Construction Of Low Environment-sensitive Flavor Carriers Via Molecular Modification Of Whey Protein Isolate

With the enhancement of consumers’ awareness of safety,the naturalness and safety of products have received more attention in addition to desirable flavor.Plant essential oils are popular among the consumers because of their unique fragrance,natural origin and additional biological activities.However,the application of the plant essential oils is limited owing to the poor solubility in aqueous matrix,high volatility,susceptibility to oxidization and inactivation.Protein/polysaccharide biopolymer nanoparticles are often used for embedding essential oils and their active components because of the good biocompatibility,degradability and safety.However,the protein/polysaccharide nanoparticles obtained by the existing preparation methods are susceptible to aggregation or dissociation under the influence of p H,ionic strength or temperature.The encapsulation and delivery of volatile flavor components can only be realized under specific conditions,thus the processing adaptability of plant essential oil and its application in more fields haven’t been achieved.Considering the environmental sensitivity of protein/polysaccharide biopolymer nanoparticles,the present study was devoted to fabricating low environment-sensitive biopolymer nanoparticles through the molecular surface modification and intermolecular interaction strengthening.As a flavor model,cinnamaldehyde was further encapsulated in the above nanoparticles to prepare p H-,salt-and heat-stable flavor ingredients.Furthermore,environmental condition changes in the process of processing,storage and application of the products were simulated to further clarify the release and stability mechanism.WPI-dextran conjugates were acquired for different durations using dry-heating conjugation.The dependence of grafting extent on time was investigated based on grafting degree(DG)and browning degree,indicating that DG of WPI-dextran conjugates increased with reaction time.The covalent grafting process of WPI-dextran conjugates was further identified by gel electrophoresis and spectroscopic tools,verifying the covalent binding of dextran to WPI.Results also showed that the grafting rate slowed down with the mounting conjugated dextran.To clarify the potential of protein molecular modification on size and stability control of nanoparticles,the structure and surface properties were characterized during conjugation.Results exhibited that the secondary and tertiary structures of WPI were remarkably altered as time increased.In detail,the α-helix proportion was enhanced from 25.4%to 29.6% while β-sheet as well as random coils reduced from 22.5% and 36.4% to 19.6% and32.7%,respectively.The microenvironment of chromophore changed to a varying extent as well.Besides,the surface hydrophobicity index decreased from 481 to 293,while surface sulfhydryl and total sulfhydryl content increased from 6.16 μmol/g and 15.50 μmol/g to 11.03μmol/g and 17.94 μmol/g,respectively.These changes are conducive to the improvement of the spatial stability of the conjugates and the fabrication of multi-scale low environment sensitive nanoparticles.The above WPI-dextran conjugates of different DG were used to prepare WPI-dextran conjugate/chondroitin sulfate nanoparticles by heat-induced gelation method.The impact of DG on mean size was analyzed by dynamic light scattering.The results showed that the average particle size of nanoparticles decreased with the increasing DG,which indicated that the nanoparticle size could be regulated by conjugation time.The sensitivity of different nanoparticles to p H,ionic strength and heat were further compared.The environmental sensitivity of nanoparticles was significantly decreased only by dry-heating for 2 d.The obtained nanoparticles remained stable under the synergetic action of p H 1-10,Na Cl concentration of 1-4 mol/L and heat(90℃ for 30 min).Additionally,Na Cl promoted the thermal stability of nanoparticles.The regular spherical structure was confirmed by the morphology characterization of nanoparticle which also indicated Na Cl counter-ion was able to enhance their heat stability by binding to the surface of nanoparticles.Then nanoparticles were applied for the encapsulation of cinnamaldehyde to explore the potential for delivering active compounds of plant essential oil.WPI-dextran conjugates(1-3 d)/chondroitin sulfate nanoparticles presented better cinnamaldehyde loading ability with encapsulation efficiency(EE)of 76%-80% and loading capacity(LC)of 19%-20%.Based on the particle size,dispersity,stability and the encapsulation ability of cinnamaldehyde,the WPI-dextran conjugated for 2 d was selected for the further development of low environment-sensitive flavor ingredients.The above low environment-sensitive nanoparticles were applied for cinnamaldehyde loading to obtain p H,salt and heat stable flavor ingredients.Based on size,polydispersity index(PDI),EE and LC,the optimal ratio(cinnamaldehyde to WPI)was determined as 1:1.The particle diameter and PDI were 185 nm and 0.22,respectively.The EE and LC were 76.57%and 19.02%,respectively.Structural characterization was used to investigate the mechanism of cinnamaldehyde entrapment,which indicated that hydrophobic interaction and hydrogen bonding promoted the entrapment of cinnamaldehyde into nanoparticles.Fluorescence spectra revealed that the strong affinity to cinnamaldehyde of WPI promoted the embedding and protection of cinnamaldehyde by nanoparticles.The stability and processing adaptability of cinnamaldehyde nanoparticles in practical application system were predicted based on food matrix processing and storage conditions simulation by p H,ionic strength and temperature alteration.The results showed that the nanoparticles effectively protected cinnamaldehyde under the combing effect of p H 1-10,Na Cl concentration 0-4 mol/L and heat(90℃,30min).Thermogravimetric analysis showed that the encapsulation significantly improved the thermodynamic stability of cinnamaldehyde,which further confirmed the effective protection of nanoparticles on cinnamaldehyde.After storage for 6 weeks in disparate environment,the diameter of nanoparticles did not change significantly and the retention rate of cinnamaldehyde encapsulated in nanoparticles was more than 80%.Based on the kinetic model fitting of cinnamaldehyde retention during storage,the release mechanism of cinnamaldehyde nanoparticles under different storage conditions is zero-order release.Besides,GC-MS and antibacterial experiment were performed to assay the activity of cinnamaldehyde in nanoparticles before and after storage.The results showed that no obvious oxidation and metamorphism of the cinnamaldehyde in the dispersion were observed during storage.Cinnamaldehyde-loaded nanoparticles had high antimicrobial effect on E-coli.A new mild method for low environment-sensitive WPI-dextran conjugate/chondroitin sulfate nanoparticles preparation was established by using enzyme-induced gelation instead of heat-induced gelation method.The effect of transglutaminase(TGase)and laccase cross-linking on the environmental sensitivity of nanoparticles were compared.TGase cross-linking remarkably ameliorated particle stability at p H 1-10,Na Cl concentration 0-4 mol/L and heating(90℃,30 min).However,the stability of nanoparticles was not improved under the action of laccase.The stabilization and destabilization mechanisms of enzymatic cross-linked nanoparticles were analyzed from the perspective of structural changes and molecular forces.It was found that a large number of isopeptide bonds were induced by TGase,with formation of some disulfide bonds,which facilitated the stability of the structure of nanoparticles.However,laccase led to protein degradation through radical-mediated reaction.The major forces involved corresponded to weak hydrogen and hydrophobic interaction with formation of tiny amount of isopeptide bond,which is not capable of increasing particle stability.Besides,the loading properties of cinnamaldehyde nanoparticles were investigated.It was found that TGase cross-linked cinnamaldehyde nanoparticles showed the smaller particle size(134 nm)and PDI(0.201).Moreover,the EE and LC were 69.68% and 17.31%,respectively,which were higher than those of laccase cross-linked cinnamaldehyde nanoparticles.Furthermore,the covalent cross-linking induced by TGase was more conducive to the protection and long-term functional performance of cinnamaldehyde.In addition,in vitro release showed that TGase cross-linked cinnamaldehyde nanoparticles exhibited better resistant to digestive attack and sustained-release performance in the gastrointestinal tract.