| The amphiphilic polysaccharides-based polymer can self-assemble to form micelles in the aqueous solutions,which has increasingly become a focus in the field of solubilization and controlled-release for loading liposoluble constituents in the food industry.However,the premise of their application in the food industry is to overcome the processing impacts,which is different for the thermal-and p H-response and stability of polysaccharides-based micelles as the delivery systems in the digestive tract,blood,and tissue fluid in the medical industry.It was reported that as one of the most common processing procedures in the food industry,the thermal or acidic/alkaline treatments,has a significant influence on the food compositions and their aggregation structures.Therefore,understanding in molecular mechanisms underlying the micellar processes of amphiphilic polysaccharides-based polymers,and the solubilization and controlled-release of liposoluble constituents by the resultant micelles,will contribute to widening their application in the food industry.By usingβ-carotene（βC）as a represent for food liposoluble constituents,the main sections of this study are as follows:the molecular mechanisms governing the effects of temperature and p H on the micellization of octenylsuccinated oatβ-glucan（OSβG）;the molecular mechanisms related to the effects of temperature and p H on the solubilization and controlled-release ofβC by OSβG micelles.The obtained results are listed as follows:（1）The micellization processes of OSβG molecules and the effects of temperature and p H on the structures of the resultant micelles were investigated,revealing the corresponding molecular mechanisms.（1）By measuring the dynamic water contact angle at the air-water interface and 1H nuclear magnetic resonance（1H NMR）,the results showed that the octenylsuccinate modification improved the amphipathicity of OSβG.During the micellization processes of OSβG molecules,their octenylsuccinate moieties were not completely oriented to the hydrophobic core,along with few parts projecting onto the surface,and this lowered the hydrophilicity of OSβG micelles surface.The 1H NMR,Fourier transform-infrared spectrum（FT-IR）and X-ray diffractometry（XRD）characterizations indicated that the micellization of OSβG molecules was triggered by hydrophobic interactions between octenylsuccinate moieties,and the core-shell structure of OSβG micelles were further stabilized via both hydrophobic forces between octenylsuccinate moieties and hydrogen-bonding interactions between oatβ-glucan moieties.（2）The effects of temperature（293-370 K）,p H（2.5-12.5）and their interactions on the structures of OSβG micelles were investigated by a combination of dynamic light scattering,1H NMR,pyrene-labelled fluorescence spectra,thermodynamic analysis(ΔG0agg,ΔH0agg andΔS0agg),and small-angle X-ray scattering（SAXS）techniques.At p H6.5,the size decreased with temperature,while the surface charge continuously increased.By increasing p H at 293 K,parabolic and U-shaped trends were observed in the size and surface charge,peaking at p H 8.5 and 6.5,respectively.At any tested p H,the size decreased with temperature.Overall,the surface charge significantly increased with temperature at each p H.The critical micelle concentration（CMC）significantly increased with p H.Except for the subtle variations at p H 2.5,the CMC significantly increased with temperature at other p H values.As temperature increased,the compactness of hydrophobic core consisting of octenylsuccinate chains increased while the compactness of hydrophilic shell consisting of oatβ-glucan backbone changed oppositely.In an acidic environment,both the compactness increased by lowering p H,while they decreased in an alkaline environment with p H.The compactness changes were co-driven by enthalpy and entropy,and they corresponded to the changes in the hydrophobic interactions in hydrophobic core,hydrogen bonds in hydrophilic shell,and electrostatic repulsions among octenylsuccinate molecules.Accordingly,this work revealed the molecular mechanisms for both OSβG micellization and temperature-/p H-regulated structure of OSβG micelles.Therefore,on the one hand,we offer a new insight into the regulation of the size and surface charge of OSβG micelles via changing the environmental temperature and p H.On the other hand,we also provide an understanding of the structural variations of OSβG micelles during various food processing conditions.（2）Molecular mechanisms relating to the effects of environmental temperature and p H on loading ofβC by OSβG micelles were deeply analyzed by investigating the solubilizing processes and their influence.（1）By measuring the variations of dynamic water contact angle at the air-water interface and 1H NMR of OSβG micelles before and afterβC loading,the data showed that the loading ofβC improved the order of octenyl residues migrating towards and completely orientating to the core of OSβG micelles,resulting in a strong hydrophilicity ofβC-loaded OSβG micelles surface.The ultraviolet visible,FT-IR,XRD,thermal characterizations and atomic force microscope showed that the loading ofβC by OSβG micelles was driven by the hydrophobic interaction betweenβC and octenylsuccinate moieties,andβC-loaded OSβG micelles was stabilized by both hydrophobic and hydrogen-bonding interactions.It was also confirmed that the loading ofβC took place in the core of the micelles and not in the outer layers.The loadingβC process and its molecular migration mechanism were further revealed by a combination of dynamic light scattering,surface tension,and confocal laser scanning microscopy.For their uptake ofβC,βC molecules were firstly adsorbed onto OSβG micelles by interacting with octenylsuccinate moieties scattered on the micelle surface.By further interacting with octenylsuccinate moieties located in the micelles shell,βC molecules travelled across the shell and finally were trapped in the hydrophobic core.Finally,the stable OSβG micelles was formed.