| Wheat gluten proteins(WP)are nutritious and cheap plant proteins which are widely used in baking area due to their unique viscoelastic properties.However,the low water solubility leads to leads to low processing performance and single application area,which significantly inhibited the application of WP.The common techniques for solubilization of WP have limitations such as high cost,poor effect and high nutrient loss,and a novel green modification technique is urgently needed.In this study,we investigated the physical structural assembly of WP and foreign amphiphiles under the induction of pH-cycle treatment,and constructed hydrophilic protein conformations by nanotechnology to realize the solubilization of WP.The molecular mechanism of structural assembly for the solubilization of WP was investigated.Based on this mechanism,the delivery performance of assembled structures for hydrophobic bioactive molecules were evaluated to expand the value of WP for their application in high value-added fields.The details of the work are as follows.The approach for the construction of water-soluble conformations by structural self-assembly of WP was established,and the effect of binding between WP and shellac on structural self-assembly and solubility of WP under pH-cycle induction was investigated.WP and shellac were co-dissolved in dilute alkaline solution at pH 12,then the solution was adjusted to pH 7prior to centrifugation to obtain soluble WP-shellac composites(WSC).SDS-PAGE analysis demonstrated that the pH-cycle treatment improved the solubility of WP to about 92%without affecting the primary structures of WP.As the neutralization reaction proceeded,the WP bound to shellac molecules,and then refolded and aggregated under the main driving effect of hydrophobic interaction,assembling into WSC nanoparticles with particle sizes around 200 nm.Fluorescence spectroscopy and Zeta-potential demonstrated that the embedding of shellac into the self-assembled structures of WP increased the Zeta-potential and decreased the hydrophobicity of WSC.Shellac molecules provided electrostatic repulsion to restrict the refolding of WP,resulting in the relatively unfolding conformations of WSC which were similar to that of molten proteins.The secondary structures of WP showed no significant changes during the self-assembly process.Therefore,the molecular mechanism of pH cycle-induced structural assembly to enhance the solubility of WP was that the electrostatically dissociated WP reassemble during neutralization,and the embedded shellac molecules provided electrostatic repulsion to achieve the self-assembly of hydrophilic protein conformations by restricting WP refolding.With WSC as the carrier,the hydrophobic curcumin molecules participated in the self-assembly of WSC by pH-cycle.Curcumin was encapsulated in WSC with an encapsulation capacity up to 124.9 mg/g and an encapsulation efficiency up to 71.3%.Moreover,encapsulation of curcumin by WSC effectively improved its oral bioavailability in animal experiments.The supramolecular co-assembly of WP with soy proteins(SP)was achieved based on the approach for structural self-assembly of WP.WP co-assembled with SP in supramolecular structures by pH cycle to form soluble spherical nanoparticles.The co-assembly resulted in complete dissolution of SP,and the solubility of WP increased up to 72.4%.Unlike the shellac,which participated in the self-assembly of WP by embedding structures,the conformational assembly of SP and WP was driven simultaneously by hydrophobic interaction,electrostatic interaction and hydrogen bonding through structural co-folding.The incorporation of SP limited the co-folding of proteins and significantly inhibited the formation of tertiary structures,thus conferring excellent water solubility to the co-assembled nanoparticles.Due to the key role of SP in promoting the solubility of WP,the isoelectric point of WP shifted to pH 4?5 after structural co-assembly,similar to that of SP.However,the co-assembled particles showed great solubility of more than 89%at pH?7.Due to the relative unfolding of protein conformations caused by limited refolding,the structural co-assembly significantly enhanced the interfacial properties of WP,and their emulsifying activity and foaming properties increased from 8.2 and160.0 cm~3/g to 15.7 and 683.3 cm~3/g,respectively.The amino acid composition was improved with the structural co-assembly.The co-assembled nanoparticles combined the nutritional advantages of both WP and SP with essential amino acid indexes(EAAI)of 58.97-64.91,which were higher than that of WP(55.95).Therefore,the co-assembled nanoparticles could meet the daily intake requirement of adults.The oil-water interface was structured using the oil-water amphiphilicity of the WP-SP co-assembled nanoparticles.The WP-SP co-assembled particles showed three-phase contact angles ranged from 68.5°to 79.7°,and were able to stabilize gel-like high-internal-phase Pickering emulsions(HIPPE)at c=1%?3%and?=0.7?0.8.Increasing the percentage of SP could significantly promote the wettability of the assembled particles on the aqueous side.In this way,the interfacial properties of the assembled particles could be adjusted by controlling the ratio of WP and SP.The rheological results showed that increasing the proportion of SP in the co-assembled particles could enhance the electrostatic repulsion between the interfaces,which effectively inhibited the agglomeration of oil droplet,and increase the storage modulus(G'value)of HIPPE gels.The HIPPE stabilized by co-assembled particles with different WP:SP ratios all had long-lasting stability during 20 d of ambient storage and great thermal stability at90°C.The structures of HIPPE were destroyed after the freeze-thaw treatment due to the influence of ice crystals on the adsorbed layer.However,the nanoparticles still maintained their interfacial properties and could be re-emulsified into gel-like HIPPE after homogenization,giving HIPPE great performance for repeated processing.Increasing c and?could improve the thickness and interconnection of adsorption layer,strengthen the and reduce the oil droplet freedom,which finally induced the enhancement of the G'value and apparent viscosity of HIPPE.Self-emulsified delivery systems(SEDS)with antimicrobial activity were constructed by the self-emulsification reaction of eugenol induced by supramolecular co-assembly of WP and SP.Eugenol could be dissolved in dilute alkali solution at pH 12 after a boiling water bath treatment.This solution could undergo self-emulsification reactions with WP-SP co-assembled structures by pH-cycle.Nano-emulsions with particle sizes ranging from 83.3 to 182.3 nm and Zeta-potentials greater than-21 m V were obtained by the self-emulsification.Cryo-SEM and TEM showed that pH-cycle induced conformational transition of SP and WP from two-dimensional to three-dimensional to form a core-shell structure with eugenol as the core and WP-SP co-assembled structures as the wall shell.Fluorescence and FTIR results indicated the mechanism of structural co-assembly-induced self-emulsification of eugenol,which was that the structures of WP and SP refolded after pH-cycle,and the binding of hydrophobic eugenol molecules to proteins could promote protein refolding,prompting the protein to encapsulate eugenol and induce the self-emulsification reaction.The self-emulsification behavior was a physical assembly process driven by multiple non-covalent forces dominated by hydrophobic interactions.The encapsulation capacity of SEDS for eugenol reached about 500 mg/g,and the self-emulsification reaction increased the solubility of eugenol for 3.5?7 times.Moreover,eugenol solubility could be further enhanced by concentrating SEDS.The storage and thermal stability of SEDS was improved as the incorporation of WP could inhibit protein aggregation by promoting protein refolding,which made SEDS meet the requirements of thermal processing in food production.The core-shell structure of SEDS allowed the slow release of self-emulsified eugenol while maintaining its inhibitory activity against Gram-negative and positive bacteria.The freeze-dried SEDS showed nanostructures with particle size of 67.6?174.6 nm after re-solubilization,and played a significant role in inhibiting bacteria and preserving freshness after be coated on the surface of rice cake,effectively extending the shelf life of food systems. |
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