Fabrication, Interfacial Behaviors And Functional Delivery Of Soy Peptide-based Nanoparticles

Enzymatic hydrolysis has been widely applied to develop soy protein-based bioactive peptides with various health benefits or to improve the functional properties of soy protein for decades.However,owing to the natural compacted internal structure of soy protein and the release of hydrophobic clusters or peptides during proteolysis,protein-peptide/peptide-peptide aggregation would unavoidably appear,leading to the formation of undesirable insoluble aggregates,which can be up to 40%by weight,significantly reducing the industrial value and hindering the sustainable development of soy protein in food industry.In this study,we focused on the insoluble aggregates formed during protein hydrolysis,aiming to develop sustainale application of soy protein hydrolysates.Firstly,insoluble aggregates were collected when the hydrolysates showed good ACE-inhibitory and antioxidant activities,the chemical and structural properties and in vitro digestibility of the obtained aggregates were determined to evaluate their potential for food application.Ultrasonication was then adopted to induce molecular self-assembly of the insoluble aggregates into multi-functional soy peptide nanoparticles.The potential of the resultant nanoparticles as interfacial stabilizer and bioactive delivery vehicle were further investigated,attempting to provide useful guidance for the high-valued application of soy protein and the development of sustainable biomaterials designed for newly functional food formulation.The main contents and results are listed as follows:1.The ACE-inhibitory and antioxidant activities of soy protein hydrolysates catalyzed by alcalase and protamex were evaluated.Results showed that along with the hydrolysis time,the DH and protein recovery of soy protein gradually increased,and when hydrolyzed for 24h,the growing rate slowed down.The ACE-inhibitory and antioxidant activity of the resultant hydrolysates all followed the manner that first increased and then decreased with hydrolysis time increasing,and the values reached the highest at hydrolysis time 24 h.However,at the same time,large amount of insoluble protein or peptide aggregates formed upon hydrolysis and thermal inactivation were obtained at the highest bioactivities,which could be up to 40%by weight,significantly reducing the enzymatic efficiency of soy protein.Driven by the green and sustainable development in food industry,we made further investigation on the insoluble aggregates formed upon the above enzymatic condition(Alcalase/Protamex,50°C,24 h).2.Insoluble aggregates of soy protein upon alcalase-and protamex-catalyzed hydrolysis and thermal inactivation were collected and nominated as SPA and HSPA respectively.The chemical and structural properties of the obtained aggregates were examined to explore the aggregation mechanism involved upon proteolysis,and the in vitro digestibility and antioxidant activity of the digests were determined to further evaluate the potential for food application.Results clearly showed that the insoluble aggregates formed upon hydrolysis were aggregated peptides,SPA was formed mainly attributed to the hydrophobic interactions between peptides with molecular weight among 10 and 18 kDa.Thermal inactivation further enhanced the protein/peptide–peptide interactions through hydrophobic forces and disulfide bonds,accelerating the aggregation,where fractions from the basic subunits of glycinin(also some acidic subunits and theβsubunit ofβ-conglycinin)were particularly involved in HSPA.Furthermore,both SPA and HSPA had a high proportion of essential amino acids and were relatively resistant to gastric digestion,while could be well digested in the intestinal tract and simultaneously exhibited good antioxidant activity due to the existence of small peptides and antioxidative amino acids released during digestion.These results indicated a potential use of the insoluble peptide aggregates as functional supplements or active delivery systems for human consumption.3.Based on a―top-down‖strategy,a new type of soy peptide nanoparticles originated from SPA and HSPA was successfully fabricated through ultrasound-induced molecular self-assembly,the resultant nanoparticles were nominated as SPN and HSPN.The particle size and morphology of SPN and HSPN were investigated.Results showed that SPN exhibited spherical appearance with a rather smooth surface,and the particle size was about103.95 nm,while HSPN formed a core-shell structure with submicron scale at 489.70 nm.SPN was mainly maintained by non-covalent interactions,especially both hydrogen bonds and hydrophobic interactions,while in the HSPN case,the predominant interactive forces were hydrophobic interactions and disulfide bonds.Moreover,based on the relative pepsin-resistance but high degradability toward pancreatin of SPN and HSPN,curcumin was then selected as a model active cargo to evaluate their encapsulation and active delivery ability.Results indicated that curcumin was efficiently encapsulated into SPN and HSPN upon sonication,showing significant improvement in water dispersity(10~4 higher).Additionally,the pepsin-resistant SPN and HSPN could lead to the controlled release of curcumin at intestinal phase,and thus significantly improve the bioaccessibility.Noticeaby,encapsulated curcumin was effective in protecting glutamate-induced toxicity in PC12 cells,where the matrix SPN could simultaneously reduce lipid peroxidation and elevate antioxidant enzymes levels,innovatively demonstrating the bifunctionality of SPN during cellular delivery.4.Further studies on the interfacial properties(involved the adsorption kinetics and dilatational rheological properties)of SPN and HSPN were carried out.Results showed that both SPN and HSPN could effectively adsorb onto the oil-water interface,leading to the formation of stable interfacial structures.For HSPN,the core-shell structure enhanced the intermolecular interaction among particles surface,promoting structural rearrangement among particles and finially forming a more stable interfacial layer.In addition,thermal treatment accelerated the intermolecular interaction of SPN particles,the resultant SPN-H showed a more stable interfacial structure than SPN.When emulsifying,though showing a rapid creaming shortly after homogenization under gravity due to larger droplet size formed,SPN exhibited good emulsion performance with excellent stability against coalescence,which showed the typical character of Pickering emulsion.This could be mainly attributed to the effective coverage of peptide nanoparticles onto the droplet surface and the stable interfacial layer formed.These results indicated that a potential use of SPN as interfacial stabilizer in food emulsion system.5.HPMC and Tween 80 were then selected to incorporate into SPN,respectively,attempting to fabricate emulsions with long-term physical stability and high lipid oxidative stability.Rresults suggested that there existed competitive adsorption between SPN and HPMC/Tween 80 at O/W interface and mixed SPN-HPMC and SPN-Tween 80 interfaces were well formed.Emulsions stabilized by the mixed interfaces further facilitated the formation of finely distributed emulsions with smaller droplet size and good long-term stability against creaming and coalescence.Moreover,lipid oxidation in emulsions stabilized by SPN and its mixed SPN-HPMC and SPN-Tween 80 complexes,as evaluated by the formation of lipid hydroperoxides and volatile hexanal,was also well suppressed,which should be mainly due to the excellent antioxidant capacity of bioactive SPN at the oil-water interface that endowed the emulsions with improved oxidative stability.All these results indicated that SPN could act as bifunctional and effective interfacial stabilizer for preparing functional emulsion systems.