| In recent years,functional foods are widely known to play a prominent role in preventing chronic diseases and improving physical or mental well-being.Many strategies have been put forward to develop foods with different health effects,among which the most effective strategy is to add healthy bioactive substances to food.However,most of nutraceuticals possess poor solubility and stability,and what's worse,low bioavailability,which greatly limits their applications in the functional food formula.Food colloid delivery technology is one of the effective strategies to break through the application bottleneck of this kind of bioactive substances.The emerging protein self-assembly technology provides a technical solution for the development of bioactive nano carriers with high encapsulation performance,by which the hydrophobic nutraceuticals are expected to be encapsulated in the hydrophobic core of the protein architectures.Such delivery system shows many advantages over other nanocarriers including high load capacity as well as better colloidal stability.Accordingly,this paper revealed the assembly process of proteins and discussed the feasibility of assembled protein particles as carriers for bioactive in-depth.The main results of the work include but not limited to as following:Firstly,a novel self-assembled core-shell nanostructured particle was prepared by urea induced 7S dissociation and recombination strategy,which was used for the efficient encapsulation of curcumin.It was found that urea concentration([U])affected the dissociation of 7S subunit.At [U] > 4 M,the structure of 7S gradually dissociated into its separate subunits(?,??,?)and even denatured.After dialysis,the dissociated subunits would reassemble into a kind of core-shell nanostructured particles,in which aggregated ?-subunits acted as the core while the shell layer was mainly composed of ?-and ??-subunits.Curcumin crystals were directly introduced into the 7S solution at high urea concentrations(e.g.,8 M)and would preferentially interact with the denatured ?-subunits.As a consequence,almost all of the curcumin molecules were encapsulated in the core part of the reassembled core-shell nanoparticles.The loading amount of curcumin in these nanoparticles could reach 18 g of curcumin per 100 g of protein,which far exceeds those reported previously.In addition,the feasibility of the dissociation and recombination strategy to prepare the carrier of active substance was verified by the assembly 7S particles induced by ethanol.By adjusting the initial ethanol concentration([E]),we can precisely adjust the size and morphology of these recombinant particles.Under the condition of [E] = 40%,the load capacity of recombinant 7S particles on curcumin was the highest.Secondly,the structure of the assembled nanoparticles was strengthened by crosslinking with genipin to improve the stability of protein-based nanoparticles in the gastrointestinal environment.The optimal cross-linking conditions were determined by ninhydrin method and amino acid analysis method,which was further confirmed and characterized by FT-IR.The transmission electron microscopy(TEM),dynamic light scattering(DLS),and electrophoretic mobility measurements revealed that genipin cross-linking significantly prevented the structural disintegration and drug leakage of assembled nanoparticles in simulated GI environment.Then,the core-shell nanoparticles were prepared using polysaccharides such as chitosan,dextran and soybean soluble polysaccharide as a replacer of genipin to study their digestion and release behavior in simulated gastrointestinal environment.It was found that the polysaccharides,whatever types,can protect 7S protein-based nanoparticles from hydrolysis by pepsin and delay the digestion rate in small intestine in an efficient way.Moreover,the surface properties,entrapment efficiency,colloidal stability and digestion release behavior of the co-assembled particles could be regulated by changing the type of polysaccharides,p H value,complex ratio(MR),solid content(SD)and initial curcumin concentration([Cur]).Finally,the in vitro and in vivo models were used to evaluate the effectiveness of the protein/polysaccharide co-assembled core-shell nanoparticles for colon-targeted therapy of ulcerative colitis(UC).To ensure that the nanoparticles will not dissolve in the upper digestive tract,genipin was used to modify the nanoparticles.The release of curcumin from co-assembled particles in stomach,small intestine and colon was investigated by simulated digestion in vitro,where the structural changes of co-assembled particles during digestion were monitored by TEM.Results showed that the "core-shell" structure of the assembled particles survived from gastrointestinal stage which led to less than 10% of curcumin release.In the simulated colon phase,the structure of the assembled particles was destroyed,and the curcumin release rate reached the peak within 8 h.Afterwards,the anti-inflammatory effects of the assembled particles in vitro and in vivo were verified by cell inflammation model and mouse colitis model respectively.Compared with the control group,the assembled particles loaded with curcumin had significant improvement effects on various inflammatory indexes.In conclusion,the preparation of nanocarriers using protein denaturants(such as urea and ethanol)to induce protein dissociation and recombination can significantly improve the watersolubility,colloidal stability,bioavailability and bioactivity of hydrophobic active substances.The proposed strategy provides a feasible and innovative technical solution for the development of protein-based nanostructures as efficient transport carriers for nutraceuticals. |
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