（2）The effects of temperature（298-318 K）and p H（4.5-8.5）on the isomerization and degradation ofβC inβC-loaded OSβG micelles were investigated by HPLC and Raman techniques.The results showed that the isomerization and degradation of loadingβC molecules inβC-loaded OSβG micelles did not occur during any temperature and p H condition,indicating that OSβG micelles could efficiently protectβC molecules.（3）By determining the solubility ofβ-carotene,surface hydrophilicity,core hydrophobicity,size,and surface charge ofβC-loaded OSβG micelles,the molecular mechanisms of effects of temperature and p H on the loading ofβ-carotene in OSβG micelles were investigated.By increasing temperature and p H,both parabolic trends of the solubility ofβC inβC-loaded OSβG micelles were observed,peaking at 308 K and p H 7.5,respectively.The size and absolute surface charge ofβ-carotene-loaded-OSβG micelles decreased with temperature,while they gave parabolic changing patterns with p H.As temperature increased,the compactness of hydrophobic core consisting ofβC molecules and octenylsuccinate chains increased while the compactness of hydrophilic shell consisting of oatβ-glucan backbone changed oppositely.As p H increased,both the compactness decreased.The influence of temperature and p H on the abovementioned properties ofβC-loaded OSβG micelles was ascribed to their surface hydrophilicity,core hydrophobicity,and core/shell compactness via regulating molecule mobility,orientation,and interactions（hydrophobic,hydrogen bonds,and electrostatic interactions）by temperature/p H.Therefore,this work reveals the affecting mechanisms of temperature and p H on the loading ofβC by OSβG micelles.On the one hand,the loading behavior ofβC in OSβG micelles can be regulated by changing the environmental temperature and p H for the purpose of constructing stable micellar delivery systems ofβC.On the other hand,it enhances the solubilization effects of hydrophobic active compounds in OSβG micelles and further widen their application scope in different food systems.（3）The controlled-release behaviors ofβC fromβC-loaded OSβG micelles in simulated gastrointestinal fluids and various release media with different temperatures（25-45 oC）and p Hs（1.2-8.5）were investigated to reveal molecular mechanisms underlying the effects of temperature and p H on their controlled-release.（1）The release kinetics ofβC fromβC-loaded OSβG micelles was assessed in the semi-continuous homeostatic gastrointestinal simulation fluids.By fitting seven typical release kinetic models,the results showed thatβC was controlled-released from OSβG micelles as an integrated consequence of its diffusion as well as the swelling and erosion of OSβG micelles.（2）By increasing temperature of the release media from 25 oC to 45 oC,the cumulative release rate ofβC showed a gradual increasing trend.By increasing p H of the release media from 1.2 to 8.5,it showed a U-shaped trend.By fitting with seven typical release kinetic models,the results indicated that theβC release depended on a combination of Fickian diffusion and the erosion-controlled mechanism due to the shrinking and collapsing ofβC-loaded OSβG micelles via the protonated effect of carboxyl groups of octenylsuccinate chains in the case of p H 1.2 and 4.5.While in the case of p H 6.8,7.4,and 8.5,it was due to the combined effect of Fickian diffusion and swelling control because of the structural relaxation ofβC-loaded OSβG micelles caused by the deprotonated effect.（3）By using dynamic light scattering,atomic force microscope and confocal laser scanning microscopy to characterize the structural changes ofβC-releasing OSβG micelles during the above release processes and combining with the abovementioned release kinetics models,the model forβC releasing processes was established.The results indicated thatβC molecules need to overcome the following three barriers successively to complete the whole migration.Firstly,it escaped the hydrophobic core due to the broken balance among hydrophobic,hydrogen-bonding,and electrostatic interactions,and then easily passed thought the shell;secondly,it diffused from the OSβG micelles surface to the bulk solution via the free octenylsuccinate chains in the aqueous continuous phase;thirdly,it come out from the dialysis bag through the membrane to the aqueous phase outside the dialysis tube.As a result,this study uncovers the molecular mechanisms underlying the controlled-release ofβC by OSβG micelles.It provides a comprehensive and detailed analysis for the structure changes and controlled-release effects ofβC-releasing OSβG micelles triggered by temperature and p H.It is also beneficial to construct efficient and stable polysaccharide-based micellar material for delivering food liposoluble compounds and to promote industrial applications of this material in the food industry.With the successive steps,this study firstly probed the influence rule of temperature and p H on the micellization of OSβG;and on the basis of this finding,the effect mechanisms of temperature and p H on the solubilization and controlled-release ofβ-carotene by OSβG micelles were further investigated.The aim is to widen the application range of OSβG micelles andβ-carotene-loaded OSβG micelles,and further achieve their efficient and stable applications in the food fields. |
