Construction Of Low Environment-Sensitive Nanoparticles Based On Whey Protein Isolate Modification And Their Stablization Mechanism

Many important bioactive compounds present in food raw materials are deteriorated during food processing,storage and transport by external environmental factors(light,oxygen,and heat),and hence lose their bioactivity.The bioavailability of bioactive compounds is very low because of their lower solubility in aqueous solution.However,these bioactive compounds can be encapsulated in nano-carriers that could significantly increase their apparent solubility,improve their bioavailability and reduce their environmental sensitivity.Protein and polysaccharide are used as wall materials for the encapsulation of bioactive compounds due to their exceptional physicochemical characteristics,including biocompatibility,biodegradability,renewability and nontoxicity.Biopolymer(protein and polysaccharide)nanoparticle stability against pH and salt has not yet been well documented,therefore,limiting their application in food industry.The objective of the present study was firstly to study the influence of modifications by chondroitin sulfate(ChS)electrostatically interacted with protein and dextran covalently conjugated to protein on the stability of whey protein isolate(WPI)nanoparticles,secondly,to elucidate the formation and stabilization mechanism of protein-based nanoparticles against pH and salt,and finally to prepare stable nanoparticles with low environmental sensitivity against pH,salt and heat through self-assembly between WPI-dextran conjugate molecules and zein molecules.The results would provide useful theoretical guidance to produce stable nanoparticles for satisfying food industry requirements.The main research contents and results are as follows:The influences of neutral polysaccharide and ionic polysaccharide on the stability of WPI nanoparticles against pH and salt were determined by turbidity and dynamic laser scattering(DLS)methods.Protein-based nanoparticles were prepared by heat-induced gelation.Results indicated that dextran(neutral polysaccharide)could not significantly enhance the stability of WPI nanoparticles,while ChS(ionic polysaccharide)could only partly improve the stability of WPI nanoparticles.After the preparation of WPI-dextran conjugates,the stability of WPI-dextran conjugate/ChS nanoparticles against pH and salt was evaluated.Results showed that the content of free amino groups of WPI decreased and the intensity of amide I-III bands of its Fourier transform infrared(FTIR)spectrum reduced after being grafted with dextran.WPI-dextran conjugate(9.7% degree of glycosylation)/ChS nanoparticles(Z-average particle diameter,~ 150 nm and polydispersity(PDI),0.08)have good stability in the entire pH range 1.0 to 8.0 and in the presence of 0.2 mol/L NaCl.The influence of lutein incorporation on WPI-dextran conjugate/ChS nanoparticles was determined by DLS,transmission electron microscopy(TEM),atomic force microscopy(AFM),and scanning electron microscopy(SEM).Results showed that lutein incorporation did not induce significant changes of WPI-dextran conjugate/ChS nanoparticles in particle size and morphology.TEM,AFM,and SEM images of lutein-unloaded and lutein-loaded nanoparticles revealed a narrow size distribution from 50 to 100 nm and a spherical shape with smooth surface.The size difference between DLS and three microscopies methods indicated that the nanoparticles contained large quantities of water,and in turn provided sufficient space for the encapsulation of core materials.The encapsulation ability of WPI-dextran conjugate/ChS nanoparticles was evaluated using extraction and spectrophotometer methods.Results showed that WPI-dextran conjugate/ChS nanoparticles could effectively encapsulate lutein with a loading capacity and encapsulation efficiency of 8.03% and 94.07%,respectively.The influences of glycosylation,ionic polysaccharide and heat treatment on the polarity and hydrophobicity of microenvironment around fluorescent groups in protein were evaluated by fluorescence spectroscopy.Results showed that both dextran covalently conjugated to protein and ChS electrostatically interacted with protein were involved in formation process of WPI-dextran conjugate/ChS nanoparticles prepared by heat-induced gelation.Zeta potential and DLS size of different nanoparticle dispersion systems were determined from various combination of WPI,dextran,WPI-dextran conjugate and ChS.Results showed that the collaborative steric hindrance from ChS electrostatically interacted with protein and dextran covalently conjugated to protein was the principal force for formation and maintaining stability of WPI-dextran conjugate/ChS nanoparticles.The major interaction forces involved in the formation process and in maintaining stability of WPI-dextran conjugate/ChS nanoparticles were evaluated by gel breaker reagents such as NaCl,urea and dithiothreitol.Results showed that electrostatic interaction was primarily responsible for promoting the formation of WPI-dextran conjugate/ChS nanoparticles.Hydrophobic interaction was the major force for the formation and stabilization of the nanoparticles,while hydrogen-bond interaction only slightly influenced the formation and stabilization of the nanoparticles.Disulfide bond partly contributed to the stabilization of the nanoparticles.In conclusion,the steric hindrance and hydrophobic interaction were key factors for formation and stabilization of WPI-dextran conjugate/ChS nanoparticles against pH and salt.Furthermore,a preparation method of self-assembled nanoparticles against pH,salt and heat was studied.The influences of glycosylation and ratio of hydrophobic protein on the stability of WPI-dextran conjugate/zein nanoparticles were investigated by DLS size and Zeta potential methods.Results showed that the steric hindrance of dextran chains of WPI-dextran conjugates significantly suppressed the aggregation of WPI-dextran conjugate/zein nanoparticles,and the strong hydrophobic interaction of zein significantly inhibited the dissociation of WPI-dextran conjugate/zein nanoparticles because of naturally hardening property of zein.The strong hydrophobic interaction of zein was the main force for promoting self-assembly behaviour between WPI-dextran conjugate and zein without heat treatment.Therefore,WPI-dextran conjugate and zein could be used as good wall materials through self-assembly for thermosensitive core materials.Particle diameter and PDI of WPI-dextran conjugate/zein nanoparticles at WPI/zein weight ratio of 2:1 were about 170 nm and 0.14,respectively.WPI-dextran conjugate/zein nanoparticles had good stability against pH(1.0-11.0),salt(0.2 mol/L NaCl)and heat at temperature range of 30-90 °C.However,particle diameter and PDI of WPI-dextran conjugate/zein nanoparticles with protein weight ratio of 1:1 were about 490 nm and 0.15,respectively.Size-controlled preparation of protein-based nanoparticles could be reached by adjusting hydrophobic protein weight ratio.A rapid,sensitive and reliable method for detecting the Raman spectrum was optimally established under the critical desiccation state,which was discriminated by a laser confocal microscopy after the preconcentration of coffee ring effect.High signal-to-noise ratio Raman spectrum could be obtained within 1 min using one microliter of low concentration of zein or WPI-dextran conjugate dispersions.The change tendency of protein secondary structures was obtained through baseline correction,normalization,curve fitting,band assignment and peak area calculation for amide I of Raman spectra of protein-based nanoparticles with different stability.Results showed that the stability of WPI-dextran conjugate/zein nanoparticles against pH and salt was positively correlated with the stability of protein secondary structures.