,interactions of phenolic derivatives with proteins under non-alkaline conditions at room temperature received little attention.The objective of this study was to characterize the interaction of whey protein?native and heat-denatured?and phenolic compounds?gallic acid and epigallocatechin gallate?,and the effect of such interaction on protein stucture,digestibility,and funtionality?interfacial,foaming and emulsifying properties?.The research was hypothesized to pursue the feasibility and nutritional value of phenolic-modified protein for potential food and beverage applications.Interaction of gallic acid?GA?and its derivative epigallocatechin gallate?EGCG?with native whey protein isolate?NWPI?at two pH values?pH 3.0 and 7.0?,and the subsequent impact on protein structure and digestion were investigated.In comparison with pH 3.0,both phenolics induced significant free sulfhydryl loss,surface hydrophobicity decrease,and greater peptic digestibility of NWPI at pH 7.0.Therefore,pH 7.0 was choosen for following experiments.Fluorometry and isothermal titration?ITC?tests showed moderately weak binding affinity of NWPI with GA but stronger binding affinity with EGCG at pH 7.0.The binding of both phenolics to NWPI was spontaneous and exothermic.The dialysis method demonstrated that NWPI bound more amounts of EGCG than GA.Differential scanning calorimetry?DSC?exhibited that both phenolics at high concentrations lowered the thermal stability of protein.In the in vitro digestion experiment,both phenolics displayed remarkable synergism with the NWPI-derived peptides when scavenging radical.Whey protein was preheated to induce structural change,and the interaction of GA and EGCG with heat-denatured whey protein?HWPI?was investigated.Preheating at 80°C for 9min was choosen as the heating condition by circular dichroism?CD?and DSC.ITC and fluorescence quenching showed similar trend that the binding of GA to HWPI was moderately weak but weaker than EGCG binding to HWPI.Binding of both phenolics to HWPI was spontaneous,exothermic,and not cooperative.Preheating contributed to the binding.However,the shift of maximal fluorescence emission wavelength in opposite directions corresponding to GA and EGCG indicated the different binding patterns.Both phenolics were able to induce free sulfhydryl loss,unordered structural increase,surface hydrophobicity decrease,and protein unfolding in HWPI.Diagonal gel electrophoresis results indicated that EGCG induced crosslinking of protein molecules while GA had a marginal effect.Both free and phenolic-bound HWPI exhibited mild radical scavenging activity.However,when they were digested,synergism was evidenced in scavenging radicals between the phenolics and whey protein peptides,with the highest synergism being on the phenolic concentration of 120?mol/g HWPI.The interfacial poperties at the air–water interface and the foaming properties of NWPI and HWPI influenced by phenolic binding under neutral pH were studied.Dependant drop analysis in combiantion with oscillation technology were used.NWPI and HWPI were able to absorb at the air–water interface to form elastic film.The adsorption process was dominated by protein unfolding and rearrangement at the interface.Preheating greatly improved the dynamic surface pressure???while decreased the dilatational elasticity?E_d?of whey proteins at the air–water interface.However,preheating enhanced foam stability due to heat-induced protein polymers.GA and EGCG induced different patterns in the time-dependent?,which were closely related with phenolic concentration.Both phenolics decreased the Ed of protein film by impairing molecular interaction of protein at the interface.However,foaming properties were significantly enhanced at all phenolic concentrations except for the 240?mol/g EGCG-treated HWPI,which was corroborated by the foam image.The interfacial poperties at the oil–water interface and the emulsion properties of NWPI and HWPI affected by phenolic binding under neutral pH were studied.Similar to their absorption at the air–water interface,NWPI and HWPI absorbed at the oil–water interface,and the adsorption was not dominated by protein diffusion from bulk silution to the surface.Preheating exerted little impact on?during aborption while decreased the dilatational Ed of whey proteins.However,HWPI exhibited a significantly increased particle size and decreased emulsion stability.The imapct of EGCG treatment on the surface activity and emulsifying activity of both NWPI and HWPI is dependant on phenolic concentration.This was due to mutiple reasons.On one hand,phenolics induced protein unfolding thus contributing to protein unfolding on the interface;however,on the other hand,phenolics decreased the penetration of hydrophobic groups on the interface.Both phenolics decreased the Ed of protein film,being consistent with the emulsion stability results.However,in overall,emulsions stabilized phenolic-treated NWPI exhibited good stability.And significantly decreased oil oxidation was observed in phenolic-treated samples.Properties of cold-set gel affected by the interaction of phenolic with preheated whey proteins at pH 7.0 were also investigated.The results showed that phenolic treatment contributed to the formation of cold-set gel and was able to lower the amount of CaCl2 for inducing gelation.The increasing dose of GA and EGCG seemed to slightly increase the particle size of protein and exerted no obvious effect on the?potential of protein.GA and EGCG treatment significantly increased the gel strength,possibly through hydrophobic interaction,inducing protein unfolding and crosslinking,as well as interaction between Ca²⁺and negative carboxylic group in GA.Improved microstructure was also observed in phenolic-treated samples,with more homogenious protein crosslinking.In conclusion,this study showed that the phenolic treatment at neutral pH value induced change in whey protein structure,in vitro digestibility,antioxdant activity,interfacial and functional properties.And the influence was closely related with phenolic structure and concentration.This new insight into the understanding in the addition of phytophenolics into protein systems will be of value to the application of phenolic-treated whey protein as potentially functional food ingredient,as well as application of plant extracts in dairy products as functional additives.