Approaches,Characteristics And Applications For Trehalose To Efficiently Fabricate Protein-based Nanoemulsions

In the last decade,nanoemulsions have received increasing attention due to their small particle size,good solubility,high transparency,stability and bioaccessibility compared to conventional emulsions.It is generally considered that nanoemulsions are better formed using synthetic small molecule surfactants as emulsifiers.However,growing evidences have been accumulating to demonstrate their poor stability and disturbing biological fate in human bodies.In response to such dilemma,food-grade nanoemulsions have been developed.Proteins,as natural amphiphilic molecules,are frequently used in the preparation of food-grade emulsions.Nevertheless,the research on protein-based nanoemulsions has not gain enough attention,mainly due to the low interfacial activity and emulsification efficiency of proteins,which make it difficult to prepare stable protein-based nanoemulsions efficiently.In attempt to overcome such limitations,a novel strategy was proposed in this work for the facile preparation of highly stable protein-based nanoemulsions by introducing trehalose.Moreover,this work also focused on investigating the mechanisms for the formation and stabilization of protein-based nanoemulsions induced by trehalose and successfully unveiled the influence of trehalose on protein emulsifying performance by studying protein aggregation properties,structural stability and interfacial adsorption behavior.On the basis of the theoretical study,the feasibility of trehalose in the efficient encapsulation,delivery and productization of bioactives in proteinbased nanoemulsions was further investigated.Firstly,protein-based nanoemulsions were prepared using soybean isolate(SPI)as a model for complex oligomeric globulin.It was found that the introduction of trehalose(5-40 wt%)could overcome the occurrence of ’over-processing’ during SPI-based nanoemulsions fabrication and produced nanoemulsions with small average particle size(～300 nm),low flocculation index and high stability at relatively low energy input(80 MPa).Moreover,such effect was positively correlated with trehalose concentration.The presence of trehalose,irrespective of concentration,was found to significantly inhibit the aggregation tendency of SPI,mainly through the formation of a ’shell layer’ around protein particles,which significantly reduced protein surface hydrophobicity,resulting in denser protein structure with lower aggregation and smaller particle size.Meanwhile,the formation of core-shell structure was very stable,which effectively inhibited the unfolding and aggregation of protein particles during high-pressure microfluidizaiton and improved protein emulsification performance to facilely fabricate nanoemulsions with smaller particle size.Secondly,nanoemulsions were prepared using soybean 7S and 11S proteins as oligomeric globulin model.Results showed that the introduction of trehalose favored the breakup of droplets and significantly improved the emulsification ability of oligomeric globulins to different degrees.The presence of trehalose significantly inhibited the aggregation and denaturation of proteins during emulsification,regardless of protein type,and allowed proteins to remain stable in secondary and tertiary structure.In addition,in trehalose-rich system,protein particles with low aggregation extent diffused more rapidly to the oil-water interface.Once adsorbed to the interface,the structural unfolding and rearrangement of proteins was inhibited to varying degrees: the 7S protein underwent subunit dissociation and the dissociated subunits were coated with trehalose-shell and could act as a kind of ’Pickering soft particles’ with high structural stability at oil-water interface;the 11 S protein only underwent limited structural unfolding and was able to stabilize the oil-water interface as a kind of natural ’Pickering particles’.Next,the nanoemulsions were prepared using bovine serum albumin(BSA)as monomeric globulin model and were found to be less stable.Introducing trehalose could not only overcome the above limitations but also produced nanoemulsions with smaller droplet size(< 200 nm).Further analyses indicated that the presence of trehalose could form a “shell” structure around protein particles to protect protein against extensive denaturation and aggregation during microfluidization and structural unfolding and rearrangement at oil-water interface,allowing monomeric globulins to perform as ’Pickering particles’.Meanwhile,the existence of aggregated trehalose in the solution not only imparted nanoemulsions with higher physical stability but also favored the fast diffusion and adsorption of proteins and contributed to more balanced protein wettability,allowing monomeric globulins to stabilize more interfaces with Pickering effect.Then,nanoemulsions were prepared using sodium caseinate(SC)as non-globulin model.It was found that the average size of nanoemulsions gradually decreased and their stability gradually increased with increasing trehalose concentration.The nanoemulsions prepared in trehalose-rich system exhibited high storage,freeze-thaw and p H stability,and could be used as effective encapsulation carriers for volatile essential oils and oxidisable oils to improve their stability.The underlying mechanisms were closely associated with the improved protein adsorption behavior by trehalose.For one thing,the existence of trehalose could favor the interfacial diffusion and adsorption of protein.For another,the anchoring and unfolding of linear protein molecules at oil-water interface was facilitated,allowing SC to cover more droplet interfaces and form a denser interfacial protein layer.Finally,protein-based nanoemulsions that encapsulated astaxanthin were prepared and spray dried.The astaxanthin enriched nanoemulsions produced in trehalose-rich system were found to be small sized,less flocculated,more stable(against light,heat,storage).In addition,the incorporation of trehalose also contributed to high encapsulation efficiency(> 90%),low astaxanthin loss and high bioavailability.Apart from that,the trehalose-rich nanoemulsions remained stable during spray drying and displayed high spray-drying yield,astaxanthin retention and water solubility(> 90%).In conclusion,this paper not only unveiled the intrinsic mechanisms for trehalose to efficiently prepare stable protein-based nanoemulsions,but also verified the effectiveness and universality of this strategy.Moreover,the strategy proposed in our work provided ideas for the development and application of protein-based nanoemulsions as encapsulation carriers which was innovative and important.