Construction Of Starch-based Double Emulsions And Its Delivery Mechanism Of Astaxanthin

With the increase of people’s awareness of health and nutrition,lipophilic functional factors which can improve the health level of the body have attracted more and more attention.However,lipophilic functional factors have poor water solubility,sensitivity to environmental conditions,and low absorption,resulting in their poor bioavailability in human body after oral intake.Lipophilic functional factors are usually embedded in the oil phase of emulsion delivery system to effectively protect and enhance their biological activity.Double emulsions,as a kind of emulsion delivery system,can effectively control the slow and continuous release of functional factors in the specific environment of gastrointestinal tract,which is conducive to the absorption and utilization of functional factors.However,most of the current studies on double emulsions focus on the control and sustained release of functional factors in the gastrointestinal tract,while ignoring the absorption and utilization of functional factors.Starches have the advantages of low cost,good biocompatibility and biodegradability,which are the ideal carrier material for constructing green and safe food-grade delivery system.In this paper,starch-based double emulsions（SDEs）were prepared with corn starch as raw material using two-step emulsification method.The formation mechanism of SDEs were clarified from the interface point of view.Secondly,the stability of SDEs were analyzed.Further,astaxanthin（AST）was as lipophilice functional factor model,and the digestion behavior in the gastrointestinal environment of AST-loaded SDEs（AST-SDEs）was investigated by in vitro simulated digestion model.In addition,the release characteristics of AST from the SDEs were further analyzed and the release mechanism was clarified.Finally,Caco-2 cells were used as the model of small intestinal epithelial cells to analyze absorption of AST in the SDEs after digestion,and to clarify their absorption mechanism.The main contents of research are as follows:Firstly,SDEs were prepared by two-step emulsification method using corn starch as water phase,soybean oil as oil phase,Tween 20 and Span 80 as emulsifiers.The morphological observation of the emulsion was measured by optical and laser confocal microscopy（CLSM）to determine the preparation process of SDEs.That is the volume ratio and total concentration of Tween 20 and Span 80 during the first emulsification of 3:2 and 2%（v/v）,respectively.When the starch concentration was 7%,5%and 3%（w/w）,W/O/W type-A,B,and C SDEs were formed,respectively.The interfacial properties of three kinds of SDEs were investigated by interfacial tensiometer,interfacial rheometer,and differential scanning calorimeter.Results showed that the SDEs were formed when the interfacial tension was less than 10 m N/m,ΔH′′<ΔH′.Also the first interfacial film was dependent on hydrophobicity,and the second interfacial film was dependent on the interaction of hydrogen bond and hydrophobicity.When the elastic modulus（Gi′）was 0.1-1 N/m,the interfacial adsorption rate of starch was 50%-70%,mainly based on type-A SDEs were formed.When the Gi′was 0.01-0.1 N/m,the interfacial adsorption rate of starch was 40%-50%,mainly based on type-B SDEs were formed.When the Gi′was 10^-3-10^-2N/m,interfacial adsorption rate of starch was 20%-40%,mainly based on type-C SDEs were formed.Secondly,the effect of temperatures,p H values,sodium chloride concentrations,and storage time on stability of A,B,and C SDEs were investigated by laser particle size analyzer,optical microscope,rheometer and low-field nuclear magnetic resonance imaging analyzer to reveal the stability mechanism of SDEs.Results showed that with the increase of temperature or the presence of sodium chloride,both type-B and type-C SDEs became single emulsion,but the droplet size distribution and microstructure of type-A SDEs remained unchanged,and the volume mean particle size(d₅₀)was the smallest,indicating that type-A SDEs had good thermal stability and salt ion resistance.Compared with type-B SDEs,type-A and C SDEs were not sensitive to p H value.After storage of 14 days at 4℃,type-A SDEs still maintained double emulsion structure and basically unchanged particle size distribution,indicating that they had good storage stability.In addition,the stability mechanism of SDEs was mainly due to the small droplet size,the viscosity of the overall system up to 100 Pa·s,the highly elastic gel network structure Tanδ<1,and the dense and elastic interfacial film.Further,AST was as lipophilic functional factor model.The storage stability of three types of AST-SDEs was explored.Result indicated that type-A AST-SDEs presented the smallest droplet size and excellent storage stability for 35 d.The AST remaining of types-A,B,C SDEs of were 90%,74%,and 69%,respectively,and the encapsulation efficiency were above 94%.The changes of droplet size,microstructure and Zeta potential of type-A,B,C AST-SDEs during digestion were investigated by in vitro digestion model.Results revealed that after simulated gastric juice（SGF）digestion,all the double emulsions structures disappeared,forming droplets of evenly mixed oil and water.The Zeta potential also increased from negative to neutral or slightly positive.The d₅₀value of type-B SDEs increased from 11.18μm to 27.93μm.The d₅₀value of type-A and type-C SDEs remained unchanged,which were 8.23μm and 21.77μm,respectively.After simulated intestinal fluid（SIF）digestion,the Zete potential value changed from neutral or slightly positive to negative.type-B and type-C SDEs formed irregular large clusters with the increasing of droplet size,while type-A SDEs were still small droplets evenly mixed with oil and water.The lipolysis efficiency of AST-SDES was in the order of type-C?type-B?type-A,and bioavailability of astaxanthin was 42.63%,37.19%,17.04%,respectively.The bioavailability of type-C AST-SDEs was four times higher than that of free AST.In addition,the release behavior and mechanism of AST in three types of SDEs after digestion was studied.In vitro release model experiment showed that release rate of AST was less than 10%after digestion in SGF,but a slow and continuous release was achieved in SIF.The release kinetics model showed that the release mechanism of AST was diffusion controlled,and both type-B SDEs at SGF and SIF stage and type-A SDEs at SIF stage followed the non-Fick diffusion release mechanism,suggesting that the diffusion of AST was combined with the surface erosion of SDEs.Both type-C SDEs at SGF and SIF stage and type-A SDEs at SGF stage follow the Fick diffusion release mechanism.In addition,the interaction between the SDEs and AST was analyzed.Results showed that AST was only dissolved in the oil phase in the type-B SDEs,while there were hydrophobic interactions and type-V complexes formation between the type-A and type-C SDEs and AST,which was beneficial to regulate the diffusion of AST.Finally,based on Caco-2 cell model,the effect of cell on the absorption efficiency of AST in type A,B,and C SDEs after gastrointestinal digestion were investigated using CLSM,high performance liquid chromatograph,and flow cytometer equipments.Results showed that compared with AST oil solution,the cell absorption rate of AST in type-A,B,C SDEs increased by 0.8,0.6 and 0.5 times,respectively.The type-A SDEs could significantly improve absorption rate of AST,mainly because part of AST and starch carrier formed evenly mixed small droplets to be directly absorbed by cells.While type-B and C SDEs were absorbed by cells through AST formed mixed micelles.The cell absorption pathway of AST in the SDEs was investigated by absorption inhibition and cell bypass experiments.Results revealed that the AST in the type-A SDEs was absorbed through clathrin and fossa protein mediated endocytosis and cell bypass pathway.The AST in type-B SDEs was absorbed through clathrin,fossa protein,and macropinocytosis mediated endocytosis as well as cell bypass pathway.While the AST in the type-C SDEs was absorbed only through cell bypass pathway.